Allergy and Allergic Diseases, 2 Volume Set

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Author: A. Barry Kay | Allen P. Kaplan | Jean Bousquet | Patrick G. Holt

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9781405157209_1_pre1.qxd 4/10/08 17:57 Page i

Allergy and Allergic Diseases

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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To our wives: Rosemary, Lee, Hélène and Barbara

Commissioning Editor: Maria Khan Development Editor: Jennifer Seward Production Controller: Debbie Wyer CD produced by: Meg Barton and iBooks Production Services

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Allergy and Allergic Diseases Editor-in-Chief

A. Barry Kay MD, PhD, FRCP, DSc, FRSE, FMedSci Emeritus Professor of Allergy and Clinical Immunology, National Heart and Lung Institute, London, UK

Editors

Allen P. Kaplan MD The National Allergy, Asthma, and Urticaria Centers of Charleston; Professor, Department of Medicine Medical University of South Carolina, Charleston, South Carolina, USA

Jean Bousquet MD, PhD CHU Montpellier, Service des Maladies Respiratoires, Hôpital Arnaud de Villeneuve, Montpellier, France

Patrick G. Holt DSc, FRCPath, FAA Head, Division of Cell Biology, TVW Telethon Institute for Child Health Research, West Perth, Western Australia, Australia

SECOND EDITION

FOREWORD BY K. FRANK AUSTEN, MD

In two volumes Volume 1

A John Wiley and Sons, Ltd., Publication

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This edition first published 2008, © 1997, 2008 by Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form Wiley-Blackwell. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Allergy and allergic diseases / edited by A.B. Kay . . . [et al.]; foreword by K. Frank Austen. – 2nd ed. p. ; cm. Includes bibliographical references and indexes. ISBN 978-1-4051-5720-9 (alk. paper) 1. Allergy. I. Kay, A.B. [DNLM: 1. Hypersensitivity–immunology. 2. Allergens–immunology. 3. Asthma–immunology. 4. Immunotherapy. WD 300 A4326 2008] RC584.A348 2008 616.97–dc22 2008000958 ISBN: 978-1-4051-5720-9 A catalogue record for this book is available from the British Library. Set in 9/12pt Meridien by Graphicraft Limited, Hong Kong Printed & bound in Singapore by Fabulous Printers Pte Ltd. 1 2008

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Contents

List of Contributors, ix

Part 2 Inflammatory Cells and Mediators

Foreword, xvii

11 Mast Cells: Biological Properties and Role in Health and Allergic Diseases, 217 Peter Bradding and Glenn Cruse

Preface, xviii

VOLUME 1 The Scientific Basis of Allergy Part 1 Immunology of the Allergic Response 1 Allergy and Hypersensitivity: History and Concepts, 3 A. Barry Kay 2 Development of Allergy and Atopy, 23 Catherine Thornton and Patrick G. Holt 3 T Cells and Cytokines in Asthma and Allergic Inflammation, 48 Chris Corrigan 4 Regulatory T Cells and Other Tolerogenic Mechanisms in Allergy and Asthma, 83 Catherine Hawrylowicz and Cezmi A. Akdis 5 IgE and IgE Receptors, 103 Brian J. Sutton, Andrew J. Beavil, Rebecca L. Beavil and James Hunt 6 Immunoglobulin Gene Organization and Expression and Regulation of IgE, 119 Hannah J. Gould and David J. Fear 7 Environmental Factors in IgE Production, 141 Anne Tsicopoulos, Catherine Duez and Andrew Saxon 8 Antigen-presenting Dendritic Cells and Macrophages, 166 Bart N. Lambrecht and Hamida Hammad 9 Innate Immunity in Allergic Disease, 187 Ian Sabroe 10 Signal Transduction in Allergic and Inflammatory Cells, 203 Rafeul Alam

12 Eosinophils: Biological Properties and Role in Health and Disease, 258 Simon P. Hogan, Helene F. Rosenberg, Redwan Moqbel, Simon Phipps, Paul S. Foster, Paige Lacy, A. Barry Kay and Marc E. Rothenberg 13 Neutrophils: Biological Properties and Role in Health and Allergic Diseases, 295 Alison M. Condliffe, Andrew S. Cowburn and Edwin R. Chilvers 14 Basophils: Biological Properties and Role in Allergic Diseases, 320 Gianni Marone, Giuseppe Spadaro and Arturo Genovese 15 Leukocyte Adhesion in Allergic Inflammation, 337 Michelle J. Muessel and Andrew J. Wardlaw 16 Airway Epithelium, 366 Pedro C. Avila and Robert P. Schleimer 17 Airway Vascularity in Asthma, 398 John W. Wilson 18 Fibroblasts and the Extracellular Matrix, 412 Lynne A. Murray, William G. Glass, Anuk M. Das and Geoffrey J. Laurent 19 Immune Complexes and Complement: Their Role in Host Defense and in Disease, 436 Michael M. Frank and C. Garren Hester 20 Bradykinin Pathways and Allergic Disease, 451 Allen P. Kaplan 21 Chemokines, 471 James E. Pease and Timothy J. Williams 22 Neurotrophins, 494 Wolfgang A. Nockher, Sanchaita Sonar and Harald Renz 23 Neuropeptides, 511 David A. Groneberg and Axel Fischer

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Contents 24 Late-phase Allergic Reactions in Humans, 524 Yee-Ean Ong and A. Barry Kay

41 Airway Smooth Muscle, 874 Stuart J. Hirst

Part 3 Pharmacology

Part 5 Allergens

25 Antihistamines, 551 F. Estelle R. Simons and Keith J. Simons

42 Biochemistry of Allergens and Recombinant Allergens, 895 Rudolf Valenta

26 Lipid Mediators: Leukotrienes, Prostanoids, Lipoxins, and Platelet-activating Factor, 566 Sophie P. Farooque, Jonathan P. Arm and Tak H. Lee 27 Theophylline and Isoenzyme-selective Phosphodiesterase Inhibitors, 634 Mark A. Giembycz 28 Adrenergic Agonists and Antagonists, 668 Tony R. Bai 29 Cholinergic Antagonists, 683 Nicholas J. Gross 30 Antileukotriene Agents, 694 Graeme P. Currie and Brian J. Lipworth 31 Glucocorticosteroids, 715 Peter J. Barnes 32 Immunomodulating Drugs, 732 Iain A.M. MacPhee

Part 4 Physiology 33 Physiologic Aspects of Asthma, 749 Philip W. Ind and Neil B. Pride 34 Aerosol Delivery Systems, 768 Thomas G. O’Riordan and Gerald C. Smaldone 35 Bronchial Hyperresponsiveness, 783 Guy F. Joos 36 Exercise-induced Bronchoconstriction: Animal Models, 794 Arthur N. Freed and Sandra D. Anderson 37 Exercise-induced Bronchoconstriction: Human Models, 808 Arthur N. Freed and Sandra D. Anderson

43 Host Responses to Allergens, 913 Wayne R. Thomas and Belinda J. Hales 44 Allergen Extracts and Standardization, 928 Ronald van Ree 45 Grass, Tree, and Weed Pollen, 942 Jean Emberlin 46 Fungi as Allergens, 963 Cathryn C. Hassett, W. Elliott Horner, Estelle Levetin, Laurianne G. Wild, W. Edward Davis, Samuel B. Lehrer and John Lacey 47 Dust Mites and Asthma, 984 Thomas A.E. Platts-Mills and Judith A. Woodfolk 48 Animal Allergens, 997 Adnan Custovic and Angela Simpson 49 Airborne Allergens and Irritants in the Workplace, 1017 Xaver Baur 50 Allergens from Stinging Insects: Ants, Bees, and Vespids, 1123 Te Piao King and Rafael I. Monsalve 51 Cockroach Allergens, Environmental Exposure, and Asthma, 1131 Martin D. Chapman and Anna Pomés 52 Food Allergens, 1146 Ricki M. Helm and A. Wesley Burks 53 Latex Allergy, 1164 Robyn E. O’Hehir, Michael F. Sutherland, Alexander C. Drew and Jennifer M. Rolland

Part 6 Animal Models of Asthma

39 Mucus and Mucociliary Clearance in Asthma and Allergic Rhinitis, 840 Duncan F. Rogers

54 Primate Models of Allergic Asthma, 1187 Charles G. Plopper, Suzette M. Smiley-Jewell, Lisa A. Miller, Michelle V. Fanucchi, Michael J. Evans, Alan R. Buckpitt, Mark V. Avdalovic, Laurel J. Gershwin, Jesse P. Joad, Radhika Kajekar, Shawnessy D. Larson, Kent E. Pinkerton, Laura S. Van Winkle, Edward S. Schelegle, Emily M. Pieczarka, Reen Wu and Dallas M. Hyde

40 Biology of Vascular Permeability, 857 Peter Clark

55 Airway Remodeling in Small Animal Models, 1202 Clare M. Lloyd

38 Sensory and Autonomic Nervous System in Asthma and Rhinitis, 823 Bradley J. Undem and Kevin Kwong

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Contents 56 Are Animal Models of Asthma Useful?, 1214 Reinhard Pabst

69 Management and Treatment of Allergic Rhinitis, 1430 Jean Bousquet and Michael A. Kaliner

Index

70 Nasal Polyps and Rhinosinusitis, 1454 Wouter Huvenne, Paul Van Cauwenberge and Claus Bachert

VOLUME 2 Allergic Diseases: Etiology, Diagnosis and Treatment Part 7 Etiology and Pathology 57 Genetics of Asthma and Atopic Dermatitis, 1225 Saffron A.G. Willis-Owen, Miriam F. Moffatt and William O.C. Cookson 58 Epidemiology of Asthma, Atopy, and Atopic Disease, 1239 Debbie L. Jarvis, Seif O. Shaheen and Peter Burney 59 The Allergy March, 1259 Ulrich Wahn

71 Ocular Allergy, 1482 Avinash Gurbaxani, Virginia L. Calder and Susan Lightman 72 Mechanisms in Allergen Injection Immunotherapy, 1510 Stephen J. Till and Stephen R. Durham 73 Allergen Injection Immunotherapy: Indications and Practice, 1522 Hans-Jørgen Malling 74 Sublingual Immunotherapy, 1543 G. Walter Canonica and Giovanni Passalacqua 75 Novel Approaches to Allergen Immunotherapy, 1555 Mark Larché

60 Outdoor Air Pollution and Allergic Airway Disease, 1266 Gennaro D’Amato

Part 10 Asthma and its Treatment

61 Indoor Air Pollution, 1279 Paul Harrison, Rebecca Slack and Sanjeev Bagga

76 Definition, Clinical Features, Investigations, and Differential Diagnosis of Asthma, 1567 Piero Maestrelli, Gaetano Caramori, Francesca Franco and Leonardo M. Fabbri

62 Molecular Immunopathology of Allergic Disease, 1290 Susan Foley and Qutayba Hamid

77 Asthma in Infancy and Childhood, 1591 John O. Warner

Part 8 Diagnosis of Allergic Disease 63 Principles and Practice of Diagnosis and Treatment of Allergic Disease, 1321 Anthony J. Frew and A. Barry Kay 64 Skin Testing in Diagnosis and Management of Respiratory Allergic Diseases, 1335 Pascal Demoly, Anaïs Pipet and Jean Bousquet 65 Allergy Testing in the Laboratory, 1346 Steven O. Stapel and Jörg Kleine-Tebbe 66 Measurement of Markers of Inflammation in Induced Sputum and Exhaled Air, 1368 Ian D. Pavord and Dominick E. Shaw

Part 9 Allergic Rhinoconjunctivitis and Immunotherapy 67 Definition and Classification of Allergic Rhinitis and Upper Airways Diseases, 1383 Wytske Fokkens and Jean Bousquet 68 Pathophysiology of Allergic Rhinitis, 1402 Peter H. Howarth

78 Pathogenesis of Asthma, 1608 Stephen T. Holgate 79 Pathology of Asthma, 1632 Peter K. Jeffery, A. Barry Kay and Qutayba Hamid 80 Management of Chronic Asthma, 1650 Peter J. Barnes 81 Anti-IgE in Persistent Severe Allergic Asthma, 1661 Marc Humbert, Stephen T. Holgate, Howard Fox and Jean Bousquet 82 Occupational Asthma, 1687 Paul Cullinan and Anthony J. Newman Taylor 83 New Drugs for the Treatment of Allergy and Asthma, 1712 Trevor T. Hansel, Ed Erin,Onn Min Kon and Peter J. Barnes

Part 11 Eosinophil-associated Disease and Hypersensitivity Pneumonitis 84 Allergic Bronchopulmonary Aspergillosis, 1743 André-Bernard Tonnel, Stéphanie Pouwels-Frys and Isabelle Tillie-Leblond

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Contents

85 Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis, 1757 Michael C. Zacharisen and Jordan N. Fink 86 Pulmonary Eosinophilia, 1779 Jean-François Cordier and Vincent Cottin 87 Hypereosinophilic Syndromes, 1802 Hans-Uwe Simon

Part 12 Allergy and the Skin

93 Food Allergy and Eosinophilic Gastroenteropathies, 1921 Scott H. Sicherer and Hugh A. Sampson 94 Drug Hypersensitivity, 1943 Werner J. Pichler 95 Hypersensitivity to Aspirin and other NSAIDs, 1966 Andrzej Szczeklik, Ewa NiLankowska-Mogilnicka and Marek Sanak 96 Insect Sting Allergy, 1980 Ulrich R. Müller

88 Atopic Dermatitis, 1813 Julia D. Proelss and Thomas Bieber

Part 14 Prevention of Allergic Disease

89 Contact Dermatitis, 1831 David I. Orton and Carolyn M. Willis

97 Prevention of Allergic Disease, 1997 Susan L. Prescott and Bengt Björkstén

90 Urticaria and Angioedema, 1853 Allen P. Kaplan

98 Prevalence of Atopic Disorders in a Developing World: Pitfalls and Opportunities, 2020 Maria Yazdanbakhsh, Taniawati Supali and Laura C. Rodrigues

91 Mastocytosis, 1878 Nataliya M. Kushnir-Sukhov, Dean D. Metcalfe and Jamie A. Robyn

Index

Part 13 Anaphylaxis and Allergy to Food and Drugs 92 Anaphylaxis, 1897 M. Rosario Caballero, Stephen J. Lane and Tak H. Lee

Companion CD-ROM A companion CD-ROM is included at the end of Volume 2 with: • the complete text of both volumes • a full text search function • over 300 of the text figures in full colour

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List of Contributors

Cezmi A. Akdis

Tony R. Bai

Swiss Institute of Allergy and Asthma Research (SIAF) Davos, Switzerland

Professor of Medicine ABC Head, Respiratory Division University of British Columbia; James Hogg iCAPTURE Center St. Paul’s Hospital Vancouver, British Columbia, Canada

Rafeul Alam Professor & Head Division of Allergy & Immunology National Jewish Medical and Research Center; University of Colorado Health Sciences Center Denver, Colorado, USA

Sandra D. Anderson Principal Hospital Scientist Department of Respiratory Medicine & Sleep Medicine Royal Prince Alfred Hospital Camperdown Melbourne, New South Wales, Australia

Jonathan P. Arm Harvard Medical School; Brigham and Women’s Hospital Boston, Massachusetts, USA

Peter J. Barnes Head of Respiratory Medicine Airway Disease Section National Heart and Lung Institute Imperial College London London, UK

Xaver Baur Head of the Zentralinstitut für Arbeitsmedizin und Maritime Medizin; Chair in Occupational Medicine University Medical Center Hamburg-Eppendorf, Germany

Andrew J. Beavil

California National Primate Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Senior Lecturer in Asthma King’s College London MRC & Asthma UK Centre in Allergic Mechanisms of Asthma The Randall Division of Cells & Molecular Biophysics London, UK

Pedro C. Avila

Rebecca L. Beavil

Northwestern University Feinberg School of Medicine Department of Medicine Division of Allergy-Immunology Chicago, Illinois, USA

Postdoctoral Research Fellow King’s College London MRC & Asthma UK Centre in Allergic Mechanisms of Asthma The Randall Division of Cell & Molecular Biophysics London, UK

Mark V. Avdalovic

Claus Bachert Chief of Clinics Head, Upper Airway Research Laboratory University Hospital Ghent Ghent, Belgium

Thomas Bieber Professor and Chair Department of Dermatology & Allergy University of Bonn Bonn, Germany

Sanjeev Bagga Institute of Environment and Health Cranfield University Cranfield, UK

Bengt Björkstén The National Institute of Environmental Medicine/IMM

Division of Physiology Karolinska Institutet Stockholm, Sweden

Jean Bousquet CHU Montpellier Service des Maladies Respiratoires Hôpital Arnaud de Villeneuve Montpellier, France

Peter Bradding Professor of Respiratory Medicine University of Leicester Glenfield Hospital Leicester, UK

Alan R. Buckpitt California National Primate Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

A. Wesley Burks Professor and Chief Pediatric Allergy and Immunology Duke University Medical Center Durham, North Carolina, USA

Peter Burney Professor of Respiratory Epidemiology and Public Health National Heart and Lung Institute Imperial College of Science, Technology and Medicine London, UK

M. Rosario Caballero King’s College London; Guy’s Hospital London, UK

Virginia L. Calder Department of Clinical Ophthalmology Institute of Ophthalmology University College London London, UK

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G. Walter Canonica Full Professor and Director of Allergy & Respiratory Diseases Clinic Department of Internal Medicine University of Genoa Genoa, Italy

Gaetano Caramori Centro di Ricerca su Asma e BPCO University of Ferrara Ferrara, Italy

Martin D. Chapman INDOOR Biotechnologies Inc. Charlottesville, Virginia, USA

Reference Center for Rare Pulmonary Diseases; Claude Bernard University Department of Respiratory Medicine Hospices Civils de Lyon Louis Pradel University Hospital Lyon, France

Respiratory Medicine Unit Department of Medicine University of Cambridge School of Clinical Medicine Addenbrooke’s and Papworth Hospital Cambridge, UK

Glenn Cruse

Department of Medicine University of Cambridge School of Clinical Medicine Addenbrooke’s Hospital Cambridge, UK

Postgraduate Research Student Institute for Lung Health University of Leicester Medical School Department of Infection, Immunity and Inflammation Division of Respiratory Medicine Glenfield Hospital Leicester, UK

Leukocyte Biology Section National Heart and Lung Institute Imperial College London London, UK

Alison M. Condliffe Respiratory Medicine Unit Department of Medicine University of Cambridge School of Clinical Medicine Addenbrooke’s and Papworth Hospital Cambridge, UK

William O.C. Cookson The National Heart and Lung Institute Imperial College London London, UK

Jean-François Cordier Professor of Respiratory Medicine University of Lyon; Head, Reference Center for Rare Pulmonary Diseases; Claude Bernard University Department of Respiratory Medicine Hospices Civils de Lyon Louis Pradel University Hospital Lyon, France

Paul Cullinan Consultant Physician Department of Occupational and Environmental Medicine Royal Brompton Hospital London, UK

Graeme P. Currie Department of Respiratory Medicine Aberdeen Royal Infirmary Aberdeen Scotland, UK

Adnan Custovic University of Manchester North West Lung Centre Wythenshawe Hospital Manchester, UK

Gennaro D’Amato Director Division of Respiratory and Allergic Diseases Department of Respiratory Diseases High Speciality Hospital “A. Cardarelli” Napoli, Italy

Chris Corrigan Allergy and Respiratory Science King’s College London School of Medicine; Department of Asthma, Allergy and Respiratory Science Guy’s Hospital London, UK

Anuk M. Das Immunobiology Centocor Radnor, Pennsylvania, USA

W. Edward Davis Vincent Cottin Professor of Respiratory Medicine University of Lyon;

Head, Allergy Department University Hospital of Montpellier and Hôpital Arnaud de Villeneuve Montpellier, France

Alexander C. Drew Andrew S. Cowburn

Edwin R. Chilvers

Peter Clark

Pascal Demoly

Ochsner Clinic Foundation Department of Allergy New Orleans, Louisiana, USA

Department of Allergy, Immunology and Respiratory Medicine Alfred Hospital Melbourne, Victoria, Australia

Catherine Duez Chargée de Recherche Inserm Institut Pasteur de Lille Lille, France

Stephen R. Durham Head, Section of Allergy and Clinical Immunology National Heart and Lung Institute Faculty of Medicine Imperial College London London, UK

Jean Emberlin Director National Pollen and Aerobiology Research Unit Institute of Health University of Worcester Worcester, UK

Ed Erin National Heart and Lung Institute Clinical Studies Unit Royal Brompton Hospital London, UK

Michael J. Evans California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Leonardo M. Fabbri Full Professor of Respiratory Medicine University of Modena and Reggio Emilia Modena, Italy

Michelle V. Fanucchi California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Sophie P. Farooque MRC & Asthma UK Centre in Allergic Mechanisms of Asthma King’s College London Guy’s Hospital London, UK

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David J. Fear

Arturo Genovese

Trevor T. Hansel

Division of Allergy, Asthma and Lung Biology King’s College London London, UK

Professor of Medicine Division of Clinical Immunology and Allergy University of Naples Federico II Naples, Italy

Medical Director National Heart & Lung Institute Clinical Studies Unit Imperial College Royal Brompton Hospital London, UK

Jordan N. Fink Medical College of Wisconsin Departments of Pediatrics and Medicine Milwaukee, Wisconsin, USA

Axel Fischer Otto-Heubner-Centre Pneumology and Immunology Charité School of Medicine Free University Berlin and Humboldt-University Berlin Berlin, Germany

Laurel J. Gershwin California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Mark A. Giembycz Department of Pharmacology & Therapeutics Institute of Infection, Immunity and Inflammation Faculty of Medicine University of Calgary Calgary, Alberta, Canada

Susan Foley Meakins-Christie Laboratories McGill University Montreal, Quebec, Canada

Paul S. Foster Centre for Asthma and Respiratory Diseases School of Biomedical Sciences University of Newcastle Newcastle, New South Wales, Australia

Howard Fox Novartis Horsham Research Centre Horsham West Sussex, UK

Francesca Franco Section of Respiratory Disease Department of Oncology, Hematology and Respiratory Disease University of Modena and Reggio Emilia Modena, Italy

Cathryn C. Hassett

William G. Glass Immunobiology Centocor Radnor, Pennsylvania, USA

Catherine Hawrylowicz

Hannah J. Gould Professor Randall Division of Cell & Molecular Biophysics Division of Allergy, Asthma and Lung Biology King’s College London London, UK

David A. Groneberg Department of Respiratory Medicine Hannover Medical School Hannover, Germany

MRC & Asthma UK Centre in Allergic Mechanisms of Asthma King’s College London London, UK

Ricki M. Helm Project Team Manager Research Support Center Office of the Vice Chancellor for Academic Affairs and Research Administration Little Rock, Arkansas, USA

Nicholas J. Gross

C. Garren Hester

Pulmonary and Critical Care Division Hines VA Hospital Hines, Illinois, USA

Laboratory Research Analyst II Duke University Medical Center Durham, North Carolina, USA

Avinash Gurbaxani

Stuart J. Hirst

Moorfields Eye Hospital London, UK

MRC & Asthma UK Centre in Allergic Mechanisms of Asthma King’s College London London, UK

Belinda J. Hales Michael M. Frank

Institute of Environment and Health Cranfield University Cranfield, UK

Tulane University School of Medicine Department of Medicine Section of Clinical Immunology, Allergy and Rheumatology New Orleans, Louisiana, USA

Wytske Fokkens Department of Otorhinolaryngology Head and Neck Surgery Academic Medical Center Amsterdam, The Netherlands

Paul Harrison

Samuel L. Katz Professor of Pediatrics, Medicine and Immunology Duke University Medical Center Durham, North Carolina, USA

Senior Research Officer University of Western Australia Centre for Child Health Research Telethon Institute for Child Health Research Subiaco, Western Australia, Australia

Arthur N. Freed

Qutayba Hamid

Director, Department of Research Sinai Hospital of Baltimore Baltimore, Maryland, USA

Meakins-Christie Laboratories McGill University Montreal, Quebec, Canada

Anthony J. Frew

Hamida Hammad

Professor of Allergy & Respiratory Medicine Department of Respiratory Medicine Brighton General Hospital Brighton, UK

Postdoctoral Fellow University of Ghent Laboratory of Immunoregulation Ghent, Belgium

Simon P. Hogan Division of Allergy and Immunology Department of Pediatrics Cincinnati Children’s Hospital Medical Center University of Cincinnati College of Medicine Cincinnati, Ohio, USA

Stephen T. Holgate Allergy & Inflammation Research Division of Infection, Inflammation & Repair School of Medicine Southampton General Hospital Southampton, UK

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Patrick G. Holt Head Division of Cell Biology TVW Telethon Institute for Child Health Research West Perth, Western Australia, Australia

W. Elliott Horner Principal Consultant Air Quality Sciences Inc. Marietta, Georgia, USA

Peter H. Howarth Reader in Medicine & Hon. Consultant Physician Southampton General Hospital Southampton, UK

Marc Humbert Professor of Respiratory Medicine Université Paris-Sud II Service de Pneumologie Respiratoire Hôpital Antoine-Béclère Clamart, France

James Hunt Postdoctoral Researcher Randall Division of Cell & Molecular Biophysics; Division of Asthma, Allergy & Lung Biology King’s College London London, UK

Wouter Huvenne Upper Respiratory Laboratory ENT-Department University Hospital Ghent Ghent, Belgium

Dallas M. Hyde California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Philip W. Ind Consultant Physician Clinical Head of Department Hammersmith Hospital; Hon. Senior Lecturer National Heart & Lung Institute at Hammersmith London, UK

Royal Brompton Hospital London, UK

Jesse P. Joad California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

John Lacey Deceased

Paige Lacy

Department of Respiratory Medicine Ghent University Hospital Ghent, Belgium

Division of Pulmonary Medicine Department of Medicine University of Alberta Edmonton, Alberta, Canada

Radhika Kajekar California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Michael A. Kaliner Medical Director Institute for Asthma & Allergy Wheaton, Maryland, USA

Allen P. Kaplan The National Allergy, Asthma, and Urticaria Centers of Charleston; Professor, Department of Medicine Medical University of South Carolina Charleston, South Carolina, USA

A. Barry Kay Emeritus Professor of Allergy and Clinical Immunology National Heart and Lung Institute Imperial College London London, UK

Te Piao King Professor Emeritus Rockefeller University New York, New York, USA

Jörg Kleine-Tebbe Allergy & Asthma Center Westend Berlin, Germany

Onn Min Kon

Senior Lecturer in Public Health Respiratory Epidemiology and Public Health Group National Heart and Lung Institute Imperial College London London, UK

National Heart and Lung Institute Clinical Studies Unit Royal Brompton Hospital London, UK

Nataliya M. Kushnir-Sukhov Emeritus Professor of Lung Pathology Senior Research Investigator Honorary Consultant Imperial College London

Johns Hopkins University Johns Hopkins School of Medicine Johns Hopkins Asthma and Allergy Center Baltimore, Maryland, USA

Guy F. Joos

Debbie L. Jarvis

Peter K. Jeffery

Kevin Kwong

Laboratory of Allergic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland, USA

Bart N. Lambrecht Professor of Pulmonary Medicine Laboratory of Immunoregulation & Mucosal Immunology University Hospital Ghent Ghent, Belgium

Stephen J. Lane Consultant Respiratory Physician & Allergist Adelaide & Meath Hospital Tallaght Dublin, Ireland

Mark Larché Professor of Medicine, Canada Research Chair Clinical Immunology & Allergy Department of Medicine McMaster University Hamilton, Ontario, Canada

Shawnessy D. Larson California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Geoffrey J. Laurent Director Centre for Respiratory Research Department of Medicine Rayne Institute University College London London, UK

Tak H. Lee Professor King’s College London; Director MRC & Asthma UK Centre in Allergic Mechanisms of Asthma Guy’s Hospital London, UK

Samuel B. Lehrer Professor of Medicine Tulane University School of Medicine Department of Medicine

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Section of Clinical Immunology, Allergy and Rheumatology New Orleans, Louisiana, USA

Estelle Levetin The University of Tulsa Department of Biological Science Tulsa, Oklahoma, USA

Susan Lightman Professor of Clinical Ophthamology Moorfields Eye Hospital London, UK

Brian J. Lipworth Asthma & Allergy Research Group Ninewells Hospital and Medical School Dundee, Scotland, UK

Clare M. Lloyd Leukocyte Biology Section National Heart and Lung Institute Faculty of Medicine Imperial College London London, UK

Iain A.M. MacPhee Senior Lecturer in Renal Medicine St. George’s, University of London London, UK

Piero Maestrelli Professor of Occupational Medicine Department of Environmental Medicine and Public Health University of Padova Padova, Italy

Hans-Jørgen Malling Allergy Clinic National University Hospital Copenhagen, Denmark

Gianni Marone Division of Clinical Immunology and Allergy Center for Basic and Clinical Immunology Research (CISI) University of Naples Federico II School of Medicine Naples, Italy

University of California Davis, California, USA

St George’s Hospital London, UK

Miriam F. Moffatt

Robyn E. O’Hehir

Reader in Human Genetics Molecular Genetics Group National Heart and Lung Institute Imperial College London London, UK

Professor and Director Department of Allergy, Immunology and Respiratory Medicine Alfred Hospital and Monash University Melbourne, Victoria, Australia

Rafael I. Monsalve

Thomas G. O’Riordan

Group Leader Allergen Chemistry and Biotechnology Department Research and Development ALK-ABELLÓ Madrid, Spain

Associate Professor of Clinical Medicine and Public Health Department of Medicine State University of New York at Stony Brook Stony Brook, New York, USA

Redwan Moqbel

David I. Orton

Division of Pulmonary Medicine Departments of Medicine and Medical Microbiology and Immunology University of Alberta Edmonton, Alberta, Canada

Consultant Dermatologist Environmental & Contact Dermatitis Unit Department of Dermatology and Allergy Amersham Hospital Amersham, UK

Ulrich R. Müller

Reinhard Pabst

Allergy Division Medical Department Spital Bern Ziegler Bern, Switzerland

Head, Functional & Applied Anatomy Medical School of Hannover Hannover, Germany

Michelle J. Muessel

Giovanni Passalacqua

Research Associate Glenfield Hospital Department of Respiratory Medicine Leicester, UK

Research Professor Allergy and Respiratory Diseases Department of Internal Medicine University of Genoa Genoa, Italy

Lynne A. Murray

Ian D. Pavord

Manager of Pharmacology Promedior Inc Malvern, Pennsylvania, USA

Institute for Lung Health Department of Respiratory Medicine and Thoracic Surgery Glenfield Hospital Leicester, UK

Anthony J. Newman Taylor Head, National Heart and Lung Institute Faculty of Medicine Imperial College London London, UK

Ewa Ni-ankowska–Mogilnicka Department of Medicine Jagiellonian University School of Medicine Kraków, Poland

Dean D. Metcalfe

Wolfgang A. Nockher


Department of Clinical Chemistry and Molecular Diagnostics Medical Faculty Philipps-Universität Marburg Marburg, Germany

Lisa A. Miller

Yee-Ean Ong

California National Primate Research Center and Schools of Veterinary Medicine and Medicine

Consultant Physician and Honorary Senior Lecturer

James E. Pease Leukocyte Biology Section National Heart and Lung Institute Faculty of Medicine Imperial College London London, UK

Simon Phipps Centre for Asthma and Respiratory Diseases School of Biomedical Sciences University of Newcastle Newcastle, New South Wales, Australia

Werner J. Pichler Division of Allergology Clinic for Rheumatology and Clinical Immunology/Allergology, University of Bern Bern, Switzerland

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Emily M. Pieczarka

Jamie A. Robyn

Edward S. Schelegle

California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA



Kent E. Pinkerton California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Anaïs Pipet Exploration des allergies Hôpital Arnaud de Villeneuve University Hospital of Montpellier Montpellier, France

Robert P. Schleimer Laura C. Rodrigues Infectious Disease Epidemiology Unit London School of Hygiene and Tropical Medicine London, UK

Duncan F. Rogers Thoracic Medicine National Heart & Lung Institute Imperial College London London, UK

Thomas A.E. Platts-Mills Professor Emeritus Asthma and Allergic Diseases Center University of Virginia Health System Charlottesville, Virginia, USA

Jennifer M. Rolland Associate Professor and Deputy Head Department of Immunology Monash University Melbourne, Victoria, Australia

Charles G. Plopper Professor Emeritus, California National Primate Research Center and Schools of Veterinary Medicine and Medicine University of California Davis, California, USA

Anna Pomés INDOOR Biotechnologies Inc. Charlottesville, Virginia, USA

Stéphanie Pouwels-Frys Department of Pneumology and Immunoallergology University Hospital Lille, France

Helene F. Rosenberg Laboratory of Allergic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland, USA

Marc E. Rothenberg Division of Allergy and Immunology Department of Pediatrics Cincinnati Children’s Hospital Medical Center University of Cincinnati College of Medicine Cincinnati, Ohio, USA

Ian Sabroe Susan L. Prescott School of Paediatrics and Child Health Princess Margaret Hospital Perth, Western Australia, Australia

Academic Unit of Respiratory Medicine School of Medicine and Biological Sciences University of Sheffield; Royal Hallamshire Hospital Sheffield, UK

Neil B. Pride Emeritus Professor of Respiratory Medicine and Senior Research Investigator National Heart and Lung Institute Imperial College London, UK

Julia D. Proelss Senior House Officer Department of Dermatology and Allergy University of Bonn Bonn, Germany

Hugh A. Sampson Professor of Pediatrics and Immunobiology Dean for Translational Biomedical Sciences Chief of Pediatric Allergy and Immunology Mount Sinai School of Medicine New York, New York, USA

Marek Sanak Department of Medicine Jagiellonian University School of Medicine Kraków, Poland

Harald Renz Professor and Chairman Department of Clinical Chemistry Philipps University Marburg Marburg, Germany

Andrew Saxon Professor of Medicine UCLA School of Medicine Los Angeles, California, USA

Northwestern University Feinberg School of Medicine Department of Medicine Division of Allergy-Immunology Chicago, Illinois, USA

Seif O. Shaheen Clinical Senior Lecturer in Epidemiology, and Asthma UK Senior Research Fellow Respiratory Epidemiology & Public Health Group National Heart and Lung Institute Imperial College London London, UK

Dominick E. Shaw Specialist Registrar Department of Respiratory Medicine Nottingham City Hospital Nottingham, UK

Scott H. Sicherer Associate Professor of Pediatrics The Elliot and Roslyn Jaffe Food Allergy Institute Division of Allergy and Immunology Department of Pediatrics Mount Sinai, School of Medicine New York, New York, USA

Hans-Uwe Simon Department of Pharmacology University of Bern Bern, Switzerland

F. Estelle R. Simons Department of Pediatrics & Child Health Department of Immunology Faculty of Medicine University of Manitoba Winnipeg, Manitoba, Canada

Keith J. Simons Faculty of Pharmacy Department of Pediatrics & Child Health Faculty of Medicine University of Manitoba Winnipeg, Manitoba, Canada

Angela Simpson Senior Lecturer Respiratory Research Group Education and Research Centre University Hospital of South Manchester NHS Foundation Trust Manchester, UK

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Rebecca Slack

Wayne R. Thomas

Laura S. Van Winkle

Institute of Environment and Health Cranfield University Cranfield, UK

University of Western Australia Centre for Child Health Research Telethon Institute for Child Health Research Subiaco, Western Australia, Australia


Catherine Thornton

Ulrich Wahn

Senior Lecturer in Newborn Immunity Institute of Life Science School of Medicine Swansea University Swansea, UK

Professor and Director Department of Pediatric Pneumology and Immunology Charité-University Medicine Berlin, Germany

Gerald C. Smaldone Chief, Pulmonary, Critical Care and Sleep Medicine Department of Medicine State University of New York at Stony Brook Stony Brook, New York, USA

Suzette M. Smiley-Jewel

Stephen J. Till


Clinician Scientist Fellow (The Health Foundation) Allergy and Clinical Immunology Imperial College London London, UK

Sanchaita Sonar

Isabelle Tillie-Leblond

Department of Clinical Chemistry and Molecular Diagnostics Medical Faculty Philipps-Universität Marburg Marburg, Germany

Giuseppe Spadaro Associate Professor of Medicine Division of Clinical Immunology and Allergy University of Naples Federico II Naples, Italy

Steven O. Stapel Head, Department of Allergy Diagnostics Sanquin Diagnostic Services Amsterdam, The Netherlands

Taniawati Supali Department of Parasitology University of Indonesia Jakarta, Indonesia

Michael F. Sutherland Department of Allergy, Immunology and Respiratory Medicine Alfred Hospital and Monash University Melbourne, Victoria, Australia

Brian J. Sutton Professor of Molecular Biophysics Randall Division of Cell & Molecular Biophysics; Division of Asthma, Allergy & Lung Biology King’s College London London, UK

Department of Pneumology and Immunoallergology University Hospital Lille, France

Andrew J. Wardlaw Institute for Lung Health Department of Respiratory Medicine Glenfield Hospital Leicester, UK

John O. Warner Department of Paediatrics Division of Medicine Imperial College London London, UK

André-Bernard Tonnel Department of Pneumology and ImmunoAllergology A. Calmette Hospital Lille, France

Anne Tsicopoulos Directeur de Recherche Inserm Institut Pasteur de Lille Lille, France

Bradley J. Undem Johns Hopkins University Johns Hopkins School of Medicine Johns Hopkins Asthma and Allergy Center Baltimore, Maryland, USA

Rudolf Valenta Christian Doppler Laboratory for Allergy Research Division of Immunopathology Department of Pathophysiology Center for Physiology and Pathophysiology Medical University of Vienna Vienna General Hospital Vienna, Austria

Paul Van Cauwenberge Upper Respiratory Laboratory Department of Otorhunolaryngology Ghent University and Ghent University Hospital Ghent, Belgium

Laurianne G. Wild Ochsner Clinic Foundation Department of Allergy New Orleans, Louisiana, USA

Timothy J. Williams Leukocyte Biology Section National Heart and Lung Institute Faculty of Medicine Imperial College London London, UK

Carolyn M. Willis Research Director Department of Dermatology Amersham Hospital Amersham, UK

Saffron A.G. Willis-Owen Postdoctoral Research Associate National Heart and Lung Institute Imperial College London London, UK

John W. Wilson Department of Allergy, Immunology and Respiratory Medicine Alfred Hospital and Monash University Melbourne, Victoria, Australia

Ronald van Ree Andrzej Szczeklik Professor and Chairman Department of Medicine Jagiellonian University School of Medicine Kraków, Poland

Head, Allergy Research Department of Experimental Immunology Academic Medical Center University of Amsterdam Amsterdam, The Netherlands

Judith A. Woodfolk Associate Professor Asthma and Allergic Diseases Center University of Virginia Health System Charlottesville, Virginia, USA

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Reen Wu

Maria Yazdanbakhsh

Michael C. Zacharisen


Department of Parasitology Leiden University Medical Center Leiden, The Netherlands

Medical College of Wisconsin Departments of Pediatrics and Medicine Milwaukee, Wisconsin, USA

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Foreword

The second edition of Allergy and Allergic Diseases is a timely, all-encompassing two-volume text that covers in detail the basic science at the heart of the specialty and its major and minor clinical components. The pathobiology links back to the basic sciences through both clinical findings and translational research. There is insightful emphasis not only on pathogenesis, but also on underlying genetic and environmental factors uncovered through genomics and epidemiology, respectively. Jean Bousquet of Montpellier, France; Patrick Holt of Perth, Australia; and Allen Kaplan of Charleston, USA, now join Barry Kay, the founding editor. The differing interests and expertise of this distinguished editorial group and their geographic diversity provides unusual editorial capacity in basic and clinical science. The figures, photographs, tables and diagrams reflect their editorial wisdom in their clarity and value to the text. Similarly, the sharing of editorial responsibilities has not only resulted in a thoughtful selection of the subject matter but

also in a superb selection of authors to assure that each presentation has depth and balance. These authors are international in distribution and widely acknowledged for their comprehension of their subject. Subjects are often covered in several chapters so as to provide history, preclinical knowledge, and clinical meaning in terms of pathobiology and intervention. For example, IgE is addressed from history, to crystal structure, to effects of indoor and outdoor pollution, to neutralization by monoclonal antibodies; leukotrienes are considered from history, to chemical and biological properties, to small molecule inhibitors; eosinophils are covered from discovery, to function, to monoclonal antibody neutralization of the chemokines/ cytokines that regulate function; and the pathobiology of many conditions is presented from in situ findings of cell types, pathways, and soluble effectors through analysis of animal models and the human condition. K. Frank Austen MD Harvard Medical School

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Preface

The scope and breadth of allergy and allergic diseases has increased enormously since the first edition of this textbook was published in 1997. Our present aim, as before, is to provide a detailed, up-to-date and authoritative source of information on both the scientific basis and clinical aspects of our subject. Barry Kay is delighted to be joined by Allen Kaplan, Jean Bousquet and Patrick Holt as co-editors of this two-volume work. We have attempted to cover all important aspects of the field. This includes growing areas on the immunological basis of allergy, particularly regulatory T cells, environmental factors in IgE production and innate immunity, as well as up to date accounts of the cells and mediators of allergic inflammation. Furthermore, the sections on pharmacology, physiology, allergens and animals models have all been fully revised. In Volume 2, the clinical section, we have dealt in detail with allergic conjunctivo-rhinitis, asthma, eosinophilic disorders, allergy and the skin, anaphylaxis, food and drug allergy and allergy prevention. We have also addressed current approaches to treatment with emphasis on internationally agreed guidelines and there is a substantial section on allergen immunotherapy, including recent advances. There are of course other textbooks in the field of “Allergy and Allergic Diseases”, many from the USA. However the present work, like the first edition, attempts to give an international perspective on the principles and practice of allergy and this is reflected in our worldwide authorship. As before, we have endeavoured, where possible, to adhere to the correct “allergy nomenclature” as urged by the late Professor Robin Coombs, who kindly wrote the Foreword to the first edition. As explained in Chapter 1 (Allergy and Concepts: History and Concepts) it has become increasingly difficult to use the term “allergy” in its original sense as conceived by Clemens Von Pirquet. However Von Pirquet’s concept of “altered reactivity” to describe a true allergic response, as opposed to intolerance or pseudo-allergy, remains as valid now as it did in 1906. The burden of allergic disease is sometimes underestimated. In addition to the obvious health effects, allergic disorders can make social interactions difficult as even simple everyday activities can pose a major health risk (House of Lords Science and Technology Committee Report, September 2007). On a

national scale, the treatment of allergy patients forms a significant part of the work of the health care providers and, in Western societies, the number of allergy-related work absences represents a large cost to the economy. Allergies affect all aspects of a patient’s life. Hayfever symptoms disrupt children’s sleep and often impair their performance at school and asthma has been associated with school absenteeism. Eating out can be highly stressful for patients with food anaphylaxis, especially in teenagers and young adults who may not want to draw attention to their condition. Allergy patients often find it difficult to live a normal life. This is especially apparent in children, where special care has to be taken whilst engaging in everyday activities which in turn induces anxiety and impairs the quality of life. In England approximately 3 million people (6% of the population) each year consult their primary care physician with conditions related to allergy and 72.6 million community prescriptions are issued (Department of Health 2006; Royal College of Physicians of London 2003). This included 38.9 million prescriptions for asthma, 4.5 million for nasal allergies and 20.4 million prescriptions for eczema. This amounted to a cost of £0.9 billion, which represented 11 per cent of the total drugs budget (compared to 27 per cent spent on cardiovascular diseases and 8 per cent on gastro-intestinal disorders). The prevalence of allergic disease has markedly increased over recent years. In the UK, by 2004, the scale of the “allergy epidemic” was such that 39 per cent of children and 30 per cent of adults had been diagnosed with one or more of asthma, eczema and hayfever, and 38 per cent of children and 45 per cent of adults had experienced symptoms of these disorders in the preceding 12 months (Gupta et al. 2004). In fact by the end of 2005, approximately one in nine people had a recorded diagnosis of “any allergic disease,” including any one of asthma, hayfever, eczema, anaphylaxis or peanut allergy. This figure represented a 28% increase in prevalence over a four year period. Most alarming has been the increase in food anaphylaxis, particularly peanut allergy. Thus there was a 117% increase in the prevalence of peanut allergy from 2001 to 2005, and it is estimated that 25,700 people in England are affected. Asthma, eczema and allergic rhinitis often occur together and this comorbidity, or multiple allergic disease,

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Preface often requires multiple referrals to different organ specialists. More than two million people in England are estimated to suffer from multiple allergic diseases, with an increase in the prevalence rate of 49% cent between 2001 and 2005. (QRESEARCH and The Information Centre for health and social care). The increase in allergy and atopy in the United Kingdom has been mirrored in many other developed countries in Western Europe, the United States, Canada, Australia and New Zealand. Until a few years ago developing countries in Africa and the Middle East reported a relatively low prevalence of allergy (although this situation is rapidly changing as a result of “Western Modernisation”). Nevertheless these different patterns of prevalence represented one of the origins of the “hygiene hypothesis”. Several chapters deal with aspects of the hygiene hypothesis. These include the protective effects of early childhood infection, the influence of bowel flora, farming and the proximity to animals and a “traditional” lifestyle and other factors which may explain rising trends in allergy such as changes in diet, allergen exposure, atmospheric pollution and tobacco smoke. Although the hygiene hypothesis has stimulated much debate interventions designed to reverse the rising prevalence have met with limited success and these attempts at primary prevention are also covered in the appropriate chapters. Producing a book of this size has been an enormous task but we have been very fortunate to have had critical comments from many colleagues on all the individual chapters submitted. Several authors have also acted as reviewers of other contributors’ chapters and for this we are most grateful. In particular we would like to thank Dr Graham Devereux, Prof Cezmi Akdis, Dr Peter Clark, Prof Tony Segal, Dr Bernhard Gibbs, Dr James Pease, Dr Richard Costello, Dr Lars-Olaf Cardell, Prof Andrew Wardlaw, Prof Sergio Bonini, Dr Christian Virchow, Prof Harald Renz, Dr Andreas Nockher, Prof Guy Joos, Dr Domenico Spina, Prof David Groneberg, Dr Ray Penn, Dr Sussan Nourshargh, Prof Tim Williams, Dr Paul Cullinan, Prof Clare Lloyd, Prof Tony Frew, Dr Moises

Calderon, Dr Veronica Varney, Dr Andrew Menzies-Gow, Dr Yee-Ean Ong, Dr Charles McSharry, Prof Duncan Geddes, Dr Nataliya Kushnir, Dr Shuaib Nasser, Prof John Warner. We are also grateful to Dr K. Frank Austen for kindly writing a Foreword and for his general encouragement during the preparation of this work. Wiley-Blackwell have given us constant help and guidance with the production of the textbook and we would like to thank Maria Khan and her team for the huge effort involved in seeing this task through to a successful completion. Finally we are particularly grateful to Miss Jennifer Mitchell, the Editorial Assistant, without whom the publication of “Allergy and Allergic Diseases” would not have been possible. Her untiring efforts, commitment and attention to detail cannot be overestimated. A. Barry Kay Allen P. Kaplan Jean Bousquet Patrick G. Holt

References Department of Health. July 2006. Review of Services for Allergy. (www.dh.gov.uk/publications). Royal College of Physicians of London. 2003. Allergy: the unmet need. QRESEARCH and The Information Centre for health and social care, Primary care epidemiology of allergic disorders: analysis using QRESEARCH database 2001–2006, 2007, pp. 69–70. (see also http://www.ic.nhs.uk/ work-with-us/research/qresearch/primary-care-epidemiologyof-allergic-disorders:-analysis-using-qresearch-database-20012006) Gupta et al. (2004) Burden of allergic disease in the UK: secondary analyses of national databases. Clinical and Experimental Allergy 34, 520–526. House of Lords Science and Technology Committee Report, September 2007 Allergy (http://www.publications.parliament.uk/pa/ ld200607/ldselect/ldsctech/166/166i.pdf)

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Immunology of the Allergic Response


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Allergy and Hypersensitivity: History and Concepts A. Barry Kay

Summary The study of allergy (“allergology”) and hypersensitivity, and the associated allergic diseases, have their roots in the science of immunology but overlap with many disciplines including pharmacology, biochemistry, cell and molecular biology, and general pathology, particularly the study of inflammation. Allergic diseases involve many organs and tissues such as the upper and lower airways, the skin, and the gastrointestinal tract and therefore the history of relevant discoveries in the field are long and complex. This chapter gives only a brief account of the major milestones in the history of allergy and the concepts which have arisen from them. It deals mainly with discoveries in the 19th and early 20th century, particularly the events which followed the description of anaphylaxis and culminated in the discovery of IgE as the carrier of reaginic activity. An important conceptual landmark that coincided with the considerable increase in knowledge of immunologic aspects of hypersensitivity was the Coombs and Gell classification of hypersensitivity reactions in the 1960s. This classification is revisited and updated to take into account some newer finding on the initiation of the allergic response.

The story of anaphylaxis Untoward reactions to external agents, which were harmless to most people, were recognized even in ancient times. The Egyptian pharaoh Menes (2641 BC) was reported to have died from a wasp (kehb) sting and is thus the first recorded case of anaphylactic shock (although interpretation of hieroglyphics is apparently controversial) (Avenberg & Harper 1980). Hippocrates (born 375 BC) is attributed with the first description of allergy to goats’ milk and cheese and Britannicus (born AD 41) was said to be afflicted by acute allergic reactions to horses. During the 19th century there were a number of reports describing violent or fatal reactions to repeated injections of Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Fig. 1.1 A commemorative postage stamp to mark the discovery of anaphylaxis by Charles R. Richet (1850–1935) and Paul J. Portier (1866–1962).

foreign proteins to various species including dogs (Magendie 1839), guinea pigs (Von Behring 1893, quoted in Becker 1999, p. 876) and rabbits (Flexner 1894, reviewed in Bulloch 1937). However it was not until the discovery of anaphylaxis by Charles R. Richet (1850–1935) and Paul J. Portier (1866– 1962) (Fig. 1.1) in 1901 that the concept of hypersensitivity reactions having a possible immunologic basis was put on a firm scientific footing. The story of the discovery of anaphylaxis is provided by Richet (1913) and goes as follows: During a cruise on Prince Albert of Monaco’s yacht, the Prince suggested to Portier and myself a study of the toxin production of Physalia (the jelly-fish known as Portuguese Man-of-War) found in the South Seas. On board the Prince’s yacht, experiments were carried out proving that an aqueous glycerine extract of the filaments of Physalia is extremely toxic to ducks and rabbits. On returning to France, I could not obtain Physalia and decided to study comparatively the tentacles of Actinaria (sea anemone) . . . While endeavouring to determine the toxic dose (of extracts), we soon discovered that some days must elapse before fixing it; for several dogs did not die until the fourth or fifth day after administration or even later. We kept those that had been given insufficient to kill, in order to carry out a second investigation upon these when they had recovered. At this point an unforeseen event occurred. The dogs which had recovered were intensely sensitive and died a few minutes after the administration of small

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Immunology of the Allergic Response ment in Vienna, he observed that some patients receiving antiserum developed a spectrum of systemic and local symptoms, notably, fever, skin rash, arthropathy, and lymph node swelling, which they termed “serum sickness” (von Pirquet et al. 1905; Wagner & von Pirquet 1968). Earlier accounts in the hospital records described similar symptoms with diphtheria and tetanus antisera. Therefore serotherapy appeared to have produced not only immunity (protection) but also hypersensitivity (or “supersensitivity” as was the then more favored word). Von Pirquet realized that in both situations an external agent had induced some form of “changed or altered reactivity” for which he proposed the term “allergy” from the Greek allos (“other”) and ergon (“work”). The critical passage from his article in Munchener Medizinische Wochenschrift (von Pirquet 1906) is as follows:

Fig. 1.2 Clemens von Pirquet (1874–1929). von Pirquet conceived the term “allergy” (see Appendix). He meant it to include any situation where there was “changed reactivity” irrespective of whether this resulted in immunity or hypersensitivity (see Chapter 2). He also introduced tuberculin skin tests in diagnosis. (From Cohen & Samter 1992, with permission.)

doses. The most typical experiment, that in which the result was indisputable, was carried out on a particularly healthy dog. It was given at first 0.1 mL of the glycerine extract without becoming ill; 22 days later, as it was in perfect health, I gave it a second injection of the same amount. In a few seconds it was extremely ill; breathing became distressful and panting; it could scarcely drag itself along, lay on its side, was seized with diarrhoea, vomited blood and died in 25 minutes.

The vaccinated person behaves toward vaccine lymph, the syphilitic toward the virus of syphilis, the tuberculous patient toward tuberculin, the person injected with serum towards this serum in a different manner from him who has not previously been in contact with such an agent. Yet, he is not insensitive to it. We can only say of him that his power to react has undergone a change.

The problem of reconciling the protective effect of antitoxin with the adverse reactions associated with the administration of foreign agents came to a climax in 1903 with three important discoveries. Firstly, Maurice Arthus (Fig. 1.3) found that repeated injections of horse serum to rabbits

It is difficult to overstate the importance and ramifications of this seminal discovery. Our present understanding of immediate-type hypersensitivity reactions, the antibody involved together with the pharmacologic mediators released, as well as the treatments for allergic diseases which have followed, have their roots in the discovery of anaphylaxis. However the concept that foreign proteins could induce hypersensitivity as well as immune reactions was difficult for early investigators to accept. It was out of this controversy that the word “allergy,” first coined by Clemens von Pirquet (Fig. 1.2) in 1906, was introduced (von Pirquet 1906).

The word “allergy” Von Pirquet and others had noticed that as well as the protective effects of passive immunotherapy with vaccinia and horse antiserum, many patients experienced adverse reactions. Thus the word “allergy” arose from his attempts to reconcile the two apparently contradictory phenomena of immunity and hypersensitivity. Working with Bela Schick in the scarlet fever wards of Escherich’s Paediatric Depart-

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Fig. 1.3 Nicholas-Maurice Arthus (1862–1945). The Arthus reaction is an experimental localized acute necrotizing vasculitis first described as local anaphylaxis. (From Cohen & Samter 1992, with permission.)

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produced hypersensitivity reactions that were antigenically specific, so challenging the currently held view that these reactions to foreign proteins were essentially toxic (Arthus 1903). Second, von Pirquet and Bela Schick (1905) observed that a child who had received a second injection of antitoxin had clinical symptoms the same day, though, on the first injection, given some time previously, her clinical symptoms appeared only after the tenth day. As a result they hypothesized that “the time of incubation is the time necessary for the formation of these antibodies.” These two observations, together with the third discovery by Hamburger and Moro (1903) of precipitating antibody in the blood during serum sickness, led von Pirquet to speculate that the diseaseproducing factor produces symptoms only when it had been changed by antibody. Thus, although not explicitly stating so in his original definition, in a later work von Pirquet (1911) made it clear that he intended the term “allergy” to be applied only to immunologic reactions. Von Pirquet also suggested that the word “allergen” should be used to describe the agent which, after one or more applications, induced this changed reactivity. Therefore von Pirquet had brilliantly laid the foundation for the modern science of immunology by appreciating that a foreign substance “sensitizes” the organism in a way that produces a different response on the second and subsequent administration. Unfortunately with the passage of time the word “allergy” became corrupted and is now used incorrectly and with a limited usage, i.e., to describe hypersensitivity mechanisms that are operative transiently, or persistently, in a limited group of conditions, particularly the IgE-mediated allergic diseases. Such a restricted meaning was not von Pirquet’s original intent and in fact it misses his point by, in a sense, merely substituting “allergy” for “hypersensitivity” (Kay 2006).

The “corruption” of allergy So why has the term “allergy” become misused, and are “allergists” themselves largely to blame? As Elmer Becker (1999) described in his erudite paper “Elements of the history of our present concepts of anaphylaxis, hay fever and asthma,” part of the confusion lay in attempts to “classify” allergy. Doerr (1914) initially divided allergy into hypersusceptibility to antigenic substances as well as altered reactivity to nonantigens in which he included morphine addiction. By 1926 Arthur Fernandez Coca (Fig. 1.4) advised the abandonment of the term “allergy” on the basis of its then numerous conflicting meanings. More confusingly he did not consider “anaphylaxis” as part of allergy because it was a phenomenon in which the antigen–antibody reaction was established. Instead Coca classified under “allergy” all those conditions where he considered an antibody mechanism had not been demonstrated, e.g., drug “idiosyncrasies,” serum sickness in

Allergy and Hypersensitivity: History and Concepts

Fig. 1.4 Arthur Fernandez Coca (1875–1959). Coca introduced the term “atopy” (now recognized as IgE-mediated hypersensitivity). (From Cohen & Samter 1992, with permission.)

man, and hay fever. In fact the view that allergy was all forms of hypersensitivity except anaphylaxis was to persist until the 1940s. By this time a medical subspecialty practiced by clinicians diagnosing and treating hay fever, asthma, serum sickness, drug reactions, etc. and involving treatment with desensitization injections was already then well established. As Becker (1999) explained: [clinicians] desired a brief, convenient, not too limiting group of terms describing what their specialty was and what it was about. As a consequence, they turned more and more to the use of “allergy”, “allergic”, etc. so that these terms became embedded in clinical usage. This was recognized by the editors of the Journal of Allergy when in the first issue they stated, “We believe that it [allergy] does not possess an established meaning in scientific usage. [They then quoted Karsner and Ecker (1921) as the source for this belief.] However, the term is very generally employed by clinicians who apply it to conditions of specific hypersensitiveness exclusive of anaphylaxis in lower animals . . . it seems the title of this journal corresponds to current medical usage.”

In hindsight the Journal of Hypersensitivity and Hypersensitivity Disorders would have been more historically correct and still have served the clinicians’ purpose. In the 1960s Robin Coombs (Fig. 1.5) and Philip Gell attempted to restore the term “allergy” to its original meaning (Coombs & Gell 1963). They pointed out that “hypersensitivity” is a general term to describe an adverse clinical reaction to an antigen (or allergen). Such an antigen could be bacterial-derived as in a classical delayed-type hypersensitivity reaction to tuberculo-protein or derived from allergen such as pollen giving rise to IgE-mediated hypersensitivity. They argued that limiting the term “allergy” to any exaggerated

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Immunology of the Allergic Response where an “immunologic” abnormality often cannot be identified (i.e., pseudoallergy). Is anything to be gained by attempting to restore von Pirquet’s word to its original meaning? Probably not. Words lose, or change, their meaning according to custom. Semantics can bury communication. We say that a T cell is “sensitized” but it would be more correct (in the von Pirquet sense) to call it an “allergized” lymphocyte. However this would only cause further confusion and so current imprecision may have to be accepted. What is important is to appreciate that von Pirquet introduced not so much a word but a fundamental biological rule which, arguably, marked the modern approach to immunology.

From reagin to IgE

Fig. 1.5 Robin A. Coombs (1921–2006). Described the antiglobulin (Coombs’) test and, with Philip Gell, classified the hypersensitivity reactions.

response of the immune system to external (antigenic or allergenic) substances was illogical as, by definition, the role of the immune system is to effect immunity. By way of example they pointed out that a deleterious effect to autoantigens should more properly be termed “autoallergic” rather than “autoimmune.” As suggested later (Kay 1997), most of this difficulty is removed if instead of “allergy” we refer to “allergic diseases” and confine the word “allergy” (as von Pirquet originally intended) to the uncommitted biological response. In the individual this uncommitted response may lead to either immunity (which is beneficial) or allergic disease (which is harmful). Therefore, the allergic response, in producing antibodies and specifically reacting (sensitized or allergized) lymphocytes, supplies a common armamentarium for both the reactions of immunity as well as those of the hypersensitivity reactions (allergic diseases). The restricted usage of the term “allergy” (allergic disease) is reflected in the practice of the clinical allergist, where physicians diagnose and treat only selected examples of hypersensitivity states rather than the wide spectrum of immunologic disorders. In many countries this is confined to the IgE-mediated (“atopic”) diseases, e.g., summer hay fever, perennial rhinitis, allergic asthma, allergy to stinging insects, food anaphylaxis, and atopic dermatitis. Other “hypersensitivity diseases” such as celiac disease and contact dermatitis are frequently managed by the relevant organ-based specialist. Furthermore, the clinical allergist also deals with patients whose signs and symptoms mimic those of true allergic disease, i.e., where there is evidence of local or generalized release of histamine and other pharmacologic reagents but

6

In the years after Portier and Richet’s discovery numerous unsuccessful attempts were made to transfer anaphylactic sensitivity to experimental animals using the serum from patients with hay fever or asthma. However in 1919, Ramirez reported that a normal nonallergic recipient of a blood transfusion 2 weeks previously from a donor sensitive to horse serum developed asthma upon being exposed to horses when riding in an open carriage in Central Park, New York (Ramirez 1919). This was the prelude to the classical experiments of Otto Carl Prausnitz and Heinz Küstner (Fig. 1.6) who, in 1921, demonstrated the presence of a tissue-sensitizing antibody in humans. Küstner was a fish-sensitive individual. When his serum was transferred to the skin of Prausnitz, a nonallergic recipient, there was a positive reaction at the skin site when this was subsequently injected with fish extract. They suggested that the sensitizing agent should be called “reagin” because they were not sure it was an antibody (Prausnitz & Küstner 1921). The eventual identification of human reagin as IgE antibody was one of the most important biological discoveries of the 20th century. For some years reaginic activity was believed to be a property of IgA that had been discovered in the 1950s. However, Mary Loveless (Fig. 1.7) in 1964 reported the presence of reagin in an individual who formed no detectable IgA (Loveless 1964). In 1966, Kimshige and Teruko Ishizaka (Fig. 1.8) (Ishizaka & Ishizaka 1966) found that reaginic activity was associated with an immunoglobulin other than IgG and IgA and went on to develop an antiserum which, after absorption with IgG and IgG subclasses, IgA, IgM, and IgD (a new immunoglobulin whose discovery had been reported by Rowe and Fahey in 1965), still precipitated protein in immunoglobulin fractions and also precipitated skin-sensitizing activity. As they stated “the results suggest the presence of a unique immunoglobulin as a carrier of reaginic activity.” The protein was tentatively designated IgE-globulin and in a series of remarkably thorough and brilliant experiments in which they laboriously checked and cross-checked their

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Fig. 1.6 (a) Otto Carl W. Prausnitz (Giles) (1876–1963) and (b) Heinz Küstner (1897– 1963). The passive transfer of immediate skin reactivity by interdermal injection of serum from an allergic patient (“reagin”) is called the Prausnitz–Küstner (or P–K) test. (From Cohen & Samter 1992, with permission.)

(a)


(b)

findings, they finally left no doubt of the validity of their conclusions (reviewed in Ishizaka & Ishizaka 1968). Gunnar Johansson and Hans Bennich (Fig. 1.9) had used a completely different approach to arrive at the same conclusion. They discovered in 1965 (but reported in 1967) a myeloma protein (IgND) which did not belong to any of the four known immunoglobulin classes (Johansson & Bennich 1967). IgND was shown to block skin-sensitizing activity and an antiserum prepared against it had the same specificity as the anti-IgE globulin prepared by Ishizaka. At an international conference held in 1968 it was agreed to call the new class of proteins to which reagins belonged “immunoglobulin E” (IgE) (Bennich et al. 1968).

Fig. 1.8 (Left) Teruko Ishizaka (1926–) and (right) Kimshige Ishizaka (1925–). Characterized reaginic antibody as IgE.

Mediator cells and mediators

Fig. 1.7 Mary Hewitt Loveless (1899–1991). Major contributions to immunotherapy including the identification of blocking antibody (with Robert Cooke) and the use of pure venoms in Hymenoptera allergy. (From Cohen & Samter 1992, with permission.)

The mast cell was discovered by Paul Ehrlich (Fig. 1.10) while still a medical student at the University of Freiburg. He was testing a new basic synthetic dye, “dahlia,” and discovered that some connective tissue cells contained large granules which avidly took up the dye and changed its color to a reddish purple (metachromasia) (Ehrlich 1877). He named these cells “mast” cells, i.e., well-fed cells, giving them this name because he believed the cell granules were products of cell overfeeding.

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Fig. 1.9 (Left) Hans Bennich (1930–) and (right) Gunnar Johansson (1938–). Identification of IgND as IgE immunoglobulin and with L. Wide developed the radioallergosorbent technique (RAST). (Courtesy of Pharmacia, Uppsala, Sweden.)

Fig. 1.10 Paul Ehrlich (1854–1915). An exceptionally creative bioscientist who, along with many other accomplishments, described the side-chain theory of antibody formation and discovered the mast cell and the eosinophil. (From Mahmoud & Austen 1980, with permission.)

James Riley and his coworker Geoffrey West (Fig. 1.11) were the first to provide convincing evidence that tissue mast cells contained histamine (Riley & West 1952, 1953; Riley 1959), and Stuart (1952) reported that anaphylaxis in the mouse, rabbit, and guinea pig was associated with degranulation of mast cells. By approximately the mid-1950s, evidence was

8

available that mast cells contained the mediators histamine, serotonin, and heparin, and that mast degranulation was related to the release of these mediators during anaphylaxis in several species. By that time, moreover, it was also evident that the tissue mast cell was not the only mediator cell. Two years after describing the mast cell, Ehrlich (1879a) noted the presence of cells with metachromatic granules in blood. Although he termed them “blood mast cells,” he proposed that unlike the tissue mast cells the blood cells were derived from bone marrow and were essentially equivalent to the neutrophil and eosinophil, cells he had also described. Later workers renamed the blood mast cell, the basophil. Eosinophils were discovered by Paul Ehrlich in 1879 and so called because they stained with negatively charged dyes including eosin (Ehrlich 1879b). He also suggested that the bone marrow was their site of origin. Some time earlier (1846) an anatomist at Charing Cross Hospital, London (Thomas Wharton-Jones) described granulated blood cells from several species using a simple compound microscope and no staining methods. His drawings indicate that these were almost certainly eosinophils (Wharton-Jones 1846). Histamine was the first substance to be considered an anaphylactic mediator. Sir Henry Dale (Fig. 1.12) demonstrated the presence of histamine in various tissues (Best et al. 1927). It was not until 1932 that others (Gebauer Fuelnegg in Dragstedt’s laboratory, Bartosch working with Feldberg and Spinelli) were finally successful in demonstrating the release of histamine during in vitro and in vivo anaphylaxis (Bartosch et al. 1932; Gebauer-Fuelnegg et al. 1932; Spinelli 1932). Histamine could explain some but not all the features of anaphylaxis. Schild (1936), for example, pointed out that a hundred times more histamine had to be administered to the guinea-pig lung than was released from shocked lungs to have the same effect in contracting the bronchi. This

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Fig. 1.11 (a) James F. Riley (1912–85) and (b) Geoffrey B. West (1916 –). Riley and West discovered that the mast cell granule was the major source of histamine in tissues. (From Cohen & Samter 1992, with permission.)

(a)

Fig. 1.12 Sir Henry H. Dale (1875–1968). Established the role of histamine in anaphylaxis and demonstrated chemical transmission of nerve impulses. (From Cohen & Samter 1992, with permission.)

and many other observations led to the discovery of slowreacting substance of anaphylaxis. Charles Kellaway and Everton Trethewie (Fig. 1.13) reported that the lungs or jejunum of sensitized guinea pigs perfused with antigen released a substance which gave a slow sustained contraction of guinea-pig ileum (Kellaway & Trethewie 1940). This was unlike the sharp short contraction given by histamine and, accordingly, they named the substance slow-reacting substance (SRS). It was not until


(b)

1953 that Walter Brocklehurst confirmed earlier work showing that perfusates of sensitized guinea-pig lungs challenged with antigen gave a slow contraction of guinea-pig ileum even in the presence of an antihistamine in the organ bath (Brocklehurst 1953, 1962). He renamed the agent SRS-A, the slow-reacting substance of anaphylaxis. SRS-A was eventually chemically characterized by Robert Murphy and Bengt Samuelsson (Fig. 1.14) as leukotriene D and E (later reclassified as LTC4, LTD4, and LTE4) (Borgeat & Samuelsson 1979; Murphy et al. 1979). Leukotrienes and many analogs were totally synthesized by E.J. Corey and with K. Frank Austen (Fig. 1.15) the range of biological activities in humans and experimental animals was established (Weiss et al. 1982). (See Clinical and Experimental Allergy Reviews, volume 1(3), November 2001 for the history of leukotrienes.) The story of bradykinin dates back to the experiments of Rocha e Silva, Beraldo, and Rosenfield who added trypsin or snake venom to serum globulin and obtained a peptide which was hypotensive, stimulated smooth muscle, and was also a vasodilator (Rocha e Silva et al. 1949). They termed the peptide “bradykinin” because it gave a contraction of smooth muscle that was somewhat slower than histamine. Beraldo (1950) demonstrated the liberation of bradykinin into the blood of dogs undergoing anaphylaxis. These results over the succeeding 10 years were confirmed and extended to other species.

Asthma and hay fever Asthma (meaning “panting”) has been recognized since ancient times. Moses Maimonides (1135–1204) (Fig. 1.16) wrote a Treatise on Asthma which described the disease and recommended certain lifestyle changes, especially diet, as

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(a)


(b)

Fig. 1.14 Bengt I. Samuelsson (1934–). Identification and chemical characterization of the SRS-A leukotrienes. (From Cohen & Samter 1992, with permission.)

beneficial. The first description of occupational asthma was made by Bernardino Ramazzini (1633–1714) (Fig. 1.17). Thomas Willis (1621– 75) (Fig. 1.18) suggested that asthma may have a nervous or neural component, a concept clearly valid to this day. Throughout the 19th and early 20th century asthma was generally believed to be due to spasm of the bronchial musculature either as result of heightened neural pathways or the result of anaphylaxis due to specific sensitization. Although John Floyer (1649–1734) (Fig. 1.19) recog-

10

Fig. 1.13 (a) Charles H. Kellaway (1889–1952) and (b) Everton R. Trethewie (1913–84). First description of a “slowreacting substance of anaphylaxis” causing smooth muscle contraction. (From Cohen & Samter 1992, with permission.)

Fig. 1.15 K. Frank Austen (1928–). Pioneered the biochemistry of mast cell mediator release and the biological properties of leukotrienes.

nized that asthma had many triggers, as well as a hereditary component, it was Henry Hyde Salter (1823–71) (Fig. 1.20) who made the first attempts to understand asthma mechanisms (McFadden 2004). Salter, himself an asthmatic, noticed that asthma attacks could be triggered by “extrinsic” factors such as exercise, cold air, laughing, coughing, sneezing, chemical and mechanical irritants, and animal and vegetable products. He also realized that other causes were operating and suggested that asthma involvedboth neural and vascular

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Fig. 1.16 Moses Maimonides (1135–1204). A prolific writer and author of a famous Treatise on Asthma. (From Cohen & Samter 1992, with permission.)

Fig. 1.17 Bernardino Ramazzini (1633–1714). First description of occupational diseases, in particular baker’s asthma. (From Cohen & Samter 1992, with permission.)

mechanisms and wrote with amazing accuracy, “The inflammation or congestionof the mucous surface appears to be the stimulus that, throughthe nerves of the air tubes, excites the muscular wall to contract.” On the other hand, Sir William Osler(1849–1919) taught that asthma was a psychoneurosis and because of his prestige this opinion was accepted by many physicians for decades after. It was many years before this view was put in perspective.


Fig. 1.18 Thomas E. Willis (1621–75). Recognized the importance of bronchial innervation in asthma; and asthma as a “nervous disease.” (From Cohen & Samter 1992, with permission.)

Fig. 1.19 Sir John Floyer (1649–1734). Recognition of asthma as a multifactorial disease with many triggers (e.g., tobacco smoke, dust, foods, exercise, emotions, environmental factors). First description of heredity in asthma. (From Cohen & Samter 1992, with permission.)

In studies on the pathology of asthma, Ernst von Leyden (1832–1910) (Fig. 1.21) described colorless, needle-like crystals in the sputum of asthmatics and used the observation to “prove asthma was not a nervous disease.” Much earlier Jean Martin Charcot (1825–93) (Fig. 1.21) had noted similar crystals from a leukemic spleen, hence Charcot–Leyden crystals.

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Fig. 1.20 Henry Hyde Salter (1823–71). Description of the various causes of asthma and of cells (now known as eosinophils) in sputum. (From Cohen & Samter 1992, with permission.)

Fig. 1.22 Francis M. Rackemann (1887–1973). Introduced the term “intrinsic asthma.” (From Cohen & Samter 1992, with permission.)

Many clinicians appreciated that asthma had an allergic component but it was Francis Rackemann (1887–1973) (Fig. 1.22) who introduced the term “intrinsic asthma” to describe patients who gave no history of “allergy” and who were skin test negative to common allergens. There was a well-recognized association between asthma and hay fever but it was John Bostock (Fig. 1.23) who in 1819 was the first to describe hay fever as a disease with distinct symptoms. Much later, in 1873, Charles Blackley (Fig. 1.24), in experiments carried out largely on himself, showed that hay

fever was due to pollen. He was the first to use conjunctival and skin tests and also showed a relationship between the number of pollen grains collected in 24 hours on sticky glass slides and the intensity of symptoms. Dunbar thought that hay fever was caused by a toxin in pollen and he produced an “antitoxin” in horses which he called “Pollatin” which was widely used therapeutically. But it was Leonard Noon (1911) (Fig. 1.25) who successfully introduced specific injection therapy for hay fever. After his premature death from tuberculosis, his colleague John

(a)

12

(b)

Fig. 1.21 (a) Jean Martin Charcot (1825–93) and (b) Ernst V. von Leyden (1832–1910). The needle-like (eosinophil-derived) crystals characteristic of asthmatic sputum are named after Charcot and Leyden. (From Cohen & Samter 1992, with permission.)

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Fig. 1.23 John Bostock (1773–1846). Described “catarrhus aestivus,” later recognized as summer hay fever. (From Cohen & Samter 1992, with permission.)

Fig. 1.24 Charles H. Blackley (1820–1900). Identified pollen as a cause of hay fever and devised methods for pollen counts and clinical challenge tests. (From Cohen & Samter 1992, with permission.)

Freeman (Fig. 1.25) continued the work (Freeman 1914) and by 1920 it was a standard method of treatment among allergists. Cooke (Fig. 1.26) and Vander Veer then recognized a hereditary component to hay fever and other conditions associated with sensitization and, later, Coca and Cooke (1923) introduced the term “atopy” (meaning “out of place”)

which they considered to be a peculiarly human condition in which there was hereditary predisposition to produce reagin but which was quite separate from anaphylaxis. Another antibody beside IgE found in human immediatetype allergic disease is the so-called “blocking antibody.” This was discovered by Cooke et al. (1935) in attempting to find

Fig. 1.25 (a) Leonard Noon (1877–1913) and (b) John Freeman (1877–1962). Noon and Freeman introduced the treatment of hay fever by immunization with pollen extracts. (From Cohen & Samter 1992, with permission.)

(a) (b)

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Fig. 1.26 Robert A. Cooke (1880–1960). Introduced the protein nitrogen unit (PNU) for standardization of allergen extracts, realized the role of hereditary factors in hay fever and described allergic drug reactions. Cooke also discovered “blocking antibody” with Mary Loveless. (From Cohen & Samter 1992, with permission.)

out why the injection treatment for hay fever was successful. The concept that the blocking activity was due to binding of the antibody with the allergen was first explicitly stated by Mary Loveless when she found that blocking activity, unlike the skin-sensitizing activity, was heat stabile, withstanding heating at 56°C (Loveless 1940). Blocking antibody seemed to offer a respectable, “scientific” reason for the efficacy of the injection treatment of hay fever, although doubt continues to be expressed as to whether the development of blocking antibody was responsible for the therapeutic relief claimed for the treatment. Many other “allergy landmarks” are listed in chronologic order in Table 1.1. All were crucial observations even though in many instances their full importance was not recognized at the time.

The Coombs and Gell classification of hypersensitivity reactions Until the 1960s there had been difficulties in relating the various models of hypersensitivity in humans and experimental animals, as well allergic diseases themselves, to some form of systematic classification in order to study disease processes in an ordered fashion. Coombs and Gell (1963) described a

14

“classification of allergic reactions which may be deleterious to the tissues and harmful to the host”. This still remains useful to practicing physicians, scientists, and students today because it relates mechanisms to disease entities. Coombs and Gell predicted correctly that in any one disease it was likely that more than one kind of allergic process was involved. They also emphasized the fact that their classification was primarily one of initiating mechanisms and not of the subsequent events or the diseases themselves. The type I–IV hypersensitivity reactions of Coombs and Gell, with some modification in the light of more recent knowledge, are shown in Fig. 1.27. The type I reaction is initiated by allergen or antigen reacting with tissue cells passively sensitized by antibody produced elsewhere, leading to the release of a wide range of biological agents including pharmacologically active substances, proteases, cytokines, and chemokines. These anaphylactic reactions include general anaphylaxis in humans and other animals as well as local manifestations of anaphylaxis, such as that observed in the skin following diagnostic skin-prick tests, and local responses in the respiratory and gastrointestinal tracts. IgE was discovered shortly after the Coombs and Gell classification was published. The high-affinity IgE receptor was discovered some 15 years later (Lanellopoulis et al. 1980; Perez-Montfort et al. 1983) and was a landmark observation leading to the later elucidation of signal transduction pathways involved in IgE-dependent mediator release. More recently it has been shown that in mice antigenspecific light chains can sensitize mast cells for subsequent antigen-induced release of mediators (Kraneveld et al. 2005). This observation is yet to be confirmed in humans. Type II reactions (cytolytic or cytotoxic) are initiated by antibody reacting either with an antigenic component of a tissue cell or with an antigen or hapten intimately associated with these cells. Complement was usually, but not always, necessary to effect the cellular damage. Examples include drug-induced hemolytic anemia in association with chlorpromazine or phenacetin and thrombocytopenic purpura caused by the now obsolete sedative Sedormid. There are many examples of type II reactions outside the province of the clinical allergist, including incompatible blood transfusion reactions and autoallergic (autoimmune) hemolytic anemia. In some instances antibodies against cell-surface receptors have cell-stimulatory (agonist) effects without necessarily being cytotoxic. An example is Graves’ disease (hyperthyroidism, autoallergic thyroiditis) in which IgG antibodies directed against the thyroid-stimulating hormone (TSH) receptor is produced. These have agonist effects by stimulating thyroid hormone production with subsequent thyrotoxicosis and goitre formation. Similarly, some patients with chronic urticaria have histamine-releasing IgG autoantibodies against the ε subunit of the high-affinity IgE receptor (FcεRIα) (Hide et al. 1993). The antibody is believed to activate normal mast cell function by receptor cross-linking

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Table 1.1 Hypersensitivity reactions: Landmark findings and theories throughout the ages (adapted from De Weck (1997)). 28th BC

Shen Nung

First reference to an anti-asthmatic plant (“ma-huang”), shown later to contain ephedrine, in the first herbal compendium Pen Ts’ao

2698 BC 2641 BC

Huang Ti Menes

First decription of asthma (“noisy breathing”) in the Nei Ching, oldest treatise of internal medicine. Egyptian pharaoh reported to have died from a wasp (“kehb”) sting (first report of an anaphylactic shock?) However, interpretation of hieroglyph controversial (Avenberg & Harper, 1980)

460–365 BC

Hippocrates

Description of asthma, eczema and allergy to goats’ milk and cheese

25 BC–AD 40

Aulus Celsus

Thorough description of dyspnoea, asthma and orthopnoea in treatise “De medica”

AD

c. 40

Marcus Terrentius Varro

Very small animals invisible to the eye, floating in the air, growing in damp places, inhaled and giving rise to serious diseases (mites?)

AD

b.41

Britannicus

Reported to be afflicted by acute allergic reactions to horses

AD

c. 60

Pedanius

Remedies for asthma in classical pharmacology treatise

Aretaeus of Cappadocia

First detailed description and coining of the word asthma

120–AD 180 AD

b.131

Galen

Description of untoward reactions to various milks (goat, cow, ewe, camel, ass): allergy?

AD

b.865

Rhazes

Description of seasonal catarrh due to roses in Persia

1135–1204

Moses Maimonides

Author of famous “Treatise on Asthma”. Physician to Sultan Saladin

b.1306

John of Arderne

Prescription of a “syrup” for asthma

1530

Thomas Moore

Report on acute skin eruption of King Richard III due to ingestion of strawberries (Shakespeare!)

1552

Girolamo Cardano

Cures asthma of Archbishop Hamilton of St Andrew by elimination of bedding feather pillows

1565

Leonardhus Botallus

Description of “rose cold” in Pavia

1570

Pietro Mattioli

First reported challenge of a cat allergic patient by stay in a room containing a concealed cat

c. 1584

Johann Schenk

Coins the term “idiosyncrasy”

b.1603

Kenelm Digby

Blister to rose petal applied on cheek of English court lady hypersensitive to roses (first patch test?)

1603

Felix Platter

Asthma due to obstruction of small pulmonary arteries or to nerve disturbances

c. 1630

Sanctorius

Description of asthma to cat hair

1656

Pierre Borel

Weakness, fainting and asthma upon contact with cats, mice, dogs and horses (particularly in Germans?) Blister upon applying egg on skin of hypersensitive patient (first skin test?)

1662

K.V. Schneider

Nasal catarrh caused by exudation from nasal mucosa, not by secretion from the brain (!)

1665

Philipp Jacob Sachs

Description of a case of urticaria caused by strawberries and of shock upon ingestion of fish

1673

Johannes Binneringus

Description of seasonal rose coryza in Basle

1675

Theophile Bonet

Idiosyncrasies to bread, strawberries and wine

1680

Thomas Willis

Studies of asthma as bronchial disease and role of bronchial innervation; asthma as nervous disease

c. 1680

Nehemiah Green

First microscopic studies of pollen grains

1682

Joan van Helmont

Description of seasonal asthma with itching skin eruption (atopic dermatitis?) and of psychosomatic asthma

1691

Jacob de Rebecque

Coryza due to rose scent but only at the end of spring

1698

John Floyer

Description of asthma causes (tobacco smoke, dust, foods, exercise, emotions, environmental factors) First description of heredity in asthma

1713

Bernardino Ramazzini

First systematic description of occupational diseases, in particular baker’s asthma

1765

Debrest

Description of sudden death by bee sting in Montpellier

1775

William Cullen

Hereditary idiosyncrasy to eggs in “Historia de Materia Medica” Continued p. 16

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Table 1.1 (Cont’d ) 1776

Johann Murray

Ipecac root (emetine), causes asthma attacks in pharmacists

1778

Stolpertus

Description of acute angioneurotic oedema following ingestion of eggs

1783

Friedrich Schademantal

Allergic urticaria due to ingestion of fresh pork meat

1783

Philipp Phoebus

Comprehensive monograph on hayfever. First epidemiological enquiry

1802

William Heberden

Description of summer catarrh and asthma; differentiation from common cold

1816

Henri Laennec

Invents stethoscope. Identifies bronchospasm as important component of asthma

1819

John Bostock

Description of 28 cases of “estival catarrh” or hay-fever, disease restricted to upper classes of society

1839

François Magendie

Description of sudden death of dogs repeatedly injected with egg albumin

1853 1886

Jean Martin Charcot and Ernst van Leyden

Description of Charcot–Leyden crystals in sputum of asthmatics

1868

Henry Hide Salter um

Description of asthma with various causes (animal emanations, foods, hayfever), intrinsic asthma, cells in sputum (later identified as eosinophils)

1872

Heinrich Quincke

First description of angioneurotic oedema

1872

Morrill Wyman

Description of autumnal catarrh in USA and identification of ragweed pollen as cause

1873–80

Charles Blackley

Experimental demonstration of role of grass pollens in hayfever, first pollen counts

1877

Paul Ehrlich

Description and staining of mast cells (1877) and eosinophils (1879)

1894

Samuel Flexner

Experimental “toxic death” whilst injecting dog serum into rabbits

1895

Josef Jadassohn

Establishment of patch tests in contact dermatitis

1895

Josef Jadassohn

Description of various types of drug reactions in the skin

1900

Solomon Solis-Cohen

Role of autonomic imbalance in allergic diseases. Use of adrenal substance in hayfever and asthma

1902

Charles Richet Paul Portier

Discovery of experimental anaphylaxis in dogs

1903

Theobald Smith

Observations of anaphylactic reactions of guinea pigs to horse serum

1903

Maurice Arthus

Experimental localized acute necrotizing vasculitis, first described as local anaphylaxis

1905

Bela Schick

First description of serum sickness disease: skin test for diphtheria susceptibility

1905

Clemens von Pirquet

Studies on serum sickness, coins the term “allergy”: introduces tuberculin skin test in diagnosis

1906

A. Wolff-Eisner

Relationship of human hayfever and urticaria to experimental anaphylaxis

1909

William Schultz

Detection of anaphylaxis by contraction of isolated smooth muscle in vitro

1909

William B. Osler

Asthma associated with neurotic disease

1910

S. Meltzer

Bronchial asthma as a phenomenon of anaphylaxis

1910

William Dunbar

Methodology for pollen extraction; first approaches to pollen immunotherapy

1911

Leonard Noon John Freeman

Wide use of immunotherapy with pollen extracts in hayfever patients

1911

Henry Dale

Role of histamine in anaphylaxis and studies on chemical transmission of nerve impulses

1911

Tomaso Casoni

Skin test in patient infected with Echinococcus

1912

Oscar M. Schloss

Use of scratch test in allergy to foods

1913

William Dunbar

Methodology for pollen extraction, identification of allergenic protein

1914

A.T. Waterhouse

Anaphylactic reactions of beekeepers to stings

16

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Warfield T. Longcope

Experimental lesions in organs from animals repeatedly injected with foreign proteins

1916

Robert A. Cooke

Standardization of allergen extracts – protein nitrogen unit (PNU), role of hereditary factors in hayfever, description of allergic drug reactions

1918

Francis Rackemann

Description of intrinsic and extrinsic asthma: asthma is not always of allergic origin

1919

M.A. Ramirez

Horse dander asthma following blood transfusion

1921

Carl Prausnitz Heinz Küstner

Passive transfer of immediate skin reactivity of fish allergen by intradermal injection of serum from allergic patient (“reagins”)

1921

Arent de Besche

Passive transfer of serum from horse asthmatics sensitized to horse proteins by injection of diphtheria vaccine

1922

Fernand Widal Pierre Abrami J. Lermoyez

Described triad of asthma, vasomotor rhinitis (with or without nasal polyps) and intolerance to aspirin and aspirin-like medicines (also known as Samter’s syndrome)

1923

Arthur Coca

Reagent for allergen extraction

1923

Arthur Coca

Coined the term “atopy”

1924

F.T. Codham

First description of mould allergy

1924

Ko Kuei Shen Carl F. Schmidt

Systematic investigations of pharmacological actions of ephedrine, the active component of “ma-huang”

1927

Thomas Lewis

Description of similarities between urticaria and skin vascular reactions to histamine (vasodilatation, flare and local oedema as triple response)

1927

Bret Ratner

Experiments on allergic sensitization in utero

1928

Storm van Leeuwen

Inhalation allergy to house dust

1928

Edward Dienes

Induction of cellular delayed hypersensitivity by protein antigens

1934

Mataso Masugi

Experimental glomerulonephritis with anti-kidney antiserum

1935

Ulf von Euler

Discovery of the activity of lipid fraction of seminal fluid on smooth muscle (“prostaglandins”)

1937

Daniel Bovet

First synthesis of antihistaminic drugs

1939

Harry H. Donally

Transmission of food allergens in breast milk

1940

Charles H. Kellaway Everton T. Trethewie

First description of “a slow-reacting substance of anaphylaxis” causing smooth muscle contraction

1940

Mary Loveless

Description of blocking antibodies arising during immunotherapy with pollen extracts. Use of pure venoms in immunotherapy for hymenoptera allergy

1941

Louis B. Jaques

Relationship between mast cells and anaphylaxis in dogs: mast cells as source of released heparin

1941

Joseph Harkavy

Bronchial asthma with recurrent pulmonary eosinophilic infiltration and polyserositis

1942

Merrill W. Chase

Transfer of tuberculin sensitivity by cells from immunized animals

1945

Merrill W. Chase

Transfer of contact dermatitis to simple chemicals by sensitized leukocytes

1945

Robin A. Coombs

Described the antiglobulin (“Coombs”) test

1949

Philip S. Hench Edward C. Kendall

Isolation of cortisone from adrenals for therapy of rheumatoid arthritis

1952

Zoltan Ovary

Development of passive cutaneous anaphylaxis (PCA) for quantification

1953

James F. Riley Geoffrey B. West

Mast cell granules as major source of histamine in tissues

1954

William Frankland Rosa Augustin

First placebo-controlled clinical trial of desensitization (allergen-injection immunotherapy)

Continued p. 18

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Ernest Witebsky

Experimental autoallergic thyroiditis: pathology and criteria of immune diseases

1958–60

Zoltan Ovary Herman N. Eisen

Elicitation of immediate anaphylactic reactions requires bridging of antibody molecules by bi- or multivalent allergen

1960

Bernard. B. Levine Alain L. de Weck

Identification of major and minor antigenic determinants in penicillin allergy

1962

Alain L. de Weck Charles W. Parker

Diagnostic skin testing for penicillin allergy with synthetic penicilloyl-polylysine polymers

1963

Robin A. Coombs Philip Gell

Classification of the hypersensitivity reactions

1963

K. Frank Austen

Biochemistry of mast cell mediator release

1963

Jack Pepys

Identification of moulds and anti-Thermosopora IgG antibodies as cause of farmer’s lung

1964

Lawrence M. Lichtenstein Abraham G. Osler

Development of allergen-specific histamine release test

1964

T.E. King

Identification of major allergen in ragweed

1966

Barry R. Bloom John R. David

Description of lymphokine-induced cell interaction: macrophage migration inhibitory factor (MIF)

1966

David Marsh

Identification of major grass allergens

1966

J.J. Curry

Asthmatics more sensitive than normals to the action of histamine on the respiratory tract

1967

Kimishige Ishizaka Teruko Ishizaka

Characterization of reagins as IgE immunoglobulins

1967

Hans Bennich Gunnar Johansson

Identification of myeloma ND as IgE immunoglobulin

1967

L. Wide Gunnar Johannsson

Development of a RAdioSorbent Test (RAST) for detection of allergen-specific IgE

1967

Reindert Voorhorst Frederick Spieksma

Identification of Dermatophagoides mites as major allergen source in house dust

1967

Bernard Halpern

Lymphocyte stimulation test in drug allergy

1967

Roger Altounyan

Discovery of sodium cromoglycate as anti-asthmatic drug

1981

K. Frank Austen

Biological properties of leukotrienes

1983

Bengt I. Samuelson

Identification of leukotrienes as slow-reacting substance of anaphylaxis, role in allergic diseases

and in this sense is cytostimulating rather than cytolytic (in which there is destruction of the cells with liberation of preformed histamine). In myasthenia gravis, on the other hand, autoantibodies directed against acetylcholine receptors have been identified. These have antagonist properties leading to a failure to sustain maintained or repeated contraction of striated muscle. Although, in both situations, the initiating event is IgG bound to cell-surface antigen, the outcome is quite different, giving on the one hand cytolytic or cytotoxic reactions, and on the other a cytostimulating hypersensitivity reaction in which there is altered cell function (or cell signaling) with IgG antibody acting either as an agonist or an

18

antagonist. For these reasons, Janeway and Travers (1995) proposed that cytotoxic or cell-stimulatory reaction are subdivided into type IIa (cytotoxic) and type IIb (cell-stimulating) responses (Fig. 1.27). Type III reactions (Arthus reactions and “immune complex” or toxic complex syndrome) occur when antigen and antibody, reacting in antigen excess, form complexes which, possibly with the aid of complement, are toxic to cells. As shown by Jack Pepys (1914–96) (Fig. 1.28), this mechanism operates, at least in part, in farmer’s lung (and other forms of extrinsic allergic alveolitis). Other examples of type III reactions include erythema nodosum leprosum, serum sickness,

Soluble

Antigen

• Certain allergic drug reactions (e.g. penicillin) • Incompatible transfusion reactions • Autoallergic (“autoimmune”) hemolytic anemia

b

Antagonist

Neutrophil-rich inflammatory response

APC

MHC Class II

TCR

Macrophage-rich inflammatory response

Type 1 cytokines

AF

CD4+ type 1

T Lymphocyte

Soluble

Antigen presentation to sensitized CD4+ type 1 T lymphocytes (also called T helper (Th) type 1 cells)

• Tuberculin • Chronic urticaria • Serum sickness reaction (Anti-FceRIa antibody • Extrinsic allergic • Contact – agonist) alveolitis dermatitis • Graves disease • Antigen-antibody • Rheumatoid (Thyroid stimulating complex arthritis antibody – agonist) (“immune complex”) • Myasthenia gravis glomerulonephritis (Anti-acetylcholine receptor antibody – antagonist)

Agonist

IgG

Microvasculature

Soluble

Antigen-antibody complexes, in and around the microvasculature, which activate complement

• • • •

MHC Class II

Chronic asthma Chronic allergic rhinitis Atopic eczema Late-phase allergic reactions (in experimental models of atopic allergic disease)

Eosinophil- and basophil-rich inflammatory response

Type 2 cytokines

APC

CD4+ type 2

T Lymphocyte

Soluble

MHC Class I

• Early-onset, insulin-dependent diabetes • Graft rejection

Cytotoxicity (apoptosis)

Target cell

CD8+ cytotoxic

T Lymphocyte

Cell-associated

Cytotoxic CD8+ T lymphocytes recognize fragments of antigen on the surface of target cells

Cell-mediated eosinophilic hypersensitivity or chronic allergic inflammation Antigen presentation to sensitized CD4+ type 2 T lymphocytes. Sensitized CD8+ type 2 T lymphocytes (also called T cytotoxic (Tc) type 2 cells) may also participate

Tissue injury by cytotoxic T lymphocytes

Th2

Th1

Type IV cytotoxic

Type IV

Type IV

Arthus type (or Classical delayedantigen-antibody type hypersensitivity complex) – often called ‘immune complex’ – hypersensitivity reaction

Type III

Receptor Antigen-antibody complexes + ligand complement

Target cell Receptor

Cell-associated

IgG cell-stimulating antibody interacting with cell surface receptors involved in cell signaling

Cell-stimulating reactions involving altered cell function (or signaling)

Type II

Fig. 1.27 A modification to the Coombs and Gell classification (1963) of hypersensitivity reactions based on more recent knowledge of the initiating events but restricted to human allergic disease. Immediate-type (type I) reactions involve soluble antigen interacting with cell-bound IgE. Type I reaction involves IgE antibodies bound to high-affinity (FceRI) IgE receptors on mast cells or basophils (and possibly macrophages or even eosinophils). Antigen (allergen) induces the release of granule-associated and membrane lipid-derived mediators of hypersensitivity as well as several cytokines including interleukin (IL)-5, IL-3, and granulocyte–macrophage colony-stimulating factor (GM-CSF). Type II reactions are subdivided into type IIa, cytolytic or cytotoxic reactions originally described by Coombs and Gell, in which antibody-sensitized cells are destroyed by complement lysis or removed by the reticuloendothelial (RE) system; and type IIb, those in which antibodies directed against cell-surface receptors cause altered cell function or signaling. In type IIb, antibody is cell-stimulating (cytostimulatory) and acts as either an agonist or antagonist. (From Janeway & Travers 1995.) The type III Arthus-type reaction, or antigen–antibody complex reaction (mostly called immune-complex reaction), is mediated by soluble antigen and involves IgG, complement, and an inflammatory reaction which initially is neutrophilrich. Type IV reactions are subdivided into (i) classical delayed-type hypersensitivity initiated by CD4+ Th1-type lymphocytes (type IV Th1), (ii) cell-mediated eosinophilic hypersensitivity or chronic allergic reactions involving CD4 + (and sometimes CD8 +) Th2-type cells (type IV Th2) and (iii) reactions in which tissue damage is evoked by CD8 + cytotoxic T lymphocytes (type IV cytotoxic).

• Acute symptoms of allergic rhinitis • General and local anaphylaxis • Early-phase allergic reactions (in experimental models of atopic allergic disease)

Complement lysis or removal by the RE system

+/– complement

IgG

Cell surface antigen

Target cell

Cell-associated

IgG antibody interacting with cell surface antigen

Cytolytic, or cytotoxic, reactions

a

CHAPTER 1

Examples in humans

Allergen

Release of granuleassociated mediators (e.g. histamine) and membrane-derived lipid mediators of hypersensitivity

IgE

Mast cell/basophil

Antigen (allergen) interacting with mast cells or basophils passively sensitized by IgE

Initiating event

Simplified scheme of the proposed mechanism

Immediate-type (IgE-dependent, or anaphylactic) hypersensitivity

Descriptive term

Type I

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Fig. 1.28 Jack Pepys (1914–96). Identified thermophilic actinomycetes as a cause of farmer’s lung. Major contributions to our understanding of the etiology and pathogenesis of allergic alveolitis, occupational asthma, and allergic bronchopulmonary aspergillosis. (From Cohen & Samter 1992, with permission.)

antigen–antibody complex glomerulonephritis, and deposition of antigen–antibody complexes at other sites such as the skin as in certain vasculitic skin rashes. Type IV reactions refer to situations where specifically sensitized T cells react with allergen or antigen deposited at the local site, as in delayed- or tuberculin-type hypersensitivity. Classical delayed-type hypersensitivity involves predominantly CD4+ T cells with antigen presented in a major histocompatibility complex (MHC) class II restricted fashion. These reactions are characterized by infiltration of T lymphocytes with a restricted cytokine profile. As described below, and in detail elsewhere (see Chapters 3 and 4), these cells preferentially produce interferon (IFN)-γ and interleukin (IL)-2 and are therefore characteristic of the T helper type 1 (Th1) lymphocyte. Contact dermatitis, an important allergic disease, is another example of a type IV reaction with a prominent Th1-type cytokine response. Th2 cells on the other hand elaborate IL-4, IL-5, IL-9, and IL-13 and are involved in atopic allergic reactions as well as parasitic helminthic disease. Some T lymphocyte-mediated hypersensitivity reactions, of which early-onset (insulin-dependent) diabetes is an example, involves CD8+ cytotoxic T cells. These recognize cell-surface antigen presented to T cells in an MHC class I restricted fashion. After cell–cell contact, programmed cell death (apoptosis) of the target is initiated. Although in health, cytotoxic T cells provide a basic “immune” mechanism for

20

dealing with viruses and other insoluble antigens, in the context of insulin-dependent diabetes and graft rejection they mediate a variant of type IV hypersensitivity, termed type IV cytotoxic (see Fig. 1.27). As stated, the effector cell in classical delayed-type hypersensitivity is the CD4 type 1 (or Th1) lymphocyte whereas allergic tissue damage is mediated by Th2 cells. For this reason it is logical that these two forms of cell-mediated hypersensitivity are referred to as type IV Th1 and type IV Th2 respectively, since the initiating event involves T lymphocytes with distinct characteristics (see Fig. 1.27). The involvement of other classes of T cells or T-cell subsets in allergic reaction is also of current interest and is described in Chapters 3 and 4. Akbari et al. (2006) found that 60% of CD4 + T cells in the airways of asthmatics were invariant natural killer (NK)T cells. This finding remains controversial since others have found low numbers of NKT cells in asthma, chronic obstructive pulmonary disease, and controls (Vijayanand et al. 2007). There is growing interest in the possible role of Th17 cells in allergic disease although their role remains ill-defined in humans. As discussed in Chapter 3, they are distinct from Th1 and Th2 cells are involved in the initiation of a predominantly neutrophil-rich inflammatory response (Romagnani 2006). Control of allergic inflammation by natural and inducible T regulatory cells is discussed in Chapter 4. Some hypersensitivity reactions do not fall neatly into the type 1–IV classification. For example, activation of the plasma cascade via factor XII, prekallikrein, and high-molecularweight kininogen leads to bradykinin formation, the critical mediator of hereditary angioedema (Fields et al. 1983).

Concluding comments The full history of allergy is long and complex and only a relatively superficial account can be given here. The story of direct relevance to atopic allergic disease ranges from the first full description of anaphylaxis in 1903 to the discovery of IgE in the 1960s. Side by side is the unraveling of the structure and biological properties of various biological agents released in the allergic cascade. The coining of the word “allergy” itself, although often misunderstood, laid the foundation for a fundamental rule of immunology, with the concept that humans and animals “alter” their reactivity to antigen when they meet it on second and subsequent occasions.

Acknowledgments I have drawn heavily on the following three excellent and important works in the preparation of this chapter: “Elements of the history of our present concepts of anaphylaxis, hay fever and asthma” by the late Elmer Becker (1999); Excerpts from Classics in Allergy by Sheldon Cohen and Max Samter

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(1992); and “A short history of allergological diseases and concepts” by Alain de Weck (1997).

References Akbari, O., Faul, J.L., Hoyte, E.G. et al. (2006) CD4+ invariant T-cellreceptor + natural killer T cells in bronchial asthma. N Engl J Med 354, 1117–29. Arthus, M. (1903) Injections répétées de serum de cheval chez le lapin. C R Soc Biol (Paris) 50, 20. Avenberg, K.M. & Harper, S.D. (1980) Footnotes on Allergy. Pharmacia, Uppsala, Sweden. Bartosch, R., Feldberg, W. & Nagel, E. (1932) Das Freiwarden eines histaminhnlichen. Stoffes bei der Anaphylaxie des Meerschweinchens. Pflugers Arch ges Physiol 230, 129–53. Becker, E.L. (1999) Elements of the history of our present concepts of anaphylaxis, hay fever and asthma. Clin Exp Allergy 29, 875–95. Bennich, H., Ishizaka, K., Johansson, S.G.O., Rowe, D.S., Stanworth, D.R. & Terry, W.D. (1968) Immunoglobulin E, a new class of human immunoglobulin. Bull WHO 38, 151–2. Beraldo, W.T. (1950) Formation of bradykinin in anaphylactic and peptone shock. Am J Physiol 163, 283– 9. Best, C.H., Dale, H.H., Dudley, H.W. & Thorpe, W.V. (1927) The nature of the vasodilator constituents of certain tissue extracts. J Physiol (Lond) 62, 397– 417. Borgeat, P. & Samuelsson, B. (1979) Transformation of arachidonic acid by rabbit polymorphonuclear leukocytes. Formation of a novel dihydroxy eicosanoic acid. J Biol Chem 254, 2643– 6. Brocklehurst, W.E. (1953) Occurrence of an unidentified substance during anaphylactic shock in cavy lung. J Physiol (Lond) 120, 16P. Brocklehurst, W.E. (1962) Slow reacting substance and related compounds. Prog Allergy 6, 539– 88. Bulloch, W. (1937) The History of Bacteriology. Oxford University Press, London. Coca, A.F. & Cooke, R.A. (1923) On the classification of the phenomena of hypersensitiveness. J Immunol 8, 163– 82. Cohen, S.G. & Samter, M. (1992) Excerpts from Classics in Allergy, 2nd edn. Symposia Foundation (Oceanside Publications), Providence, RI. Cooke, R.A., Barnard, S.H., Hebald, A.T. & Stull, A. (1935) Serological evidence of immunity with co-existing sensitization in a type of human allergy, hay fever. J Exp Med 62, 733–50. Coombs, R.R.A. & Gell, P.G.H. (1963) The classification of allergic reactions responsible for allergic reactions underlying diseases. In: Gell, P.G.H. & Coombs, R.R.A., eds. Clinical Aspects of Immunology. Blackwell Scientific Publications, Oxford, pp. 217–37. De Weck, A. (1997) A short history of allergological diseases and concepts. In: Kay, A.B., ed. Allergy and Allergic Diseases. Blackwell Scientific Publications, Oxford, chapter 1. Doerr, R. (1914) Neuere ergebnisse der anaphylaxieforschung. Ergebnisse Immunittsforsch Hyg Bakteriol Exp Ther 1, 257–76. Ehrlich, P. (1877) Beitrage zur Kenntnis der Anilifarbungen und ihrer Verwedung in der Mikroskopischen Technik. Arch Mikr Anat 13, 263–77. Ehrlich, P. (1879a) Beitrage zür Kenntniss der granulirten Bindegewebszellen und der eosinophilen Leukocythen. Archiv Fur Anatomie und Physiologie: Physiologische Abteilung 166–9.


Ehrlich, P. (1879b) Ueber die specifischen Granulationen des Blutes. Archiv fur Anatomie und Physiologie: Physiologische Abteilung, 571– 9. Fields, T., Ghebrehiwet, B. & Kaplan, A.P. (1983) Kinin formation in hereditary angioedema plasma: evidence against kinin derived from C2 and in support of “spontaneous” generation of bradykinin. J Allergy Clin Immunol 72, 54–60. Freeman, J. (1914) Vaccination against hay fever: a report of results during the last three years. Lancet 183 (4730), 1178–80. Gebauer-Fuelnegg, E., Dragstedt, C.A. & Mullenix, R.B. (1932) Observations on a physiologically active substance appearing during anaphylactic shock. Proc Soc Exp Biol Med 29, 1084–6. Hamburger, F. & Moro, E. (1903) Ueber die biologisch nachweisbaren Veränderungen des menschlich Blutes nach den Seruminjektion. Wien Klin Wochenschr 16, 445–7. Hide, M., Francis, D.M., Grattan, C.E.H., Hakimi, J., Kochan, J.P. & Greaves, M.W. (1993) Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria. N Engl J Med 328, 1599–604. Ishizaka, K. & Ishizaka, T. (1966) Physicochemical properties of human reaginic antibody. I. Association of reaginic antibody with an immunoglobulin other than gA or gG globulin. J Allergy 37, 169– 85. Ishizaka, K. & Ishizaka, T. (1968) Human reaginic antibodies and immunoglobulin E. J Allergy 42, 330–63. Janeway, C. & Travers, P. (1995) Immunobiology, 2nd edn. Garland Press, London, chapter 11. Johansson, S.G.O. & Bennich, H. (1967) Immunological studies of an atypical (myeloma) immunoglobulin. J Immunol 98, 381–94. Karsner, H. & Ecker, E. (1921) Principles of Immunology. Lippincott, Philadelphia, p. 308. Kay, A.B. (1997) Concepts of allergy and hypersensitivity. In: Kay AB, ed. Allergy and Allergic Diseases, Vol. 1. Blackwell Science, Oxford, pp. 23–35. Kay, A.B. (2006) 100 years of “Allergy”: can von Pirquet’s word be rescued? Clin Exp Allergy 36, 555–9. Kellaway, C.H. & Trethewie, E.R. (1940) The liberation of a slowreacting smooth muscle stimulating substance in anaphylaxis. Q J Exp Physiol 30, 121–45. Kraneveld, A.D., Kool, M., van Houwelingen, A.H. et al. (2005) Elicitation of allergic asthma by immunoglobulin free light chains. Proc Natl Acad Sci USA 102, 1578–83. Lanellopoulis, J.M., Liu, T.Y., Poy, G. & Metzger, H. (1980) Composition and subunit structure of the receptor for immunoglobulin E. J Biol Chem 255, 9060. Loveless, M.H. (1940) Immunological studies of pollenosis. I. The presence of two antibodies related to the same pollen-antigen in the serum of treated hay fever patients. J Immunol 38, 25–58. Loveless, M.H. (1964) Reagin production in a healthy male who forms no detectable b2A immunoglobulins. Fed Proc 23, 403. McFadden, E.R. Jr (2004) A century of asthma. Am J Respir Crit Care Med 170, 215–21. Magendie, F. (1839) Lectures on the Blood. Harrington, Barington and Hasswell, Philadelphia. Mahmoud, A.A.F., Austen, K.F. (eds) (1980). The Eosinophil in Health and Disease. Grune & Stratton, p. 5. Murphy, R.C., Hammarstrom, S. & Samuelsson, B. (1979) Leukotriene C. A slow reacting substance from murine mastocytoma cells. Proc Natl Acad Sci USA 76, 4275–9.

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Noon, L. (1911) Prophylactic inoculation against hay fever. Lancet 177 (4580), 1572–3. Perez-Montfort, R., Kinet, J.-P. & Metzger, H. (1983) A previously unrecognized subunit (gamma) of the receptor for immunoglobulin E. Biochemistry 22, 5722. Prausnitz, C. & Kustner, J. (1921) Studien uber die Ueberempfindlichkeit. Zentralbl Bakteriol Mikrobiol Hyg 1 Abt Orig 86, 160–8. Ramirez, M.A. (1919) Horse asthma following blood transfusion. JAMA 73, 984–5. Riley, J.F. (1959) The Mast Cells. E.D. Livingston, Edinburgh. Riley, J.F., West, G.B. (1952) Histamine in tissue mast cells. J Physiol 117, 729–39. Riley, J.F. & West, G.B. (1952) Histamine in tissue mast cells. J Physiol (Lond) 172, 72–3. Riley, J.F., West, G.B. (1953) Mast cells and histamine in normal and pathological tissues. J Physiol 119, 44P. Rocha e Silva, M., Beraldo, W. & Rosenfeld, G. (1949) Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and trypsin. Am J Physiol 156, 261–73. Romagnani, S. (2006) Regulation of the T cell response. Clin Exp Allergy 36, 1357–66.

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Rowe, D.S. & Fahey, J.L. (1965) A new class of human immunoglobulins. I. A unique myeloma protein. J Exp Med 121, 171–99. Schild, H.O. (1936) Histamine release and anaphylactic shock in isolated lungs of guinea pigs. Q J Exp Physiol 26, 165–77. Spinelli, A. (1932) Demonstration de la mise en libert de substance type histamine du pumon isole du cobaye en choc anaphylactique. Bull Sez Ital Soc Int Microbiol 14, 257–64. Stuart, E.G. (1952) Mast cell responses to anaphylaxis. Anat Rec 112, 344. Vijayanand, P., Seumois, G., Pickard, C. et al. (2007) Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease. N Engl J Med 356, 1410–22. von Pirquet, C. (1906) Allergie. Munchen Med Wochenschr 53, 1457. von Pirquet, C. (1911) Allergy. Arch Intern Med 7, 259–88, 382–8. von Pirquet, C. & Schick, B. (1905) Die Serum Krankheit. (Serum Sickness, English translation 1951). Williams and Wilkins, Baltimore. Wagner, R. (1968) Clemens von Pirquet. His Life and Work. The Johns Hopkins Press, Baltimore. Weiss, J.W., Drazen, J.M., Coles, N. et al. (1982) Bronchoconstrictor effects of leukotriene C in humans. Science 216, 196–8. Wharton-Jones, T. (1846) The blood-corpuscle considered in the different phases of development in the animal series. Memoir I. Vertebrata. Philos Trans R Soc Lond 136, 63–87.

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2

Development of Allergy and Atopy Catherine Thornton and Patrick G. Holt

Summary The development and manifestation of IgE-mediated allergic diseases reflects a complex interaction between genetic and environmental factors operational at discrete maturational stages. Central to understanding the evolution of allergic disease in childhood is an appreciation of both immunologic activities and tissue-specific factors that have key roles in disease manifestation. The immune system and tissues affected by allergic disease, especially the airways, continue to mature after birth, so critical features of the host’s genotype and the environment that impact on postnatal maturation and function of these physiologic systems need to elucidated. In particular, factors that regulate the kinetics of postnatal maturation of innate and adaptive immune functions, which are delayed in children at high risk of allergic disease, are poorly characterized and merit more detailed investigation. The epidemiologic observation of an inverse association between family size and risk of developing allergy contains important clues relevant to this issue, and the resultant “hygiene hypothesis” has emerged as a prominent candidate to explain the negative impact of the Western lifestyle on asthma and allergic disease. The current incarnation of the hypothesis relates the innate immune response to immunoregulatory mechanisms and suggests that environmental signals regulate the maturation and pattern of immune responsiveness in early life and that deficiencies in this signaling underlie the increasing prevalence of allergic (and other) diseases. Central to this is the premise that the host response to microbial stimuli has changed as a consequence of alterations in the nature, timing, or dose of microbial signals received postnatally, or possibly even prenatally.

Introduction Numerous studies support the widely held tenet that IgEmediated allergic diseases increased in prevalence during the second half of the 20th century, most dramatically among Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

children (Butland et al. 1997; Kosunen et al. 2002; Braback et al. 2004; Burr et al. 2006). While there is now debate about whether a plateau in the prevalence of allergic disease has been reached, these observations are restricted to developed countries that have seen the most dramatic increase in allergic disease prevalence over the last 50 years. The most recent ISAAC survey (Phase III; 66 centers in 37 countries) of worldwide trends in the prevalence of allergic disease symptoms found that increases in prevalence of asthma, allergic rhinoconjunctivitis, and eczema among 6–7 and 13–14 year olds were more common than decreases, especially in the younger age group. However, among 13–14 year olds, asthma had tended to decrease in centers that previously had a high prevalence (Asher et al. 2006). Irrespective of whether a plateau/decline in disease prevalence has occurred in developed countries, allergic diseases still create an enormous social and economic burden and will continue to do so. In the UK treatments for asthma and other allergic disorders account for 10% of primary-care prescribing costs and combined with other direct National Health Service (NHS) costs, such as hospital admissions, the costs of managing allergic problems in the UK are estimated at over £1 billion per annum (Gupta et al. 2004). In the USA the direct medical costs of asthma in 2004 were $US11.5 billion, of which $US5 billion was for prescription drugs and $US3.6 billion related to hospital care (National Heart, Lung, and Blood Institute 2004). Although we are focused on the dramatic increase in allergic disease prevalence over the last 50 years, it is worth noting that other immune-mediated disorders have also increased in prevalence. Notably, there has been a marked increase in type 1 diabetes, with a greater relative increase in children aged under 5 years old compared with all other age groups (EURODIAB ACE Study Group 2000). The dramatic changes in allergic and autoimmune disease prevalence rates among children in particular has prompted much interest in potential shared factors operative in early life that increase the risk of developing these diseases. Interest in the early-life origins of allergic disease has generated a wealth of published data that can be divided broadly into three categories: the immunology of allergy (including tissue-specific aspects of immune development),

23

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environmental factors that increase/decrease the risk of developing allergic disease, and genetic variation that can predispose to the development of allergy. These are discussed below with a focus on the immunology of allergy and critical environmental factors.

Immunology of allergy Pivotal to the development of allergic disease is sensitization of the host to one or more allergens leading to the production of IgE. Therefore, the early immunologic mechanisms that predispose to the production of IgE on allergen encounter are of great interest. Key effector cells include T lymphocytes, B lymphocytes, dendritic cells, eosinophils, and various populations within affected tissues (e.g., skin keratinocytes and airway epithelial cells). Whereas these populations are relatively well studied in adults, practical and ethical considerations limit the study of these in infants and children. Nevertheless, there is a growing appreciation of the contribution of these same cell populations and the soluble mediators they produce to the initiation and maintenance of atopic disease in early life.

Dendritic cells and Toll-like receptors Dendritic cells are central to the initiation of specific immunologic reactivity to allergens and other antigens. Contiguous networks of dendritic cells are present in the epithelium of the gut, skin, and airways, where they have pivotal roles in surveillance for environmental antigens. In particular, dendritic cells modulate the development of different effector T-cell populations, namely Th1, Th2, or regulatory T cells. Dendritic cells orchestrate antigen-specific activity by T cells

ia ter

ve

iti pos

Gram negative bacteria (including endotoxin) RSV F protein

?

bac

via the provision of major histocompatibility complex (MHC)/ antigenic peptide, costimulatory molecules, and cytokines. Dendritic cell expression of costimulatory molecules, such as the B7 family, and cytokine production are tightly regulated. Expression is particularly controlled by microbial products that interact with receptors on the dendritic cell (Schnare et al. 2001; Barton & Medzhitov 2002). Recent years have seen growing interest in an evolutionarily conserved, germlineencoded family of pattern recognition receptors known as Toll-like receptors (TLRs). There are 10 known functional members of the human TLR family and they recognize a growing list of pathogen-associated molecular patterns (PAMPs) (Fig. 2.1). Interaction between PAMPs and TLRs initiates a signaling cascade on recruitment of an adapter protein (e.g., MyD88), leading to activation of members of the MAP kinase family, NF-κB, and interferon (IFN) regulatory factors among others. There is particular interest in the patterns of cytokines produced by dendritic cells on encounter with microbial stimuli as these cytokines drive the development of effector T-cell populations (Kalinski et al. 1999). Interleukin (IL)-12p70 production by dendritic cells has a pivotal role in induction of Th1 responses and IL-10 is emerging as a key mediator for induction of regulatory T cells; and while prostaglandin (PG)E2 is one of a number of mediators postulated to drive the development of Th2 effector cells, critical mediators of Th2 effector cell development are not well characterized. Different TLR ligands are reported to preferentially favor Th1- or Th2-biased adaptive immune responses. TLR2 ligands generally favor Th2 responses whereas immunostimulatory viral/bacterial DNA (immunostimulatory sequence oligodeoxynucleotides, ISS-ODNs, also called CpG) that signal via

Flagellated bacteria

m Gra gi u F n

TLR10

TLR4

TLR6

TLR5

TLR2

Plasma membrane

TLR1 Viral ssRNA Viral/bacterial DNA Viral dsRNA

TLR7 TLR8 TLR3

24

TLR9

Intracellular vesicle membrane

Fig. 2.1 Toll-like receptors (TLRs) are expressed at the cell surface or on the membranes of intracellular vesicles where they interact with various microbial ligands. Note that TLR10 has been grouped with TLR1, 2 and 6 because of its close phylogenetic relationship to these three TLRs. This diagrammatic representation does not take into account the homodimerization and heterodimerization required to initiate signaling activity. RSV, respiratory syncytial virus. (See CD-ROM for color version.)

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TLR9 inhibit Th2 effector activities and there is growing interest in the use of these in allergy vaccines (Horner 2006). Activation of TLR4 with lipopolysaccharide (LPS; endotoxin) can promote either Th1 (high doses; MyD88-dependent) or Th2 (low doses; MyD88-independent) responses (Horner 2006). Little is known about the Th1/Th2 biasing properties of TLR3, TLR5, TLR7, and TLR8 but as TLR3, TLR7 and TLR8 are activated by viral RNA, the consequences of activating these receptors on immunologic reactivity especially during early childhood is likely of importance. Consequently there is much interest in the capacity of newborns and infants, especially those at low versus high risk of developing allergic disease, to respond to TLR ligands particularly for the production of Th1-trophic cytokines such as IL-12. TLR responsiveness at birth is generally diminished in comparison with the adult, with the exception of TLR8 (Levy et al. 2006) and IFN-γ-primed TLR2- and TLR4stimulated IL-12p70 production both increase from birth to 5–12 years of age even though at 12 years of age adult-like levels have not been attained (Upham et al. 2002). Although the capacity to produce IL-12 matures postnatally, reduced IL-12p70 production in the perinatal period has been associated with stronger neonatal Th2 responses and weaker postnatal allergen-specific Th1 responses, indicating that variation in antigen-presenting cell function in early life might contribute to atopic sensitization in infancy (Prescott et al. 2003). Maternal allergy has also been associated with downregulated expression and responsiveness via TLR2 and TLR4 but the long-term consequences of this remain undetermined (Amoudruz et al. 2005). Importantly, this fetally programmed diminution in the capacity to support Th1 responses postnatally might determine the ability of the host to respond to key environmental factors reputed to alter the risk of developing allergic disease (discussed below).

T lymphocytes: Th1/Th2 and regulatory T cells Th1/Th2 cells The Th1/Th2 paradigm has been an overarching feature of the immunology of allergy for over a decade. A cardinal feature of this paradigm is that Th2 cells produce IL-4 (isotype switching of B cells for IgE synthesis), IL-5 (eosinophil growth and differentiation), IL-9 (mast cell differentiation), and IL-13 (IgE synthesis and airways hyperreactivity) that initiate and amplify the allergic inflammatory response. The ability of Th1 cells to counterregulate Th2 cells bestowed them with a putative beneficial role in allergic disease. Recent years have seen a growing appreciation that the Th1/Th2 paradigm, or at least the way it has been widely interpreted in the context of atopy pathogenesis, is an oversimplification. Key observations that have led to this reappraisal include (i) the biphasic nature of the Th1/Th2 contribution to the initiation and maintenance of eczematous lesions (Th2-skewed in the acute phase and Th1-skewed in the chronic phase); (ii) the observation that the preval-

Development of Allergy and Atopy

ence of Th1-mediated disorders has also increased over recent decades (EURODIAB ACE Study Group 2000; Stene & Nafstad 2001); and (iii) the inverse association between helminth infection (which generates a Th2-biased response involving eosinophils and IgE) and allergic disease. Despite clear evidence that delayed postnatal maturation of Th1 function is strongly associated with risk of developing the Th2-polarized memory responses that underlie allergic sensitization during childhood (Holt et al. 1992; Holt & Macaubas 1997), the observation that both Th1- and Th2mediated diseases have increased in prevalence over recent decades indicates that a general shift in the overall Th1/Th2 polarity of the immune system in the population at large is unlikely to be the primary determinant of changes in disease prevalence. Instead, imbalance between Th1 and Th2 cytokines at crucial stages during the evolution of individual allergen-specific immune responses is more likely to be a downstream component of the sensitization process, driven by whatever the primary mechanism might be. So while there is growing speculation that shared environmental exposures underlie the increases in both Th1- and Th2mediated diseases (Bach 2002), events upstream of the consolidation of T-helper memory are now considered to play key roles in these changing disease patterns. Until recently there was much enthusiasm for the possibility that allergen-specific T-cell memory was initiated during intrauterine development. This was supported by observations made by many groups of proliferative and cytokine responses by umbilical cord blood mononuclear cells to food and inhalant allergens. However, an increasing number of studies have failed to find an association between proliferative response to allergen at birth and maternal (therefore fetal) exposure to the same allergen during pregnancy or various measures of allergic outcome in infancy (Smillie et al. 2001; Marks et al. 2002). These observations raised questions about the significance of cord blood responses to allergens and disease outcomes. More recently, the biological mechanism underlying the ability of cord blood mononuclear cells to respond to allergens has been elucidated. The bulk of this responsiveness has been shown to represent a non-specific response by functionally immature recent thymic emigrants rather than conventional memory T-helper cells (Thornton et al. 2004). This non-specific responsiveness is presumably initiated at around 20–21 weeks of gestation and seems to persist for at least the first 3 months of life, with specific memory responses emerging after 6 months of age (Jones et al. 1996; Szepfalusi et al. 2000; Bottcher et al. 2006). It is the emergence of Th2-biased allergen-specific responses from 6 months of age, rather than reactivity in cord blood, that correlates with sensitization (Rowe et al. 2007). Clarification that the response to allergen at birth is not allergen-specific and therefore not related to maternal allergen exposure during pregnancy questions the scientific basis for existing recommendations for allergen avoidance by high-risk women

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during pregnancy (e.g., peanuts) (Committee on Toxicity of Chemicals in Food 1998). Recognition that the response to allergens measurable at birth is not indicative of the development of T-cell memory does not discount the various studies linking proliferative and cytokine responses at birth with family history of allergic disease or disease outcomes in childhood. Rather than informing us about the development of allergen-specific memory in the perinatal period, they highlight the fact that variation in immune function at birth is linked to subsequent immunologic responsiveness in infancy, which includes the development of allergen-specific IgE and disease outcomes. Numerous studies now support the postulate that the capacity to produce a variety of both Th1 (typically IFN-γ), Th2 (typically IL-13), and regulatory (IL-10) cytokines is diminished at birth in children who are at genetic risk of developing allergic disease or who develop disease during childhood (Holt et al. 1992; Tang et al. 1994; Prescott et al. 1998; Williams et al. 2000). Subsequently, these same children consolidate Th2 responsiveness [phytohaemagglutinin (PHA)-stimulated IL-4, IL-5, IL-13] while failing to adequately upregulate Th1 responsiveness (PHA-stimulated IFN-γ) at 6 months and 1 year of age. This Th2-biased responsiveness during the first year of life is associated with atopic outcomes including sensitization to egg, eosinophilia, and increased IgE by 1 or 2 years of age (Prescott et al. 1999; van der Velden et al. 2001; Neaville et al. 2003). Altered cytokine production patterns at birth might predict susceptibility to particular immunologic sequelae during the first 1–2 years of life. For example, reduced IFN-γ production in early infancy has been associated with increased frequency of viral respiratory infections (Copenhaver et al. 2004) and early-onset wheezing in the first year of life (Guerra et al. 2004a), and reduced IL-13 at birth has likewise been associated with early respiratory syncytial virus (RSV)-induced wheezing (Gern et al. 2006). However, the link between viral infection in early life and the development of asthma remains controversial (as discussed below). Recently, current wheeze/ asthma at 5 years of age has been associated with wheezy and/or febrile lower respiratory tract infection in the subgroup of high-risk children sensitized prior to 2 years of age (Kusel et al. 2007). Thus, accumulating evidence links susceptibility to viral infection and atopy to a common set of transient developmental defects in cellular immune function in early infancy. Lower IFN-γ and IL-13 at birth might therefore be indicators of an overall functionally immature immune system with diminished antiviral capability. Failure to efficiently upregulate IFN-γ might also be the critical step in the development of atopic sensitization in infancy, as a reduced capacity to produce IL-12 at birth has been associated with reduced number of IFN-γ-producing cells at this time, weaker allergen-specific Th1 responses, and the development of skin-prick reactivity and atopic dermatitis during the first 2 years of life (Prescott et al. 2003; Nilsson et al. 2004).

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Of interest now is identification of the genetic and environmental factors that program this pattern of responsiveness. Maternal allergic disease has been associated with reduced IFN-γ and IL-12 at birth but the mechanisms explaining this remain unknown (Prescott et al. 2000; Gabrielsson et al. 2001). The Th2-inhibitory effects of IFN-γ are generally restricted to the early phase of polarization and the capacity to annul commitment of Th1 or Th2 populations is lost progressively with long-term exposure to polarizing stimulation (Murphy et al. 1996). A comparable situation may occur in humans: attenuated IFN-γ production is a hallmark of the immune response at birth in children at increased risk of atopic sensitization whereas (older) children who are sensitized have mixed Th1/Th2 responses (variation in which can be related to different patterns of disease) and even hyperproduction of IFN-γ (Macaubas et al. 1999; Smart & Kemp 2002; Heaton et al. 2005). Thus while there has been a focus on the potential of Th1-biased responsiveness to regulate disease this may only be feasible at the disease induction phase.

Regulatory T cells Recent years have seen a dramatic resurgence in the investigation of T-cell populations that regulate/suppress immunologic reactivity. Initially identified by their ability to suppress autoimmune disease, regulatory T cells have since been shown to modify a broad range of immunologic activities associated with inflammatory and infectious diseases, cancer, and transplantation (Read & Powrie 2001). Moreover, abnormalities in the activity of these cells have been implicated in susceptibility to many conditions with underlying immune etiology, including allergy. Several types of regulatory T cells have been described and these can be broadly subdivided into natural and inducible regulatory T cells. Natural CD4+ regulatory T cells can be identified by the expression of CD25 and low expression of CD127 (IL-7 receptor α chain) (Seddiki et al. 2006). CD4+/CD25+/CD127lo regulatory T cells express FoxP3, which has been described as the master regulator of development and function of this population (Fontenot & Rudensky 2005), and their functional properties have been variously attributed to cytotoxic T-lymphocyte antigen (CTLA)-4, transforming growth factor (TGF)-β, and IL-10 but this remains an area of ongoing investigation and controversy. There are an increasing number of studies investigating the ontogeny of this population from fetal life onwards. A notable contrast with mice, in which many studies of regulatory T-cell populations have been performed, is that T cells with regulatory properties are present in the periphery from very early in human fetal development, whereas in mice these cells do not leave the thymus until after birth. A major difference between human adult and neonatal natural regulatory T cells is the predominance of CD45RA+/CD45RO– “naive” cells among the neonatal population. While activity typical of natural regulatory T cells has been described in umbilical

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cord blood, additional maturation/education of these cells is likely required in the periphery to attain adult comparable functionality (Wing et al. 2002, 2003, 2005; Seddiki et al. 2006; Rowe et al. 2007). The fetal lymph nodes seem to be the site of peripheral education as CD4+CD25+ regulatory T cells within human fetal lymph nodes have acquired a primed/memory phenotype and are highly suppressive of CD4+CD25– T cells (Cupedo et al. 2005). The relative ease of identification of natural regulatory T cells in peripheral and umbilical cord blood has accelerated investigation of the role of these cells in health and disease. There is a burgeoning literature on the contribution of these cells to allergic disease, especially as the phenotype associated with mutations in FoxP3 includes eczema, eosinophilia, elevated serum IgE, and enhanced Th2 responses (Chatila et al. 2000). Notably there has been a shift in the paradigm relating Th2-skewed adaptive immune responses and allergic disease (Fig. 2.2). While there are increasing numbers of publications identifying perturbations in the activity of CD4+CD25+ T cells in allergic adults, a direct causal relationship between these cells and allergic inflammation in humans has not been demonstrated. There have been only a few studies attempting to relate the function of natural regulatory T cells in early life with allergic outcomes in childhood and beyond. In a study examining regulatory T-cell activity both before and after an in vivo milk challenge in children (aged 6–56 months) who had either outgrown or retained their non-IgE mediated cows’ milk allergy, differences in the ability of peripheral blood mononuclear cells to respond to β-lactoglobulin could be explained by enhanced suppressive activity of CD4+CD25+

OLD MODEL Th1

NEW MODEL Th2

Th1

Th2

Treg

Dendritic cell

Dendritic cell

Microbial stimulation

Microbial stimulation

Fig. 2.2 The “old” Th1/Th2 paradigm model to explain the role of the microbial environment in the development of allergen-specific Th2 memory and the “new” model which incorporates the postulated role of regulatory T cells. In the “old” model, microbial exposure of dendritic cells induces a cytokine profile that favors Th1- or Th2-biased activity and further crossregulation by Th1 and Th2 cells prohibits either Th1- or Th2-biased activity depending on the nature of the initial microbial exposure. The “new” model incorporates regulatory T-cell populations and the microbial exposure prevalent at the time of exposure to allergen determines the balance between these effector T-cell populations. (See CD-ROM for color version.)


T cells in the tolerant children (Karlsson et al. 2004). Also, a greater proportion of CD4+CD25+ T cells in the tolerant children expressed CD45RO. Of even greater interest is the possibility that perturbations in regulatory T-cell activity could be identified and targeted prior to disease onset, i.e., at birth. Elevated IL-10 (protein) and FoxP3 (mRNA) expression after stimulation with a TLR2 ligand has been described in cord blood mononuclear cells of neonates born to nonatopic mothers versus those born to atopic mothers (Schaub et al. 2006). Similarly, it has been suggested that the ability to generate regulatory T cells in response to antigen and LPS is impaired in newborns at risk of developing allergic disease (Haddeland et al. 2005). It is tempting to speculate, therefore, that children born to atopic mothers, who are at increased risk of developing allergy, might have an impaired ability to generate regulatory T-cell activity in early life. However, in the absence of definitive phenotypic and functional data about the cells of interest in each of these studies, this is not possible. However, these studies do highlight the extensive interest in this area and the need for detailed studies relating the phenotype and function of regulatory T-cell subsets to family history and, more importantly, clinical manifestation of allergic disease. Additional regulatory T-cell populations are also of interest but are comparably underinvestigated, and these include CD8+CD25+ regulatory T cells, inducible regulatory T cells, and natural killer (NK)T cells (Akbari et al. 2003). CD8+CD25+ human thymocytes with similar phenotypic and functional characteristics to the CD4+CD25+ population have been described (Cosmi et al. 2003) but there is as yet no information about these in relation to allergy at any age. Similarly, both CD4+ and CD8+ inducible regulatory T-cell subsets have been described and while there is great interest in the therapeutic use of these there are no data concerning the activity of these cells in childhood (Horwitz et al. 2003). Of these additional regulatory T-cell subsets, NKT cells have received the most study in relation to allergy although there are few studies of these in childhood. NKT cells are a T-cell population that have a very narrow T-cell repertoire and express cell surface markers characteristic of NK cells. Unlike conventional T cells, they recognize glycolipid antigen in the context of the monomorphic antigen-presenting molecule CD1d. Interest in their regulatory activity is driven by the capacity of NKT cells to rapidly produce large amounts of Th1 and Th2 cytokines and the postulate that they support Th2 responses in allergy (Akbari et al. 2003; van der Vliet et al. 2004). While adult NKT cells produce both IL-4 and IFN-γ, neonatal NKT cells preferentially produce IL-4 and can produce more IL-4 than adult NKT cells in contrast to conventional neonatal T cells that are comparably poor producers of IL-4 (Kadowaki et al. 2001). Thus a role for them in the initial polarization of antigen-specific Th2 cells is feasible. As discussed below, consideration of the impact of microbial exposures on allergic outcomes relates these to the interplay

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between innate and immunoregulatory mechanisms. The role of TLRs and other relevant receptors on dendritic cells has already been discussed, although regulatory T-cell populations also express TLRs and are therefore responsive to microbial stimuli. CD25-expressing regulatory T cells express TLR2, TLR4, TLR5, and TLR8 and activation via these receptors has variable effects on survival, proliferation, and the suppressive activity of these cells (Sutmuller et al. 2006). There are no data concerning the expression and activity of TLRs on neonatal and pediatric regulatory T-cell populations but this would clearly be of great interest and might potentially have therapeutic implications. The next few years should see some exciting developments in our understanding of regulatory T-cell activity in early childhood and its impact on IgE sensitization and allergic disease outcomes. Other cell types with key roles in the development and manifestation of allergic disease include B cells, as the source of IgE and IgG, and eosinophils.

B lymphocytes, IgE and IgG Potential relationships between IgE and/or IgG and the development of allergic disease have been explored by numerous research groups. As the source of these immunoglobulins, B cells are therefore of interest. B cells can be detected early in fetal life and appear sequentially in the yolk sac (3 weeks), the paraaortic splanchnopleure (5 weeks), the liver (8 weeks), and the bone marrow (12 weeks), with the bone marrow the major site of B-cell generation from mid gestation onwards (Nunez et al. 1996; Holt & Jones 2000). Mature B cells can also be detected in the fetal intestine from around 14 weeks of gestation (Spencer et al. 1986; Golby et al. 2002). Circulating B cells at birth have an immature phenotype, with the majority of cells expressing IgM and CD1c, but postnatal maturation of circulating and tissue B cells and how this might relate to allergic disease outcomes is not extensively studied.

IgE There is much debate about the usefulness of elevated umbilical cord blood IgE as a predictor of the development of atopic disease in childhood and it is now generally well accepted that while it is a strong risk factor for atopic sensitization, it lacks the predictive sensitivity to be useful for identification of those newborns who should be targeted for disease preventive measures (Magnusson 1988; Edenharter et al. 1998). The source of IgE in the fetus/neonate remains unclear, although in the absence of placental transfer of IgE it is presumed that circulating levels in the neonate represent endogenous production by the fetus/newborn (Schreyer et al. 1989; Avrech et al. 1994; Saji et al. 1999). IgE synthesis can be induced from fetal tissues as early as 11 weeks of gestation (Miller et al. 1973) and circulating IgE can be detected as early as 25–27 weeks of gestation (Thornton et al. 2003). VDJCε transcripts can be detected in fetal liver from the second trimester and cord blood from the third trimester but

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become more common in the infant’s blood after 9 months of age (Lima et al. 2000). Sterile Iε transcripts can be detected as early as 8 weeks of gestation, indicating that many fetuses are primed from isotype switching to IgE production by the end of the first trimester of pregnancy. Notably, although numbers were insufficient to achieve statistical significance, the ability to detect early transcription of Iε and VDJCε (and IL-4Rα) was absolutely associated with the presence of a polymorphism in IL-4Rα known to be associated with a high risk of atopy (Lima et al. 2000). Discrepancies between levels of IgE in the circulation and in local tissues are described frequently. During helminth infection IgE is abundant in the gut and circulating levels are only 0.5–1% of total IgE (Negrao-Correa et al. 1996) so local production is not mirrored by circulating levels. Thus IgE levels in the gut might be more useful in very early life but there are few studies on this, although fecal IgE levels at 1 month of age are more often detectable in children with a family history of atopy and are associated with atopic outcome at 18 months of age (Sasai et al. 1994).

IgG Findings from animal models support a key role for maternally derived IgG (either prenatally via the placenta or postnatally via breast milk) in modulation of the infant’s immunologic development. In a mouse model, immunization of the mother against house-dust mite either during pregnancy (Melkild et al. 2002) or prior to conception (Victor et al. 2003) protected the offspring against the development of allergy. Although the mechanism by which this occurs is unknown, maternal IgG has been shown to suppress IgE responses by the offspring (Jarrett & Hall 1983). High IgG anti-IgE levels at birth have been suggested to protect children at increased risk of allergy from the development of disease and/or reduce the severity of symptoms (Vassella et al. 1994). Therefore there has been much interest in how patterns of allergen-specific IgG might relate to the development of allergic disease. IgG in the newborn’s circulation is almost entirely of maternal origin. While IgG transfer from mother to fetus via the placenta begins at around 16 weeks of gestation, much of this IgG is acquired during the last 4 weeks of pregnancy. Maturation of IgG production by the newborn occurs slowly and adult-like levels are not reached until around 3 years of age. Expression of IgG receptors (Fcγ receptor isoforms and the neonatal Fc receptor [FcRn]) by cells of the placenta have a central role in IgG transfer during fetal development (Saji et al. 1999; Thornton & Vance 2002). Expression of FcRn within the gastrointestinal tract also facilities interaction with IgG in breast milk (and amniotic fluid) and this receptor has been implicated in mucosal immune responses to gastrointestinal bacteria via its role in facilitating secretion of IgG into the gut lumen and the delivery of antigen to mucosal dendritic cells (Yoshida et al. 2004, 2006).

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Sensitization to hen’s egg is a recognized risk factor for the later development of inhalant sensitization and asthma (Hattevig et al. 1987, 1993; Nickel et al. 1997) and egg-specific IgG has also been identified as a marker of an underlying allergic process: high levels of egg-specific total IgG, IgG1, and IgG4 are associated with allergic sensitization and disease (Okahata et al. 1990; Eysink et al. 1999). Furthermore, a dichotomous pattern of egg-specific total IgG and subclasses has been observed at 6 months of age in subsequently atopic and nonatopic children, implying that specific IgG responses might predate IgE responses and the clinical manifestation of disease (Jenmalm & Bjorksten 1999). Elevated ovalbumin (OVA)-specific IgG1 at 1 year of age can predict the subsequent development of asthma, with a predictive performance that outranks that of total IgE measurements either at birth or in infancy (Vance et al. 2004). While this might simply mark those children with atopic eczema, which is a wellknown risk factor for the later development of asthma (Kjellman & Hattevig 1994; Bergmann et al. 1998), the measurement of OVA-specific IgG and subclasses in infancy might be a useful serologic adjunct to clinical evaluation and skin-prick testing for the early identification of those children at particular risk of developing later allergic sequelae.


(Halmerbauer et al. 2000; Gore et al. 2003). Cord blood eosinophilia has been associated with the development of eczema at 1 month of age, the earlier development of atopic dermatitis, and the development of wheezing disease (Matsumoto et al. 2005). Nasal lavage fluid from children at high risk of developing allergy had elevated levels of ECP and EPX at 4 weeks of age, suggesting activation of eosinophils at the respiratory epithelium (Halmerbauer et al. 2000), although the transient or persistent nature of this nasal eosinophilia in very early life remains to be determined. However, peripheral blood but not nasal eosinophilia at 3 months and persisting until 18 months of age was observed in children who developed active atopic disease by 18 months of age, and if coupled with elevated IL-4 was associated with disease persisting until 6 years of age (Borres et al. 1995).

Cytokines and chemokines Cytokines As noted above there is particular interest in Th1/Th2 cytokines (principally the prototypic Th1 cytokine IFN-γ and the Th2 cytokines IL-4, IL-5, IL-9, and IL-13) and, more recently, regulatory cytokines (namely IL-10 and TGF-β). The contribution of Th1, Th2, and regulatory cytokines to allergic disease development has already been discussed.

Eosinophils Blood and tissue eosinophilia are hallmarks of allergy and asthma in adults and children. As umbilical cord blood contains more mature eosinophils than adult blood, neonates have a propensity to high eosinophil counts. Coupled with recognized differences in eosinophil trafficking in neonatal life, this has been postulated to have a physiologic role during colonization of the skin and mucosal surfaces during early extrauterine life (Patel et al. 1994). Additional or alternative roles for eosinophils during early infancy are supported by increased adhesion molecule expression and transmigration activity in neonatal versus adult eosinophils (Moshfegh et al. 2005). Eosinophils are reported to initiate and amplify antigenspecific Th2 cell responses (MacKenzie et al. 2001) so eosinophils present at mucosal surfaces in early life might contribute to the initiation of Th2-biased reactivity to allergens to which the infant is being exposed; in particular, eosinophils within Peyer’s patches cooperate with lymphocytes in the development of mucosal immune responses (Mishra et al. 2000). Although there are no studies of eosinophil phenotype and function in infancy, numerous investigators have sought to determine whether blood eosinophilia at birth or in infancy might be a predictive marker of atopy development. As eosinophilia is supported, among other mediators, by the Th2 cytokine IL-5, postnatal variation in maturation of the Th1 and/or Th2 populations might manifest as increased circulating and/or tissue eosinophils (Neaville et al. 2003). Many studies in children use surrogate markers of eosinophilia such as elevated eosinophil cationic protein (ECP) and eosinophil protein X (EPX) in serum, lavage fluids, or urine

Chemokines Chemokines are small secreted molecules that regulate the trafficking of leukocytes throughout the body by triggering integrin activation, firm arrest to the vascular endothelium, and then localization within tissues. Chemokines therefore have a critical role in the initiation and amplification of inflammation within the gut, skin, and airways. The role of chemokines in the development of allergic disease is best studied in the skin. Keratinocytes (and other cells in the skin) produce a unique profile of chemokines (e.g., TARC/CCL17 and CTACK/ CCL27) recognized to have a role in the development of eczema. These chemokines preferentially attract subsets of cutaneous lymphocyte antigen (CLA)-positive memory T cells via interaction with the chemokine receptors CCR4 and CCR10 (Morales et al. 1999). Serum thymus and activationregulated chemokine (TARC), macrophage-derived chemokine (MDC), cutaneous T-cell attracting chemokine (CTACK) levels are significantly higher in children (aged 4.9 ± 3.3 years, range 2 months to 14 years) with atopic dermatitis and correlate with disease severity in children with either atopic or nonatopic eczema. Levels of TARC and CTACK decline with age, indicating that they are at their highest during the period of greatest likelihood of developing eczema (Song et al. 2006). Understanding the regulation of chemokine/chemokine receptor expression during early life is likely to be crucial in relation to elucidation of mechanisms of allergic disease development in childhood. Chemokine receptor expression by T cells differs dramatically between adults and neonates (Sato et al. 2001) and T-cell trafficking is more promiscuous

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during fetal life as naive T cells can traffic through peripheral tissues and even recirculate through the thymus during this period (Cahill et al. 1999). Presumably this has a critical role in the development of peripheral tolerance but how long these differences persist postnatally remains to be determined. Expression of CCR4 and CCR10 has not been well studied in childhood but the percentage of T cells bearing CLA increases with age from birth, where expression is virtually absent, to adulthood when over 10% of T cells express CLA (Campbell & Kemp 1999). This is likely to reflect the acquisition of memory T cells from birth to adulthood. The role of CLA in childhood atopic dermatitis is unclear, although an increased percentage of T-cell receptor Vβ segments related to Staphylococcus aureus superantigens has been described in CLA-positive cells from children with atopic dermatitis (Torres et al. 1998), and other surface markers might have a more important role.

Tissue-specific aspects of immune development Understanding the maturation of hematopoietic and other cells in the tissues affected by allergic disease is critical to elucidating the underlying mechanisms of disease development.

Skin Atopic eczema is a common, chronic, relapsing, itchy, inflammatory skin condition associated with epidermal barrier dysfunction and cutaneous hyperreactivity to environmental triggers. It is characterized by typically distributed eczematous skin lesions with lichenification, pruritic excoriations, severely dry skin, and susceptibility to cutaneous infection. The clinical presentation of eczema represents complex interactions between susceptibility genes, the environment, defective skin barrier function, and immunologic responses. Around 50% of all atopic eczema develops in the first year of life and 80% develops by 5 years of age. There is a link between food allergy and atopic dermatitis. Food allergens can induce eczematoid skin lesions and nearly 40% of children with moderate to severe atopic eczema have food allergy (skin-prick test positive or circulating IgE to various foods, particularly eggs, milk, wheat, soy, and peanuts) (Eigenmann et al. 1998; Laan et al. 2000). Moreover, food allergen-specific T cells can be cloned from skin lesions of patients with atopic eczema and in mouse models of atopic eczema, oral sensitization with foods (cows’ milk and peanuts) results in the elicitation of eczematous skin lesions on repeat oral food challenges (Li et al. 2001). Children with atopic eczema are more likely than those with nonatopic eczema to have their eczema persist into adulthood and are more likely to develop other atopic disorders, primarily asthma. Indeed, atopic eczema is often the first manifestation of atopic disease in infancy and is generally considered to be the initial cutaneous mani-

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festation of a systemic disorder that leads to other atopic diseases including food allergy, asthma and allergic rhinitis, the so-called “atopic march” (see Chapter 57). Evidence of Th2-biased activity can be found among peripheral blood T cells, and subjects with atopic eczema can also have blood eosinophilia and elevated circulating total IgE. Perturbation of barrier function of the skin is an overriding feature of eczema, whether it is atopic or nonatopic. Skin barrier function is conferred by the stratum corneum, the outermost layer of the epidermis. During fetal development the skin progresses from a single-layered ectoderm to a multilayered keratinized epidermis. Keratinization commences at around 22–25 weeks of gestation (time of initiation varies with anatomic site) and the transitory layer that serves as an interface between the epidermis and amniotic fluid (periderm) is sloughed off into the amniotic fluid at this time (Yosipovitch et al. 2000). Barrier formation thus coincides with the current lower limit of viability of the preterm infant and is completed many weeks before normal term birth. However, the stratum corneum barrier must still adapt to extrauterine life. Epidermal Langerhans cells (the contiguous network of dendritic cells peculiar to the skin) are phenotypically mature in the second trimester, although numbers increase (e.g., density of Langerhans cells up to 23 weeks’ gestation is 10–20% of adult levels) and dendritic processes develop gradually with age (Foster & Holbrook 1989; Fujita et al. 1991). Peptide antibiotics present in the vernix caseosa (the cream-like substance present on the infant’s skin at birth) and skin of the healthy newborn have a role in innate immune protection during fetal and neonatal life (Marchini et al. 2002).

Gastrointestinal tract From birth onwards the gastrointestinal tract is continuously exposed to food antigens and microbial products from the commensal microflora and invading pathogens. In normal healthy humans the gut immune system is in a state of continual high activity, centered on the organized lymphoid tissues in the small intestine (Peyer’s patches), although relevant cell types and even isolated lymphoid follicles are dispersed throughout the lamina propria (MacDonald 2003). Peyer’s patches are the principal site for induction of antigen-specific tolerance to food and commensal flora antigens, including the preferential development of IL-10-secreting T cells with suppressive activity (regulatory T cells) (Fujihashi et al. 2001; Jump & Levine 2002). CD103 expression by dendritic cells within gut-associated lymphoid tissues has a central role in the maintenance of the healthy balance between effector and regulatory T-cell activity within the gut (Annacker et al. 2005). The intestine contains the largest pool of T cells in the body and is a unique immunologic compartment in which hematopoietic (and other) cells come into close contact with the intestinal microflora and food antigens. The neonate is born with a sterile gastrointestinal tract but acquisition of the commensal gut flora, exposure to potential pathogens, and

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introduction of food antigens commences at birth. The gut in particular serves as an interface between the developing immune system and microbial antigens, and the intestinal microbiota has a role in immune maturation (discussed below). The epithelial layer, together with intraepithelial and lamina propria immunocompetent cells, coordinate local innate and adaptive immune responses to the microbial challenge. The gastrointestinal commensal flora shapes both mucosal and systemic immune function throughout life and is implicated in the induction of protective IgA and oral tolerance, including induction of regulatory T cells, and immune deviation. Consequently, the host is highly adapted to its own commensal flora (Macpherson & Harris 2004). Functional heterogeneity and organization of cells within the gastrointestinal tract occurs prior to birth and in the absence of dietary and bacterial antigens (Spencer et al. 1987; Howie et al. 1998). MHC class II-positive cells can be detected in the human fetal gut as early as 11 weeks of gestation: initially these cells are dispersed throughout the lamina propria but aggregates can be found in the forming lymphoid follicles from around 16–19 weeks of gestation. T cells can be detected as early as 12–14 weeks of gestation (Spencer et al. 1986) and the numbers of these cells increase rapidly so that by 19–22 weeks the density is approximately one-third of that in the adult intestine (Howie et al. 1998). B cells can be detected as early as 14 weeks of gestation (Spencer et al. 1986). Once lymphoid follicles and aggregates emerge, clusters of T cells and B cells as well as dendritic cells can be observed within these clusters and costimulatory molecules are expressed by these populations (Spencer et al. 1986; Jones et al. 2001). The gut immune system maintains a balance between remaining relatively unresponsive to dietary and commensal flora antigens yet making a response to pathogens. There is increasing evidence that perturbing the homeostasis between gut antigens and host immunity is a critical determinant in the development of gut inflammation and allergy (MacDonald & Monteleone 2005).

Airways Alterations to lung development during intrauterine and early extrauterine development mediated by genetic or environmental factors might have long-term consequences for airflow limitation and airways disease. The structure and proportions of the infant’s airways and lungs differ to that of the adult and combined with relatively greater chest wall compliance in childhood might accentuate functional differences (Jeffery 1998). The pattern of airway branching is completed by 16 weeks of gestation and the airways continue to increase in size as lung volume increases (Hislop et al. 1972). The diameter of the airways increases linearly from 22 weeks until term and continues to increase after birth, doubling or tripling in diameter and length between birth and adulthood (Hislop & Haworth 1989). During fetal development the fetal airways are relatively


devoid of hematopoietic cells (Alenghat & Esterly 1984; Grigg et al. 1993) and seeding with resident populations of these cells must occur after birth. Because little change in the leukocyte profile in the lower airways of children is evident after 3 years of age, the first 3 years of life must therefore be the critical period. Notably there is rapid acquisition of an abundant resident population of alveolar macrophages in the first 2 years of life, with a gradual decline to 3 years of age (Grigg et al. 1999). Conversely, lymphocytes are relatively absent from bronchoalveolar lavage fluid collected prior to 2 years of age and increase after this time (Riedler et al. 1995). Thus developmental changes in the lower airway cell profile are relatively restricted to the first few years after birth. The factors that drive the expansion and maturation of these cell populations are unknown but are likely to include microbial stimuli (particularly viruses) and genetic factors are also likely to be involved. There is now much interest in the contribution of the bronchial epithelium to the local immunomodulatory milieu but this has not yet been studied in infancy. Studies of airways dendritic cell distribution and function in early life are generally conducted using animal models. Between birth and weaning dendritic cells are present in very small numbers in the airway wall and express only low levels of surface MHC class II Active immune suppression within the lung microenvironment might be a feature of early life as neonatal airways dendritic cells are hyporesponsive to inflammatory stimuli and activation signals (Nelson et al. 1994; Nelson & Holt 1995). Similarly mature dendritic cells are rare in the airway mucosa of humans prior to the first birthday (Stoltenberg et al. 1993; Tschernig et al. 2001) unless the infant has had a respiratory tract infection (Tschernig et al. 2001). Further evidence that viruses have a critical role in development of this network is the observation of increased dendritic cell numbers in nasal lavage fluid from children aged less than 15 months who have a viral, including RSV, infection. A decrease in these same populations in the blood occurred simultaneously, indicating mobilization of these cells to the mucosa (Gill et al. 2005). Thus, unlike the gut and skin, the respiratory tract dendritic cell network develops almost entirely postnatally.

Environment Studies revealing dramatic differences in allergic disease prevalence in genetically similar populations living in discrete environments (e.g., former East and West Germany, migrant and nonmigrant populations of the same ethnicity; von Mutius et al. 1994; von Hertzen & Haahtela 2004) indicate a central role for environmental factors, especially those related to a Western lifestyle, in the development of allergy and allergic disease. The environmental factors postulated to have a role in modifying the development of allergic disease can be divided broadly into two categories: those that relate to the hygiene hypothesis and those that do not (Table 2.1).

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Table 2.1 Environmental risk factors associated with the development of allergy and allergic disease in childhood. Hygiene hypothesis related Family size, birth order Infectious diseases Vaccinations Antibiotic use Farming Endotoxin Pet ownership Other Breast-feeding Parental smoking Diet and nutrition Obesity Perinatal factors

Hygiene hypothesis Since the inverse association between family size and birth order and the risk of developing allergy was highlighted (Strachan 1989), the hygiene hypothesis has emerged as a prominent candidate to explain this relationship. The current incarnation of the hypothesis relates the innate immune response to immunoregulatory mechanisms and suggests that microbial signals regulate the maturation and pattern of immune responsiveness in early life and that deficiencies in this signaling underlie the increasing prevalence of allergic (and other) diseases (Romagnani 2004; von Hertzen & Haahtela 2004). Central to this is the premise that the host response to microbial stimuli has changed as a consequence of alterations in the nature, timing or dose of microbial signals received postnatally, or even prenatally. A multitude of relevant microbial signals and environmental sources have now been investigated and a variety of epidemiologic, and to a lesser extent experimental, evidence has been provided under the umbrella term “hygiene hypothesis.”

Sibling effect, family size, and birth order Interest in the so-called “sibling effect” was initiated by the observation that the prevalence of hay fever was lower in children from larger families and while both the number of older and younger siblings exerted an effect, this was stronger for the older siblings (Strachan 1989). These observations lead to the (hygiene) hypothesis that the presence of older siblings transmits infections in early life, providing protection against the development of disease. The immunologic mechanism underlying this protective effect was later postulated to be the downregulation/prevention of Th2-skewed reactivity to allergens as a consequence of the ability of infectious organisms to favor Th1 reactivity.

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Numerous investigators have since explored the relationship between family size, birth order, and allergic outcomes. A review of the relevant literature (1965–2000) highlighted that while nearly all studies report an inverse relationship between the number of siblings and eczema, hay fever and atopic sensitization, the relationship is less robust for asthma, with only around 70% of studies reporting an inverse association for asthma/wheezing (Karmaus & Botezan 2002). The contradictory results surrounding the birth-order effect in asthma are likely to reflect the age at which outcome measures are made. For example, an older sibling in the home is associated with more frequent wheezing before the age of 2 years but children of higher birth order are less likely to have frequent wheezing after this age (Ball et al. 2000; McKeever et al. 2001). A major ambiguity in the birth order and allergic outcomes literature lies in the nature of the sibling effect, as authors variably relate their outcome measures to birth order, number of siblings, family size, number of older siblings, number of younger siblings, number of brothers, and even just having an older brother (Karmaus & Botezan 2002). The causal factors underlying the sibling effect remain unknown but the hygiene hypothesis prevails. However, family size is likely to be an indirect measure of some as yet undetermined biologically relevant factor(s) that increases with family size and in other (non-Western) environments with low prevalence of allergic disease (Strachan 2000). Two alternative, but not mutually exclusive, formulations of the birth order/family size effect on allergy and allergic disease have also been considered but to a much lesser extent: (i) the maternal microbial burden changes with each pregnancy that results in a live birth (each successive child increasing this burden) (Hersoug 2006); and (ii) hormonal and other changes in each pregnancy differentially affect the mother’s own allergic disease and the immunologic development of each successive fetus/child (Doull 2001; Rangaraj & Doull 2003). Each of these is postulated to impact on the developing fetal immune system with downstream consequences for the development of allergic disease. In contrast to the prevailing interpretation of the birth order effect (i.e., hygiene hypothesis), there are relatively few studies exploring the impact of changes in maternal immune function in successive pregnancies on the health and atopic status of either the mother or child. The hypothesis that microbial exposures via siblings might account for the protective effect on atopic outcomes as discussed above has now evolved to take into consideration environmental sources that provide or modify patterns of microbial exposure in infancy, such as farming, daycare, vaccinations, and antibiotics. Investigators have also tried to pinpoint the critical exposures that might provide protection against the development of allergic disease and these include infectious diseases of childhood, endotoxin, and the gastrointestinal flora.

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Early life infections While there have been a number of studies addressing the possibility that the dramatic reduction during the 20th century in the incidence of childhood diseases such as measles, mumps, rubella, and chickenpox underlies the increasing prevalence of atopic disease, there are insufficient data to support a critical role for these life-threatening childhood diseases in postnatal maturation of immune, gastrointestinal, skin and/or airways function. Moreover, the microbial burden in general rather than single infections has been suggested to be of greater importance for the regulation of immune responses in early life (Martinez 2001). This has shifted interest to the common respiratory and gastrointestinal bacterial and viral infections that form much of the infectious burden of children growing up in westernized countries. Bacterial, fungal, gastrointestinal, or urinary tract infections in the first year of life were not associated with wheeze between 4 and 7 years of age but repeated viral, other than lower respiratory tract, infection in early life might reduce the risk of developing asthma/wheeze up to 7 years of age and atopy up to at least 5 years of age (Illi et al. 2001). In contrast repeated lower respiratory tract infections (LRTI) with wheezing in the first 3 years of life have a positive association with wheeze up to 7 years of age but this is likely to reflect reverse causation (children predisposed to asthma might be more susceptible to LRTI) (Illi et al. 2001). The observed effects were strongest for infections in the first year of life, with the addition of infections from the second and third year of life having little impact on the results. Thus the nature and timing of infection as well as the host response to infection are critical. Consequently, discussions of the effects of infections on the pathogenesis of allergic disease have become increasingly complex. While a variety of epidemiologic evidence now supports a protective role for infection, viruses trigger around 85% of asthma exacerbations (Openshaw et al. 2004). However, it is the role of viruses in the onset of disease that is of greatest interest and there is a growing appreciation of the complex relationship between viral respiratory tract infections and the development of asthma in particular. RSV infection in infancy has been associated with the later onset of asthma by numerous groups (Openshaw et al. 2004). However, it remains unknown if those infants who suffer RSV-induced bronchiolitis at around 2–6 months of age are the subgroup at risk of developing asthma or if the virus modifies the local environment, both physiologically and immunologically, during this critical period of development. Susceptibility to RSV-associated wheeze is heavily dependent on age of infection and thereby developmental status of the immune system (Culley et al. 2002). Similarly, RSV or influenza/parainfluenza virus favors Th2 responses in the nose (elevated eosinophils and IL-4 in nasal lavage fluids) if infection occurs before 3 months of age (Kristjansson et al. 2005). Thus the nature of the host response, which is likely


to be developmentally and genetically programmed, is emerging as a key contributor to the impact of microbial exposure on allergic disease development. TLR4 and CD14 have been identified as receptors for RSV (Kurt-Jones et al. 2000) and variation in the expression of these receptors could impact on the response to RSV. Moreover, RSV upregulates TLR4 expression by airways epithelial cells and makes the cells sensitive to endotoxin (Monick et al. 2003). Children who attended daycare, especially daycare centers rather than family daycare homes, have a reduced risk of becoming atopic, with the likelihood of developing atopy decreasing as age of first attendance decreases (Kramer et al. 1999; Haby et al. 2000). However, children who attend daycare centers are more likely to suffer serious respiratory infection, including pneumonia and bronchitis, have a history of repeated respiratory illness, otitis media and the common cold, and suffer from nightly cough and blocked/runny nose without common cold (Celedon et al. 1999; Nafstad et al. 1999; Haby et al. 2000; Slack-Smith et al. 2002; HagerhedEngman et al. 2006). In contrast with atopy, the risk of developing these increases with decreasing age of first attendance at child care. While there are reports of increased risk of current wheezing, doctor-diagnosed asthma, current rhinitis, doctor-diagnosed hay fever, and food allergy among children who attended daycare, this tends to peak prior to 4 years of age (Nafstad et al. 1999; Ball et al. 2000; Hagerhed-Engman et al. 2006) and these children are less likely to suffer these disorders after 4–6 years of age (Ball et al. 2000; HagerhedEngman et al. 2006). So while the burden of common childhood diseases is greatest among children who attend daycare, the long-term outcome appears to be protection against the development of asthma and this protection is presumed to reflect the protective effects of early daycare attendance on the development of atopic sensitization (Ball et al. 2000). While there remains much interest in the impact of respiratory viral infections on postnatal lung development and wheezing/asthma outcomes, there has been another shift in the “hygiene hypothesis” away from the infectious microbial burden to the environmental/commensal microbial burden.

Farm environment The favorable effects of an early childhood spent on a farm have now been demonstrated in many countries and include a reduction in atopic sensitization in children aged 6–15 years (Braun-Fahrlander et al. 1999); reduced prevalence of hay fever, asthma, and skin-prick reactivity among 8–10 year olds (Riedler et al. 2000); lower prevalences of hay fever, asthma, and wheeze in 5–7 year olds (Von Ehrenstein et al. 2000); protection against IgE sensitization in 7–12-year-old children (Downs et al. 2001); lower risk ratio for ever having asthma and/or allergic rhinoconjunctivitis but not skin-test reactivity in children aged 7–8 years (Klintberg et al. 2001); and significantly less current asthma symptoms and current seasonal rhinitis but no difference in eczema (Perkin &

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Strachan 2006). The benefits of farming seem to relate to the presence of livestock, especially barns, stables and unpasteurized milk, and the greatest benefits occur if exposures occur during early life and even prenatally. The critical component of the farming environment postulated to protect against the development of allergy and allergic disease is exposure to microbial antigens in barns, stables, farmhouses, and unpasteurized milk leading to the stimulation of Th1-biased reactivity. Notably elevated levels of bacterial endotoxin, and other microbial products such as mold glucans and fungal extracellular polysaccharides, are a feature of farming households, barns, and stables (Von Mutius et al. 2000; Braun-Fahrlander et al. 2002; Schram et al. 2005; Perkin & Strachan 2006). The timing and duration of exposures are crucial for the protective effect: exposure of children aged less than 1 year to stables and unpasteurized milk was associated with lower frequencies of asthma and atopic sensitization compared with 1–5 year olds, although continual long-term exposure to stables until 5 years was associated with the lowest frequencies of asthma, hay fever, and atopic sensitization (Riedler et al. 2001). The children of farmers have elevated CD14, TLR2, and TLR4 gene expression compared with the children of nonfarmers (Lauener et al. 2002) and the expression of these genes might be fetally programmed in response to maternal exposures in pregnancy as each additional farm species to which the mother was exposed during pregnancy increased the expression of CD14, TLR2, and TLR4 (Ege et al. 2006). Unpasteurized milk consumption, even when infrequent and unrelated to farming status, has been associated with reduced risk of being skinprick test positive, less current eczema, lower total serum IgE, and higher whole blood IFN-γ responses to PHA in childhood (Perkin & Strachan 2006).

Endotoxin The majority of studies report an inverse association between household (farming and nonfarming) levels of endotoxin

and the development of eczema, hay fever, and atopic sensitization in infancy and childhood (Gereda et al. 2000; BraunFahrlander et al. 2002). However, the relationship between endotoxin and asthma/wheezing is far more complex, with reports of no association, an inverse association with atopic but not nonatopic wheeze, and a positive relationship. These studies differ greatly in the times at which endotoxin was measured, the site at which household dust was sampled, the family history of the children, and when disease outcomes were determined. For example, endotoxin levels measured at 3 months of age in the living room but not the mattress were inversely associated with physician-diagnosed asthma during the first 4 years of life (Schram et al. 2005), whereas there was no relationship between living room floor endotoxin levels measured at 5–10 years of age and a diagnosis of asthma at the same age (Gehring et al. 2002). Variation in the effect of/response to endotoxin is likely to reflect the duration, type, and level of exposure and to be determined by inherited predisposition. In particular, exposures immediately after birth might have different effects to exposures that occur once allergen-specific T-cell memory has been established. Animal models support this: exposure to endotoxin early in the sensitization process protects against the development of sensitization, whereas exposure after sensitization exacerbates the inflammatory response in the airways but has no effect on sensitization (Meri et al. 2000) (Fig. 2.3). In humans, household endotoxin levels correlated positively with the percentage of CD4+ T cells producing IFN-γ in infancy (Gereda et al. 2000), whereas among 6–13 year olds LPS-stimulated tumor necrosis factor (TNF)-α, IFN-γ, IL-10, and IL-12 production was inversely related to household endotoxin levels, indicating that by this age endotoxin tolerance had occurred (Braun-Fahrlander et al. 2002). The beneficial effects of early LPS exposure might extend prenatally since maternal exposure to LPS was associated with increased IFN-γ response by neonatal mice and, in OVAsensitized animals, prenatal LPS exposure was accompanied

ENDOTOXIN

Dendritic cell

Th1

Dendritic cell

Allergen

Th2

Th1

Allergen

Th2 Allergic inflammation Tissue remodeling

No allergic disease

Epithelium

ENDOTOXIN Allergic disease exacerbation

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Fig. 2.3 Beneficial and detrimental effects of endotoxin reflect the timing and dose of exposure as well as genetic and other environmental determinants. Early exposure to lipopolysaccharide is postulated to favor Th1 responses and downregulation of Th2-trophic activity prevents the development of allergenspecific IgE. However, once allergen-specific Th2-trophic activity is established and tissue remodeling/inflammation has occurred, exposure to endotoxin exacerbates this inflammation. (See CD-ROM for color version.)

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by reduced OVA-specific IgG1 and IgE and reduced bronchoalveolar lavage fluid eosinophils but had no effect on airway hyperreactivity (Blumer et al. 2005).

Domestic pets Interest in the contribution of cats and dogs to the development of allergy and allergic disease stem from their contribution to both the indoor allergen load and the household endotoxin burden, and even homes without pets contain detectable cat and dog allergens (Nafstad et al. 2001). Controversy continues to surround whether furred pets have a beneficial or detrimental effect on atopic sensitization and the development of allergic disease. As for other environmental exposures, the timing, duration, and level of exposure, as well as family history of allergic disease, are likely to have critical roles in determining allergic outcomes. However, in general cat and/or dog exposure in early life is beneficial whereas current cat (but not dog) ownership or high community cat ownership are significant risks for both sensitization and disease manifestation. The level of exposure in early life might be of critical importance: exposure to cats in the child’s bedroom, which might mark intensive exposure to cats, but not at other sites in the house was associated with reduced prevalence of atopic asthma at 5–7 years of age (Oberle et al. 2003); the highest exposure to cat was associated with decreased prevalence of sensitization to cat and higher prevalence of IgG (including IgG4) to Fel d1, with nonsensitized children exhibiting an IgG (including IgG4) response but not an IgE response (Platts-Mills et al. 2001; Lau et al. 2005). Thus if exposure leads to the production of only specific IgG a child will not develop disease, whereas if IgE and IgG develop the child is at risk of developing wheeze/ asthma (Lau et al. 2005). This relationship might be peculiar to cat allergen (Platts-Mills et al. 2001) and the level of exposure required to achieve this effect is likely to vary between populations.

Vaccinations Routine vaccinations in early life offer essential protection against potentially fatal disease. However, comprehensive vaccine strategies in Western countries have been postulated to contribute to the increase in allergy and allergic disease over recent decades. Vaccinations might have a protective effect on the development of atopy as they could directly stimulate Th1-like immunity or might favor IgE production and allergic disease by reducing the incidence of some infections that indirectly prevent “natural” Th1-like immunity (Gruber et al. 2001). Consequently there are now many studies published that consider the potential relationship between early vaccine exposures (including DTP, MMR, HBV, HIB) and allergic outcomes. While there are reports of an association between some vaccinations and eczema, asthma, and hay fever (Farooqi & Hopkin 1998), most of the available data, including those from large (> 100 000 subjects) multi-


center studies, suggest that there is no association between any common childhood vaccinations (including individual vaccinations, total doses received, number of different vaccines received, and age at which received) and the development of IgE sensitization or allergic disease up to at least 16 years of age (Anderson et al. 2001; Koppen et al. 2004). Childhood vaccination remains an essential part of “well child” health programs throughout the world and there is no evidence to suggest that this should change, although the immunostimulatory capacity of vaccines could be modified to provide optimal stimulation of the infant’s immune system. (Koppen et al. 2004). However, there is growing interest in the possibility that genetic risk for atopy influences the capacity to respond to vaccinations during infancy (Holt et al. 2003).

Antibiotics Children born between 1980 and 1995 received more antibiotic dispenses and had their first exposure at a younger age than children born in the previous decade (Mullooly et al. 2007). The use of antibiotics in the first year of life has been associated variably with increased risk of developing asthma, hay fever, and eczema and allergic sensitization in childhood, although the association between antibiotic use and wheezing/asthma outcomes is the most robust relationship. Indeed metaanalysis of studies in which antibiotic usage over first year of life was related to physician-diagnosed asthma between 1 and 18 years of age (four retrospective and four prospective) revealed that exposure to at least one course of antibiotics in the first year of life is a risk factor for the development of childhood asthma, although the authors highlighted the need for large-scale prospective studies to confirm this association (Marra et al. 2006). The timing and level of antibiotic exposure might be critical as the adjusted odds ratio for developing asthma was greater for antibiotic use in the first year of life than antibiotic use only after the first year of life or never used and for three or more courses in the first year of life versus zero, one, or two courses (Wickens et al. 1999). However, there is concern that the relationship between antibiotic use and wheezing/asthma outcomes might simply reflect reverse causation. In studies in which antibiotic use for LRTI was excluded, there was no effect of antibiotic use in the first year of life on asthma, recurrent wheezing, allergic rhinitis, or eczema (Illi et al. 2001; Celedon et al. 2002). Moreover while there was a relationship between antibiotic prescriptions prior to 5 years of age and self-reported asthma, especially in those sensitized to aeroallergens, antibiotics in this group were largely prescribed for LRTI (Cullinan et al. 2004) and children with LRTI or environmental tobacco smoke exposure are more likely to have antibiotics prescribed prior to 6 months of age (Droste et al. 2000). Thus early and repeated antibiotic usage might be a surrogate marker for the potential role of infections in the modification of immunologic maturation in early life and identify the

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children most susceptible to the development of LRTI and wheezing/asthma (Mullooly et al. 2007). Any link between antibiotic use and allergy and allergic outcomes might reflect the impact of oral antibiotic use on the gastrointestinal flora. As the composition of the maternal commensal flora late in the third trimester of pregnancy will influence that of the newborn, this could also explain the negative effect of maternal antibiotic use in pregnancy on wheezing/asthma by the child (McKeever et al. 2002a; Jedrychowski et al. 2006; Rusconi et al. 2007). Recent epidemiologic studies and experimental research, as summarized above, have highlighted that the microbial environment and exposure to microbial products in infancy might modify immune responses and enhance tolerance to ubiquitous dietary and environmental allergens. The intestinal microflora are a major external determinant of immune system maturation after birth and are requisite for the normal development of oral tolerance (Bjorksten 2004). Indeed the currently predominant version of the hygiene hypothesis, the “microbial exposure” hypothesis, reflects a critical role for altered patterns of subclinical, commensal, or environmental microbial exposure in early postnatal life (Bjorksten 2004; Noverr & Huffnagle 2005).

Gastrointestinal flora Studies of the fecal microflora in children generally indicate that decreased lactobacilli and bifidobacteria and increased clostridia are particularly associated with an allergic phenotype that typically relates to IgE sensitization, food allergy, and eczema. Differences in microflora composition might even extend to the species level and a more detailed analysis of clostridial species present in feces of allergic versus nonallergic children is warranted (Tonooka et al. 2005). Few studies have attempted to relate the commensal gut flora in childhood to wheezing/asthma outcomes but in keeping with the negative impact of elevated clostridia, increased circulating Clostridium difficile-specific IgG was more common at 1 year of age in atopic children with a history of recurrent wheeze compared with nonatopic children with no wheezing history (Woodcock et al. 2002). However, the prevalence of lactobacilli and bifidobacteria did not differ in stool samples collected from children ≥ 3 years old who were atopic/wheezy or nonatopic/nonwheezy (Murray et al. 2005). Nevertheless, as allergy-associated variations in fecal microflora can exist as early as 3 weeks of age and precede the manifestation of allergic disease, the composition of the gut microflora prior to the first birthday might have a central role in determining disease outcomes. The variations described to date are likely only the tip of the iceberg. The complex ecosystem of the intestinal microflora is estimated to contain approximately 400 microbial species, mostly bacteria. Studies of bacterial colonization in the gut are based almost entirely on conventional culture methods of fecal samples, which can only detect a small frac-

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tion of the commensal gut flora. While feces is the sample of choice, it does not fully represent the complex microflora as species distributions vary throughout the gut and fecal sampling does not give a direct indication of commensal microbiota composition, especially in the upper gastrointestinal tract. Of particular relevance, a molecular analysis of feces obtained during the first week of life showed that microbiotic diversity changes rapidly over the first few days after birth, with the acquisition of unculturable bacteria expanding rapidly after the third day (Park et al. 2005). Can alterations in gastrointestinal flora impact on allergic disease? The gastrointestinal commensal flora shapes both mucosal and systemic immune function throughout life and is implicated in the induction of protective IgA and oral tolerance, including induction of regulatory T cells, and immune deviation (Macpherson & Harris 2004). Germ-free mice develop antigen-specific Th2-biased reactivity. Reconstitution of the intestinal microflora of such mice with Bifidobacterium infantis (a predominant organism of the commensal flora) restores oral tolerance in neonatal but not adult mice (Sudo et al. 1997). Disruption of the normal gastrointestinal commensal microflora has been used as a strategy in mouse models to induce allergic airways disease characterized by increased levels of eosinophils and mast cells, increased Th2 cytokine production, and increased circulating IgE levels (Noverr et al. 2004, 2005). Gastrointestinal infection with helminths has also been shown to prevent the development of allergic airways disease in murine models of asthma, although total and specific IgE levels remain unchanged or increased (Wohlleben et al. 2004; Wilson et al. 2005; Kitagaki et al. 2006). Thus manipulation of the gastrointestinal flora can potentially dramatically affect systemic reactivity to allergens and allergic airways disease.

Other environmental factors Diet and obesity The fall in the consumption of saturated fat while the amount of polyunsaturated fat in the diet has increased parallels changes in allergic disease prevalence. Similarly, the prevalence of obesity in Western populations has increased concurrently with asthma and other allergic diseases. These observations have piqued interest in a link between dietary factors, obesity, and allergic disease.

Diet Of particular interest is maternal intake of various dietary components during pregnancy. Maternal zinc intake during pregnancy has been inversely associated with wheezing to 2 years of age (Litonjua et al. 2006) and asthma ever and current asthma at 5 years of age (Devereux et al. 2006). Low maternal vitamin E intake during pregnancy has been associated with increased proliferative responses by cord blood mononuclear cells (Devereux et al. 2002), increased likelihood of wheezing to 2 years of age (Martindale et al.

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2005; Litonjua et al. 2006), and an increased likelihood of asthma/wheezing symptoms during the first 5 years of life (Devereux et al. 2006). The child’s own nutrient intake was not associated with any of the outcomes measured. Dietary intake of polyunsaturated fatty acids (PUFAs) has also attracted interest as there is the possibility that manipulating the levels of these in the diet might have therapeutic benefits. Conversely to zinc and vitamin E intake, the intake of fatty acids by the child might be particularly relevant. A high dietary intake of PUFAs has been associated with increased risk of recent asthma in 3–5-year-old children (Haby et al. 2001), although the n-3 and n-6 fatty acid profile at birth was not associated with atopic sensitization or eczema and asthma outcomes up to 4 years of age (Newson et al. 2004). Despite this latter observation, maternal dietary supplementation during pregnancy with n-3 PUFAs has been associated with appropriate changes in the n-3/n-6 fatty acid content of umbilical cord blood erythrocytes (Dunstan et al. 2004) and modifications of immune function at birth (Dunstan et al. 2003). The long-term therapeutic benefits of such a strategy remain to be determined. Mechanisms for the impact of dietary factors on allergic status and disease manifestation include the following: 1 Modification of fetal airways development and dietary manipulation studies in rodent models support this hypothesis (Islam et al. 1999). 2 The PUFA linoleic acid, a precursor of arachidonic acid, can be converted to PGE2, which favors Th2 responses and IgE production (Black & Sharpe 1997), although animal models indicate that PUFAs alter the Th1/Th2 balance via inhibition of Th1 cytokine production (Wallace et al. 2001). 3 Methylation and/or chromatin conformation can be affected by environmental factors including diet and these would likely have long-term health effects (Vercelli 2004).

Obesity Obesity precedes asthma and persists after controlling for diet and physical activity (Weiss 2005). The mechanisms whereby obesity influences the asthma phenotype remain unknown but are postulated to include mechanical function of the lungs, changes in immune or inflammatory response, sex-specific effects of hormones, maternal diet, and fetal programming. Obesity likely potentiates and worsens asthma via specific inflammatory mechanisms. There is particular interest in the appetite-regulating peptides leptin and ghrelin that are both recognized to have additional immunomodulatory properties, with ghrelin downregulating the proinflammatory effects of leptin (Fantuzzi & Faggioni 2000; Dixit et al. 2004). Overweight children with asthma have twofold greater serum leptin levels than overweight children without asthma and leptin levels correlated positively with circulating IFN-γ levels (Mai et al. 2004). Obese children had significantly higher IgE levels and serum ghrelin levels inversely correlated with IgE levels (Matsuda et al. 2006).


These observations highlight the need to ascertain the impact of mediators of neuroendocrine function on relevant immunologic mechanisms.

Perinatal risk factors and anthropometric measurements Mode of delivery The potential negative impact of cesarean section delivery on allergic outcomes has received much interest. However, while two small studies found (i) a slightly increased risk of wheezing with respiratory tract infection and food allergen sensitization up to 2 years of age in children delivered by cesarean section (Negele et al. 2004) and (ii) that delivery by cesarean section was associated with increased cumulative incidence of asthma at 7 years of age and increased risk of being skin-prick test positive (Kero et al. 2002), four very large birth cohort studies in the UK, Italy and the USA indicate that mode of delivery is not a risk factor for the development of allergic disease including allergic sensitization and wheeze/asthma (McKeever et al. 2002b; Maitra et al. 2004; Juhn et al. 2005; Rusconi et al. 2007).

Maternal atopy Maternal atopy has been revealed to be a greater risk than paternal atopy for the development of allergic disease in the offspring by many investigators and is particularly associated with an increased likelihood of the child developing eczema in infancy and an earlier age of onset of eczema. As this is deemed the first step on the atopic march, understanding this relationship is critical and maternal and/or placental factors have been postulated to impact on the development of the fetal immune system and thereby allergy and allergic disease. In a canine model, the offspring of allergic parents developed elevated total and specific IgE and IgG and increased pulmonary resistance to histamine and allergen as well as increased eosinophils in bronchoalveolar lavage fluid (Barrett et al. 2003). The authors argued that the maternal component was the primary parental contributor via maternal factors that influence the development of the fetal/infant immune system. Thus some component of the maternal/ placental immune response, presumably related to the mother’s own elevated total IgE/allergen-specific IgE regulates the development of Th2-biased reactivity to allergen by the offspring and the clinical manifestation of atopic disease in the first year of life. To this end, detectable concentrations of IFN-γ, IL-4, and TNF-α in the neonatal circulation at birth have been associated with a lower risk of physiciandiagnosed asthma, current asthma, current wheeze and/or atopy at 6 years of age (Macaubas et al. 2003). Maternal smoking in particular was associated with reduced levels of IFN-γ and IL-4 and increased risk of wheeze at 6 years of age. Although the cellular sources of circulating cytokines at birth were not determined, the trophoblast and other cell populations of the placenta are probable sources. Thus the same

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attenuated cytokine response extends beyond the neonatal T-cell compartment to the placenta (a fetally derived tissue).

Fetal growth/anthropometric measurements A possible relationship between fetal growth and measures of atopy/atopic disease was first suggested by the observation that raised serum total IgE levels at 50 years of age were associated with increased head circumference at birth. This relationship was independent of adult physique, maternal pelvis size, and maternal parity. This study sparked numerous investigations exploring the relationship between anthropometric measurements at birth and various atopic outcomes. However, rather than considering the relationship between individual anthropometric measures and atopic outcomes, it is probably best to consider what these measures mean and how they relate to the outcome of interest. Thus markers of overnutrition, including increased birth weight, increased gestational age, and increased ponderal index (a measure of the relationship between birth weight and birth length), tend to be associated with atopic sensitization, eczema, hay fever, and asthma. Fetal undernutrition has also been linked to current wheezing (Bolte et al. 2004) but this is likely to reflect the relationship between reduced lung function in infancy to low birth weight (Dezateux et al. 2004; Hoo et al. 2004). Notably, early postnatal growth fails to compensate for this and higher rates of weight gain in early infancy might actually have a negative impact (Lucas et al. 2004). Fetal growth is determined by a variety of factors including maternal health and nutrition, placental function, and fetal growth potential. The fetal origins of child/adult health and disease hypothesis suggests that fetal growth and maturation characteristics influence the likelihood of several diseases later in life and early adverse events operating at a critical period of fetal development result in an increased risk of disease (fetal programming; Barker 2004). Thus intrauterine programming of the developing immune and other organ systems pivotal to the development of allergic disease might occur secondary to fetal overnutrition or other events that impact on fetal health and well-being.

Breast-feeding Human milk contains an array of nutrients, hormones, growth factors, and immunoactive molecules that influence the growth, development, and immune status of the newborn. Immunoactive mediators orchestrate the development of the mucosal immune system, especially at the gastrointestinal tract, and probably serve to compensate for developmentally programmed downregulation of many immune functions during infancy (Jones & Warner 2000). Breast-fed versus bottle-fed babies have been noted to have a lower incidence of gastrointestinal infections and inflammatory conditions as well as allergic diseases. However, the protective effect of breast-feeding on the development of allergic diseases remains divisive. Metaanalyses of prospective studies found that exclus-

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ive breast-feeding for the first 3 months after birth protects against the development of allergic rhinitis in childhood from both allergic and nonallergic families (Mimouni et al. 2002) and the development of atopic dermatitis and asthma particularly in children from at-risk families (Gdalevich et al. 2001a,b). Still, studies that find a positive effect, no effect, or even a negative effect of breast-feeding on atopic sensitization, eczema, allergic rhinoconjunctivitis and/or asthma continue to be published. Ambiguity surrounding what comprises breast-feeding, including duration, exclusivity, weaning age, weaning food, and family history, might account for some of these discrepancies. Moreover, the well-known potential benefit of breast milk might contribute to the controversy as women who have a child at risk of developing allergy or who have a child that develops allergy very early in life are likely to keep breast-feeding for longer, thereby masking the protective effect of breast-feeding (Lowe et al. 2006). Protection via breast-feeding has been variously ascribed to a number of mechanisms, in addition to those related to hygiene and to milk components such as maternal immunocompetent cells, immunoglobulins, antimicrobial peptides, oligosaccharides, growth factors, cytokines, lysozyme, lactoferrin, complement, and nutrients. The relationship between allergic disease and breast-feeding has prompted investigations of the biological mediators in breast milk that might explain this. Thus investigators have sought to identify how levels of critical mediators might differ in breast milk fed to children who do and do not develop disease. These critical mediators include dietary fatty acids, IgA, TGF-β, soluble (s)CD14, and IL-10 among others. Instead of providing an overview of findings relating to each of these, sCD14 has been chosen as an example. Breast milk sCD14, as well as other yet unidentified constituents of human milk (LeBouder et al. 2006), have been postulated to have a critical role during bacterial colonization of the gut immediately after birth and to modulate gastrointestinal innate and adaptive immunologic responsiveness during this time (Labeta et al. 2000). sCD14 is also a lipid carrier so could influence production of fatty acid-derived eicosanoids and link microbial and dietary exposures (Yu et al. 1997). sCD14 is produced by mammary epithelial cells and is abundant in human milk but not infant formulas (Labeta et al. 2000). Maternal milk and circulating sCD14 levels do not correlate and levels appear to be regulated differentially by the same single nucleotide polymorphism: CD14/–550T is associated with high breast milk sCD14 but low plasma sCD14 levels (Guerra et al. 2004b). A number of studies have associated reduced breast milk sCD14 with eczema in the first year of life (Jones et al. 2002) and atopic symptoms and sensitization by age 4 (Savilahti et al. 2005), but elucidating the role of a single candidate molecule in a complex disease process is fraught with difficulties (Laitinen et al. 2006). The link between maternal atopy and increased disease risk of the offspring and the potential detrimental effect of

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prolonged feeding by atopic women (Wright et al. 1999) has prompted interest in the possibility that variation in the composition of breast milk from allergic versus nonallergic mothers might explain some of the controversy surrounding the beneficial effects of breast-feeding. However, with exception of TGF-β1 (Rigotti et al. 2006), no significant effect of maternal allergy on breast milk composition that could be related to allergic disease outcomes by the child have been described. The many benefits of breast-feeding warrant its continued recommendation. Elucidating the interaction between specific defense factors in milk, the duration of breast-feeding, and genetic predisposition and how these might modulate the development of allergic disease should be the focus of investigation.

Genetics The interplay between genes and the environment has emerged as a key area of interest in understanding the development of allergy and allergic disease. However, few studies take into account the additional interaction with the developmental stage of the host, i.e., age as a confounder. Additionally, it has become apparent that the effect of an environmental factor or an intervention strategy on allergic outcomes must take into account the host’s genotype. The genetics of allergy and allergic disease are discussed in detail


elsewhere (see Chapter 55), although we briefly discuss some of those so far revealed of relevance to the early life origins of allergic disease.

IgE production Elucidating the underlying genetic variation that accounts for elevated IgE production has generated many studies of IL-4 and IL-13 and their common signaling pathway. However, most of these studies are of single genes and it is only recently that investigators have attempted to study combined extended haplotypes to better gauge the impact of genetic variation in this critical pathway. Combinations of genetic alterations in IL-4, IL-13, IL-4Rα (the shared receptor chain) and the intracellular signal transducer and activator of transcription (STAT6, the shared signaling molecule) significantly influence total serum IgE levels and the development of asthma at 9–11 years of age (Kabesch et al. 2006).

Innate immune response Interest in the interaction between innate immune function and the development of effector T cells with Th1, Th2 or regulatory activity has driven studies investigating the relationship between polymorphisms in relevant genes, disease outcomes and, on occasion, appropriate immunologic activity. The first of these was CD14. Membrane and soluble CD14 support cellular responses to various bacterial components including LPS (Fig. 2.4). The role of CD14 in promoting IL-12

mCD14+ cells

Membrane CD14-negative cells

Fig. 2.4 Postulated role of soluble and membrane CD14 in modification of innate immune responsiveness in early childhood. Soluble CD14 facilitates the delivery of lipopolysaccharide (LPS) to TLR4 on membrane CD14-positive cells (e.g., monocytes/macrophages) and CD14negative cells (e.g., dendritic and epithelial cells). Soluble/membrane CD14 delivers LPS to TLR4/MD2 to enable intracellular signaling and thereby changes in cytokine production and costimulatory molecule expression. (See CD-ROM for color version.)

Signal

LPS

LBP

CD14

TLR4

MD2

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production and thereby Th1 activity prompted investigation of the relationship between genetic variation in CD14, circulating levels of CD14, and disease phenotypes. The first study to explore this revealed a single nucleotide polymorphism, a C→T transition at –159 from the major transcription start site (CD14/–159), that was associated with serum sCD14 and total IgE levels in 11-year-old non-Hispanic white children (Baldini et al. 1999). TT homozygotes had significantly higher serum sCD14 levels than children with CC or CT genotypes and, among skin-prick test-positive children, lower total IgE levels and fewer positive skin-prick tests. An inverse relationship between serum sCD14 levels and concanavalin-A stimulated IL-4 production by peripheral blood mononuclear cells was also noted. While a similar relationship between this genetic variation and allergic disease severity has also been reported in adults (Koppelman et al. 2001), there have been contradictory findings (Kabesch et al. 2004). However, serum sCD14 levels increase dramatically after birth so that they exceed adult levels by 6 months of age, presumably reflecting changes in the prevailing microbial burden of the host (Jones et al. 2002). So it is unsurprising that CD14/–159 might have different effects on serum total and specific IgE in different environments such as a farming environment with high endotoxin levels and exposure to various animals (Eder et al. 2005) and have a role in childhood rather than adult onset of atopy and allergic symptoms (O’Donnell et al. 2004). A natural progression from these studies of CD14 has been to consider the contribution of polymorphisms in other receptors and signaling molecules involved in innate immunity to the development of allergic disease. There is particular interest in how relevant genotypes might interact with environmental stimuli to determine disease outcomes. As discussed above, growing up on a farm is now recognized as protective against the development of allergy and allergic diseases. Farmers’ children, but not children from the same rural community who did not live on a farm, carrying one or two T alleles of a polymorphism in TLR2 (TLR2/–16934) were less likely to have a diagnosis of asthma, current asthma or hay fever, and atopy. Also a polymorphism in TLR4 (TLR4/+4334) while not differentially expressed in farming versus nonfarming children or associated with symptoms was associated with a reduced likelihood of being atopic among children exposed to high endotoxin levels (Eder et al. 2004). Within the same population a strong protective effect of a farming environment on allergies was only found in children homozygous for the T allele (CARD4/–21596) in CARD4 (caspase recruitment domain protein 4), an intracellular pattern recognition receptor (Eder et al. 2006). The functional significance of genetic polymorphisms are even less well studied but a TLR4 polymorphism (Asp299Gly) has been related to decreased LPS-stimulated IL-12p70 by peripheral blood mononuclear cells of 8- and 14-year-old children and to an increased prevalence of asthma but not allergic rhinoconjunctivitis (Fageras Bottcher et al. 2004).

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ALLERGIC DISEASE IgE sensitization

Environment

Tissue-specific factors

Development

Genetics

Fig. 2.5 The “allergy pyramid” summarizes the interaction of the environment, genetics, and developmental stage on IgE sensitization and tissue-specific factors in the skin, gut and airways and, ultimately, on the manifestation of allergic disease. (See CD-ROM for color version.)

Tissue-specific genes There is increasing evidence that variation in genes operational at a tissue-specific level also contribute to variations in allergic disease phenotypes. These genes typically have been identified using family linkage studies followed by positional cloning, an approach which has revealed genes that would not have been considered as candidate genes and which are of wide-ranging function (Ober 2005). However, few studies have examined the interplay between environmental risk factors operational during early life and genetic risk for asthma and allergic disease. The importance of such studies is highlighted by the observation that polymorphisms in one of these identified “asthma genes” (ADAM33) predicts impaired lung function at 3 and 5 years of age but not allergic sensitization or physician-diagnosed asthma (Simpson et al. 2005).

Conclusion The key immunologic, environmental, and genetic factors that contribute to the development of allergy and allergic disease have been discussed to highlight that the development of allergy during early childhood is a complex multifactorial process (Fig. 2.5). A pivotal factor determining the longterm consequences of the relevant gene-by-environment interactions driving disease pathogenesis is the maturational status of a variety of cellular functions both within and exterior to the immune system, at the time these interactions occur. As an increasing number of investigators attempt to resolve these complex interactions, our understanding of the development of allergy and allergic diseases will improve and additional therapeutic strategies should be devised.

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T Cells and Cytokines in Asthma and Allergic Inflammation Chris Corrigan

Summary The past 20 years have seen considerable advances in T-cell biology. There is increased understanding of the shaping of the T-cell antigenic repertoire, and the recognition that this might be susceptible to manipulation early in life. The concept of active, antigen-specific suppression of immune responses by T cells has also turned full circle from skepticism to acceptance, although what were named “suppressor” T cells have now been renamed “regulatory” T cells. The recognition that such cells may limit immune responses to both self and external antigens provides considerable promise for future therapeutic maneuvers. In this same time-frame, the Th1/Th2 concept has ignited, exploded, and now somewhat simmered down. New triggers for Th2 T-cell differentiation, such as the costimulatory molecule OX40, and new cytokines like thymic stromal lymphopoietin are being uncovered. Concurrently, attention has switched from attempts to “skew” polarized Th2 T-cell responses in allergic disease to recognition that induction of long-lived, allergen-specific T regulatory cells may be a better strategy. In this regard, the cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β have received increasing attention as mediators of this regulation, while tumor necrosis factor (TNF)-α is being increasingly singled out as a key proinflammatory cytokine in both Th1- and Th2-mediated inflammatory responses. The T-cell hypothesis of asthma has blossomed in these 20 years, and few investigators would now seriously contest that T cells and some of their cytokine products play some sort of role in asthma. There is still, however, a great deal of uncertainty about the mechanisms of this process. The concept that the effects of cytokines are mediated through “effector” cells such as eosinophils in the bronchial mucosa has been seriously challenged (but not killed outright) by therapeutic experiments with agents such as humanized monoclonal antibodies. At the same time, there has been increasing recognition that cytokines can bring about many of the bronchial mucosal changes associated with asthma, collectively termed “remodeling,” without the necessity for intervention of inflammatory granulocytes. Above all, what has remained stubbornly elusive is any glint of light in the chasm of uncertainty as to how the observable immunologic and structural changes in the airways caused by these cytokines actually cause the clinical symptoms of asthma. This chapter contains a


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summary of new advances in T-cell biology, particularly as applied to asthma and allergy, an up-to-date account of the known or suspected role of cytokines in the asthma process, and some speculations on new approaches to therapy in the future.

Development and properties of T lymphocytes T lymphocytes (T cells) play a fundamental role in the orchestration of immune responses, against both external invading allergens and self allergens in autoimmune diseases. Through their antigen receptor complexes they are able to recognize peptides derived from the sequence of antigenic proteins when these are presented on the surface of class I or II major histocompatibility (MHC) antigens (Rudolph et al. 2006). Following activation by peptides, T cells produce a range of protein mediators called cytokines which, through their actions on other immune cells, shape the development of the subsequent immune response. T-cell-derived cytokines play a major role in determining whether an immune response will be largely cell-mediated or humoral (mediated by antibodies). With the exception of certain antigens, which can bind to conserved structural regions of the T-cell antigen receptor directly and thereby activate T cells polyclonally, antigen recognition depends on the presentation to T cells of processed peptides. While all cells express class I MHC molecules, class II MHC molecules, which present to CD4+ T cells, are restricted in their expression. Antigen presentation to CD4+ T cells is brought about generally by professional antigen-presenting cells, particularly dendritic cells, which express MHC class II antigens and other essential costimulatory molecules (see below) following activation. However, other cells may express MHC class II, particularly in conditions of inflammation, and act as antigen-presenting cells. The process of uptake and degradation of antigen into peptides, and association of the peptides with MHC class I or II molecules is a highly ordered process depending on lysosomal degradation and ordered assembly of the MHC–peptide complexes within the endoplasmic reticulum, and subsequently the outer cell membrane. While immature B cells express antibodies with low affinity for antigens, they also require

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cognate interaction with T cells in order to proliferate. During this process, B cells expressing antibody with high affinity for the stimulating antigen are selected. This process is known as affinity maturation, and depends on somatic hypermutation of the genes encoding the variable regions of antibody heavy and light chains, which together bind to the antigens. Consequently, it can be said that all immune reactions are initiated by T cells. A fundamental distinction between T-cell and B-cell antigen recognition is that, unlike antibodies produced by B cells, which recognize antigens by their three-dimensional shape, T cells do not recognize the shape of antigens but particular amino acid peptide sequences derived from proteolytic processing of the antigens. Such sequences typically comprise approximately 10–13 amino acids and are known as T-cell “epitopes.” Each antigenic peptide has variable numbers of T-cell epitopes. Since epitopes are presented on the surface of MHC molecules, different epitopes from a single antigen activate T cells variably well in different individuals because of their differing MHC haplotypes. The epitope–MHC complex specificity of T cells is determined by random association of the V, D, and J gene segments encoding the α and β chains of the T-cell antigen receptor. There are many families of these genes and their random association results in T-cell receptors with a potential epitope repertoire that is more than enough to cover epitopes from external antigens, as well as self antigens. Thus, unlike B cell-derived antibodies, the structures of which “mature” during B-cell proliferation by somatic hypermutation, the epitope specificity of any T cell is determined from the moment of the expression of its antigenic peptide receptor, and is immutable. T cells are divided into two principal functional phenotypes signaled by the expression of the surface marker complexes

T Cells and Cytokines in Asthma and Allergic Inflammation

CD4 and CD8. CD4+ T cells recognize antigenic peptides in the context of MHC class II, and are producers of cytokines that govern the nature of inflammatory responses. CD8+ T cells, which recognize antigens in the context of MHC class I, function principally as cytotoxic cells that kill target cells invaded with foreign intracellular organisms such as viruses, or tumor cells. Since most of the studies of the involvement of T cells in asthma concern CD4+ T cells (the few exceptions are mentioned later), most of this chapter is concerned with CD4+ T-cell biology and regulation.

Early T-cell development Early developing T cells in the thymus express both CD4 and CD8, and their epitope specificity, as mentioned, is determined at random. During thymic development, those T cells whose receptors happen by chance to recognize peptides derived from “self” antigens must be silenced or eliminated, otherwise they would attack the host (Fig. 3.1). The fact that autoimmune diseases do exist demonstrates that this silencing system, while ingenious and very efficient, is not perfect. Developing T cells in the thymic epithelium that happen to bind self-peptide–MHC class I or II complexes presented by thymic cortical epithelial cells proliferate: this process is called primary positive selection (Starr et al. 2003). The remainder of the cells, which have no affinity for self antigens whatsoever, die by neglect. Surviving T cells migrate on to the thymic medulla. T cells with a very high affinity for self antigen begin to proliferate but, in the absence of costimulatory signals provided by dendritic cells, undergo activationinduced apoptotic cell death. This is reflected by surface expression of Fas (CD95) and its ligand (CD95L) which interact and activate caspase enzymes that direct cell death. Mutations in Fas, its ligand, and caspase genes result in autoimmune CD4+ CD8+

Fig. 3.1 Model of central thymic tolerance. In primary positive selection, nonself-reactive T cells in the thymic cortex die by neglect. In the thymic medulla, cells with low self affinity may escape selection and become conventional naive T cells. Very high self-affinity cells undergo apoptosis when binding antigen expressed by thymic medullary epithelial cells and presented by immature dendritic cells (DC) (the AIRE factor facilitates expression of promiscuous self antigens). Intermediate affinity cells undergo secondary positive selection to become T regulatory cells (Treg). This is facilitated by ingress of dendritic cells bearing peripheral self antigenic peptides from the periphery and primed by thymic stromal lymphopoietin (TSLP) made in Hassall’s corpuscles (HC) of the thymic medulla. (Adapted from Liu et al. 2006.)

Immature T cell

Thymic cortical epithelium Primary positive selection

CD4+

CD4+

Intermediate anti-self affinity

High anti-self affinity

HC

TSLPactivated DC

TSLP

Secondary positive selection

AIRE

Non-self reactive T cells eliminated

CD4+

Immature DC

Thymic medullary epithelium CD4+

CD4+ CD25+ FoxP3+

Low-affinity anti-self T cells

Negative selection

Naive T cells in the periphery

Death

Treg cell in the periphery

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lymphoproliferative syndrome in humans (Worth et al. 2006). Although this process, called negative selection, is a major one for establishment of self-tolerance, many T cells with medium/high affinity for self antigens do not die but undergo a process called nondeletional central tolerance, or secondary positive selection, in which they give rise to immunosuppressive CD4+ T cells, now known as naturally occurring T regulatory cells (Sakaguchi 2004). These cells are characterized by expression of CD25 and FoxP3, one of the forkhead family of DNA-binding transcription factors. This factor likely plays a critical role in the development of naturally occurring T regulatory cells, since congenital mutations of the FoxP3 gene result in major disruption of immune regulation, in particular the IDEX (immune dysregulation, polyendocrinopathy, enteropathy X-linked) syndrome in humans (Bennett et al. 2001). T cells with low-affinity receptors for self antigens escape all these processes completely and enter the periphery as conventional naive T cells (Fig. 3.1). Only about 3% of all T-cell precursors entering the thymus survive selection and migrate to the periphery and colonize secondary lymphoid organs such as the spleen and lymph nodes. They bind self antigenic peptides with low avidity, yet constitute the population of T cells that will deal with foreign peptides presented by dendritic cells in the binding grooves of MHC molecules. The removal by these mechanisms of T cells with high affinity for self antigenic peptides, occurring as it does solely in the thymus gland, is a remarkable feat. Several mechanisms have been proposed to explain how this phenomenon occurs. There is evidence, for example, for promiscuous expression of a huge variety of self antigens from outside the thymus in thymic medullary epithelial cells, so that T cells can be exposed to the entire repertoire of self antigenic peptides. This is partly regulated by a gene called “autoimmune regulator” or AIRE (Su & Anderson 2004). Mutations in AIRE result in immune polyendocrinopathy syndrome type 1 (Peterson & Peltonen 2005). This mechanism may be principally involved in removal of self-reactive T cells by negative selection (again by antigen presentation by dendritic cells in the absence of costimulation), since in mice AIRE deficiency results in compromised negative selection of T cells recognizing self antigenic peptides but does not compromise the development of CD4+CD25+FoxP3+ T regulatory cells (Anderson et al. 2002). Another process that may be important in self tolerance involves the IL-7-like cytokine thymic stromal lymphopoietin (TSLP) which, as its name suggests, is produced by stromal cells such as epithelial and endothelial cells as well as inflammatory leukocytes. Recent studies show that TSLP is selectively expressed in Hassall’s corpuscles in the thymic medulla in association with an activated subpopulation of myeloid dendritic cells (Watanabe et al. 2005). These dendritic cells, on activation by TSLP, have the capacity to induce the production of T regulatory cells in the thymus, since they have the ability to express the T-cell costimulatory molecules CD80 and CD86, ligands for CD28 on the T-cell surface. Develop-

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ment of T regulatory cells in the thymus depends on CD28 signaling (Watanabe et al. 2005). There is evidence that these dendritic cells migrate into the thymus from the periphery (Donskoy & Goldschneider 2003), and could sample peripheral self antigens and present them to developing T cells within the thymus, resulting in self tolerance. Thus, while many T cells recognizing self antigenic peptides are eliminated in the thymus, others acquire a T regulatory phenotype and actively suppress anti-self responses. Reversal of this phenotype in particular circumstances may give rise to autoimmune disease, while there is some evidence that gaps in the repertoire of naturally occurring T regulatory cells may also cause exaggerated responses to external antigens, including allergens (see below). T cells in human neonates developing in utero and soon after birth are almost certainly exposed to external antigens, including allergens, at the time that the natural T regulatory cell repertoire evolves, providing a mechanistic rationale for the influence of early life exposure to allergens in regulating subsequent immune effector responses. The mechanisms of action of these cells are not well characterized. Some studies suggest that cell–cell interaction is important, and they exert their suppressive functions on potential effector T cells by membrane-bound TGF-β and its receptor CD152 (Nakamura et al. 2001). Alternatively, other studies suggest that these cells can interfere with the direct contact of naive T cells with dendritic cells, thus preventing their activation (Bluestone & Tang 2005; Tadokoro et al. 2006).

T-cell differentiation While CD8+ T cells recognize and kill cells expressing antigenderived peptides on MHC class I molecules derived from intracellular invading organisms such as viruses, and new proteins expressed by tumor cells, CD4+ T cells play a major role in directing other inflammatory responses. Immune responses have been broadly divided into “cell-mediated” and “humoral” immune responses, depending on whether invading foreign antigens are attacked principally by other immune leukocytes such as macrophages, neutrophils or eosinophils, or whether the response is mediated principally by antibodies, resulting in neutralization of foreign antigens, activation of complement, and clearance by the reticuloendothelial system. This broad division of immunologic responses into cellmediated and humoral is reflected in a somewhat similar functional division of CD4+ T cells and is reflected in the cytokines they produce. Over 20 years ago, it was observed that activated CD4+ T cells could be classified into two polarized groups, called Th1 and Th2, according to the “signature” cytokines they produce (Fig. 3.2). Th1 cells are characterized most constantly by the production of and interferon (IFN)γ, a cytokine with particular functions in activating cellmediated immunity. They also favor the production of IgG1 and IgG3 opsonizing and complement-fixing antibodies, thus being very useful for protection against intracellular parasites

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Th2

Th1

Signature cytokines

IFN-g

IL-4, IL-5, IL-13

IL-12 receptor b2-chain

+++

+/−

Chemokine receptors

CXCR3 CCR5

CCR3 CCR4 CXCR4 CCR8

Chandra protein

+

−

Costimulatory molecule ICOS

−

+

CRTH2

−

+

TIM (mice)

TIM-3

TIM-2

Signaling

STAT4 T-bet

STAT6 GATA3 c-Maf

Fig. 3.2 Phenotypic differences between Th1 and Th2 T cells. See text for further discussion and definition of abbreviations. For further information on Chandra protein, see Venkataraman et al. (2000).

(Abbas et al. 1996). On the other hand, Th2 T cells produce IL-4, IL-5, IL-9, and IL-13. These cytokines are also concerned with the activation of B cells and with the class of antibodies produced in immune responses. Thus, broadly, Th2 cells favor humoral immune responses, and are generally poorly protective against the majority of infectious agents, except perhaps some nematodes (Abbas et al. 1996). Th2type cytokines are also implicated in allergic and asthmatic inflammation, since these cytokines also influence the functions of effector leukocytes such as eosinophils and mast cells implicated in these diseases. Furthermore, IL-4 and IL-13 are the only two cytokines that can induce B-cell class switching to the production of IgE antibody, which is the basis of the atopic phenotype. The process of differentiation of naive CD4+ T cells into Th1 and Th2 cells is influenced by a number of factors, but the most important determinant is probably that of interaction with antigen-presenting cells, particularly dendritic cells. Further, the outcome of an encounter between a T cell and a dendritic cell appears to depend on maturation signals received by the dendritic cells at a relatively immature stage. Th1 differentiation appears to be activated as a result of production of IL-12 by dendritic cells during the process of presentation of antigenic peptides to naive T cells. IL-12 production is in turn stimulated by direct interaction of immature dendritic cells with microbial products. Dendritic cells and other immune leukocytes, including mast cells, T cells, and monocytes, express a series of pattern-recognition receptors,


of which the Toll-like receptors (TLRs) are best characterized in humans (Duez et al. 2006). These receptors recognize pathogen-associated molecular patterns (PAMPs) derived from microorganisms. So far, 10 TLRs have been described in humans. TLR1, TLR2, TLR4, TLR5, and TLR6 recognize constituents of extracellular bacterial pathogens. For example, TLR2, associated with TLR1 or TLR6, recognizes lipoproteins from the cell wall of Gram-negative bacteria, whereas TLR4 recognizes lipopolysaccharide from the same origin. TLR5 recognizes flagellin from flagellated bacteria. TLR3, TLR7, TLR8, and TLR9 are expressed intracellularly and recognize viral and bacterial nucleic acid fragments. TLRs signal through mitogen-activated protein kinase (MAPK), STAT1, and NF-κB. Once IL-12 production is established in dendritic cells as a result of exposure to PAMPs, it promotes Th1 differentiation of T cells by sustaining expression of the β2 chain of the IL-12 receptor (Smits et al. 2001). This receptor is therefore a “signature” cytokine receptor of Th1 cells (see Fig. 3.2). Molecules that recognize fragments of invading intracellular microorganisms should, through their actions on dendritic cells, promote Th1 T-cell differentiation, which in turn promotes cell-mediated immunity. It is less clear what initiates Th2 T-cell differentiation. While Th2-type cytokines such as IL-4 encourage propagation of established Th2 cells, there is no clear counterpart of IL-12 produced by dendritic cells that performs this function during Th2 cell differentiation. Some studies suggest that other receptors on dendritic cells may drive Th2 differentiation. For example, interaction of the Notch receptor with the Jagged ligand on dendritic cells has been suggested as a possible trigger for Th2 differentiation since it induces production of IL-4 by T cells (Amsen et al. 2004). In some parasitic infections, IL-4 may be produced by a still undefined cell type (non-T, non-B, c-kit+, FcεRI −) in response to IL-25 produced by macrophages or mast cells (Fallon et al. 2006), but the generality of this phenomenon is in doubt. TSLP is also a possible prototype candidate. While activation of dendritic cells by both TLRs and TSLP results in increased expression of MHC class II molecules, as well as the T-cell costimulatory molecules CD80, CD83 and CD86, TSLP in contrast to TLRs does not induce dendritic cells to produce IL-12 or any members of the IL-12 family of cytokines such as IL-23 or IL-27 (Soumelis et al. 2002). However, TSLP does induce expression of the TNF superfamily protein OX40 ligand (OX40L) on dendritic cells. A recent study has shown that OX40L signaling through its receptor OX40 on T cells directly induces Th2 lineage commitment by inducing the transcriptional regulatory factor NF-ATc1, which triggers IL-4 production and then IL-4-dependent transcription of GATA-3 (So et al. 2006). This process is overridden in the presence of IL-12, which may provide a mechanism for inhibition of Th2 cell differentiation in the presence of IL-12-inducing PAMPs, which is a possible mechanistic explanation for the hygiene hypothesis. This scenario suggests that OX40L represents the

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original critical polarizing signal for Th2 development and that IL-4, conventionally considered the trigger factor for Th2 development, plays a secondary, autocrine Th2-stabilizing function (Ito et al. 2005). TSLP expression has been documented within epidermal keratinocytes in lesional skin of patients with atopic dermatitis (Soumelis et al. 2002), while we have demonstrated its expression in the bronchial mucosa in human asthma (Ying et al. 2005). Triggers for its production in these compartments remain to be defined, although mast cells activated through engagement of the FcεRI receptor, for example through cross-linking by allergen, expressed TSLP (Soumelis et al. 2002), which could form a link between IgE-mediated allergic inflammation and the setting up of Th2 polarized inflammatory responses locally within target organs of allergic disease. Th1 and Th2 cells are characterized by differential expression of transcriptional regulators, reflecting the distinct pattern of cytokines they express. Th1-type cytokine expression is initiated by engagement of the T-cell receptor (TCR) with antigen by dendritic cells along with production of a STAT1 stimulatory signal, which is provided principally by IL-12 as mentioned above, but also by other members of the IL-12 cytokine family such as IL-27 and the interferons, receptors for which are expressed on T cells. STAT1 signaling induces production of the transcriptional regulator T-bet, which can be regarded as a “master regulator” of Th1-cell cytokine expression (Szabo et al. 2000; Lametschwandtner et al. 2004). In turn, T-bet upregulates expression of IFN-γ and the β2 receptor chain of IL-12 and the α chain of the receptor for IL-18, expression of which enables IL-12 signaling through STAT4 and responsiveness to IL-18. Th2 differentiation is induced by engagement of the TCR in concert with signaling through IL-4 or OX40, as described above. This in turn activates the STAT6 signaling pathway, causing low-level expression of the transcriptional regulatory factor GATA-3, which can be regarded as the “master regulator” of Th2 cell differentiation (Zheng & Flavell 1997). GATA-3 induces expres-

sion of the IL-4, IL-5, and IL-13 genes while suppressing factors critical to Th1 differentiation including STAT4 and the IL-12 receptor β chain (Zhu et al. 2004). A consequence of these mutually antagonistic signaling systems in Th1 and Th2 cells is that T-cell differentiation is somewhat plastic, and that Th1 cytokines inhibit the development of Th2 cells and vice versa. For example, Th2 responses can be shifted, at least in vitro, to a Th1 profile by antigen stimulation in the presence of IL-12, which seems to reflect persistent expression of the β2 chain of the IL-12 receptor even in polarized Th2 cells (Smits et al. 2001). Conversely, IL-4 inhibits the development of Th1 cells and shifts them to a less polarized phenotype (Skapenko et al. 2004). Chemokines, the receptors for which are differentially expressed on Th1 and Th2 cells (see below), can also affect T-cell polarization. For example, IP-10/CXCL10 promotes the production of Th1-type cytokines and inhibits that of Th2-type cytokines, while PF-4/CXCL4 does the reverse (Romagnani et al. 2005).

Th17 T cells The IL-17 family comprises six homologous cytokines (Table 3.1). The cytokines IL-17A, B, C, D, and F were identified through database searches and degenerative polymerase chain reaction (PCR) strategies (Aggarwal & Gurney 2003; Kawaguchi et al. 2004; Kolls & Linden 2004). IL-17E was independently identified and named IL-25 (Fort et al. 2001). IL-25 is least well related to the other members of the IL-17 family, showing only 16% identity with IL-17A at the primary amino acid sequence in humans. IL-17A and IL-17F are syntenic, with tightly linked expression in all species examined to date. The remaining family members each map to different chromosomes. The family as a group shows no sequence homology with other known human proteins. The IL-17 family is a cysteine knot family of homodimeric proteins, so named for their unusual pattern of inter- and intra-chain disulfide bonds. The receptors for these cytokines also form a

Table 3.1 The IL-17 cytokine family. Name

Alternative names

Cellular sources

Receptor

IL-17A

IL-17, CTLA-8

IL-17RA (IL-17R)

IL-17B IL-17C IL-17D IL-17E

CXI, NERF CX2 IL-27 IL-25

Th17 cells, CD8 T cells, NK cells, gd T cells, neutrophils ? ? ? CD4 T cells, B cells ? Th2 cells, eosinophils, mast cells

IL-17F

ML-1

Th17 cells, T cells, NK cells, gd T cells, neutrophils, mast cells, basophils

?, cellular sources/receptor(s) not yet clear.

52

? ? ? IL-17RB (IL-17RH1) (IL-25R) IL-17RA, IL-17RC (IL-17RL)

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unique family of proteins (see Table 3.1). IL-17R (or IL-17RA) is the founding member, along with IL-17RB (or IL-17RH1 or IL-25R), IL-17RD (or SEF or IL-17RLM), IL-17RC (or IL17RL), and IL-17RE. They are predicted to be single-pass transmembrane structures with extracellular amino-termini and large intracellular tails (Moseley et al. 2003). Only IL-17RA and IL-17RH1 have been shown to bind to the IL-17 family of cytokines. The cytokines IL-17A and IL-17F are produced by a recently described functional subset of T cells distinct from Th1 and Th2 cells called Th17 cells (Kolls & Linden 2004; Weaver et al. 2006). They are also produced by CD8 T cells, γδ T cells, natural killer (NK) cells, and neutrophils. The IL-17A and IL-17F genes are expressed coordinately as mentioned, and may thus represent “signature cytokines” for Th17 cells. The receptor for these cytokines, IL-17RA, is very widely expressed and they have been described as acting on epithelial and endothelial cells, fibroblasts, osteoblasts, and macrophages. Depending on the cellular target, these cytokines induce the production of colony-stimulating factors, CXC chemokines, metalloproteinases, and IL-6. They thus have important activities in the recruitment and activation of neutrophils (Oda et al. 2005). In contrast, IL-25 (IL-17E) induces the production of CC chemokines, such as CCL5 (RANTES) and CCL11 (eotaxin-1), as well as Th2 cytokines in appropriate target cells (Fort et al. 2001). It also acts on airway smooth muscle cells (Letuve et al. 2006). It is thus more implicated in allergic inflammation and the recruitment and mobilization of eosinophils. IL-17A has been implicated in limiting allergic inflammation in a mouse model of asthma by inhibiting the production of Th2-attracting chemokines such as eotaxin (CCL11) and TARC (CCL17) by dendritic cells. Its production in this model is in turn limited by the Th2 cytokine IL-4 (Schnyder-Candrian et al. 2006). Th17 cells were first discovered as a subset of T cells induced by Borrelia burgdorferi infection in mice (Infante-Duarte et al. 2000) producing IL-17A, granulocyte–macrophage colonystimulating factor (GM-CSF), TNF-α, and IL-6 but not IFN-γ or IL-4. It was later discovered that the subset is stimulated by IL-23, a member of the IL-12 family comprising the p40 chain of IL-12 and a unique p19 chain (Aggarwal et al. 2003). Th17 cells appear to play a particular role in certain models of autoimmune disease in mice (e.g., collagen-induced arthritis). They probably represent a distinct Th1- and Th2independent lineage of development from naive CD4 T-cell precursors, since Th17 development in mice is unimpaired in the presence of IFN-γ or T-bet deficiency (Park et al. 2005); indeed interferons, which activate STAT1-induced expression of T-bet and Th1 development, as well as IL-4, strongly inhibit Th17 development. Although IL-23 amplifies Th17 cell development, it is not absolutely required for Th17 commitment, and does act directly on naive CD4 T cells (Iwakura & Ishigame 2006). Instead, it appears that the cytokines TGFβ and IL-6 act cooperatively and nonredundantly to achieve


Th17 commitment. TGF-β appears to be a critical factor for Th17 commitment, while IL-6 acts to deviate TGF-β-driven development of FoxP3-expressing T regulatory cells toward Th17 (Bettelli et al. 2006). Thus, antagonistic effects of TGF-β/ IL-6 versus IFN-γ signaling early in activation of naive T cells deviates lineage development toward Th17 or Th1 respectively, with concomitant upregulation of IL-23 or IL-12 receptor components, respectively. This requirement for TGF-β in Th17 development is shared with adaptive T regulatory cells, which default to FoxP3 induction in the absence of IL-6, induced by pathogen-induced activation of innate immune cells by TLRs. The orphan nuclear receptor retinoic orphan receptor (ROR)γT may be a “master regulator” of Th17 development (Ivanov et al. 2006), although its putative ligand remains unidentified. The cytokine IL-27, another member of the IL-12 family that is a heterodimer with chains homologous to the IL-12 p40 and p35 chains and which is produced by cells similar to those producing IL-12 and IL-23 (dendritic cells, macrophages), seems to play an important role in curbing Th17 responses by limiting development of Th17 effectors. Its receptor, a heterodimer composed of a unique IL-27R α chain and gp130, shared by several members of the IL-6 receptor family, is expressed on naive T cells (Stumhofer et al. 2006). It acts directly on naive T cells to suppress the development of Th17 effectors through a STAT1-dependent, T-bet-independent mechanism. IL-25 (IL-17E) is produced by memory Th2 cells and amplifies Th2 responses. It is not detected in naive T cells, Th1 cells, B cells, dendritic cells, mast cells, or endothelial cells. In vivo its expression appears most abundant in mucosal tissues (Fort et al. 2001). Administration of IL-25 to mice recapitulates many of the features of Th2-type immunity, at least partly by induction of Th2-type cytokines such as IL-4, IL-5 and IL-13, along with elevated serum IgE, IgG1 and IgA, epithelial hyperplasia, goblet cell hypertrophy, and eosinophilia. Reports suggest that these effects occur independently of lymphocytes (Fort et al. 2001; Hurst et al. 2002) and may reflect the actions of IL-25 on an as-yet unidentified innate immune cell of hematopoietic origin (Hurst et al. 2002). Thus, IL-25 may amplify Th2-type inflammation by acting on both adaptive (memory Th2) and innate immune cells.

T-cell costimulatory molecules When CD4 T cells interact with antigenic epitopes presented on MHC class II molecules by antigen-presenting cells such as dendritic cells, their function is critically regulated not only by production of cytokines by the dendritic cells but also by two-way communication between the dendritic cells and the T cells involving surface receptors called costimulatory molecules. T-cell costimulatory molecules may modify T-cell function in a positive or negative fashion. Recently a series of these molecules has been identified (reviewed by Kroczek et al. 2004). Many of these molecules are constitutively expressed (Table 3.2), but others are expressed de novo

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Table 3.2 T-cell costimulatory molecules and their receptors.

CD28 CD27 HVEM BTLA† ICOS‡ OX40 (CD134)‡ CD30‡ 4-1BB (CD137) SLAM† CTLA-4 (CD152) PD-1

Naive T cells

Activated Th2 cells

Memory T cells

Other cells*

+++ ++ +++ +++ − − − − + − −

+++ +++ ++ +++ +++ +++ ++ +++ +++ +++ ++

+++ +++ +++ ++ + − − − − − −

Yes Yes Yes Yes No No Yes Yes No No No

Counterreceptor(s)

Action

CD80, CD86 ? BTLA HVEM ICOS-L OX40L ? 4-1BB-L

+ + +? − + + + + + − −?

CD80, CD86 PD-L1, PD-L2

* Cells other than T cells. † More expressed on Th1 cells. ‡ More expressed on Th2 cells. HVEM, herpesvirus entry mediator; BTLA, B- and T-lymphocyte attenuator; CTLA-4, cytotoxic T-lymphocyte antigen-4; PD-1, programmed death-1; PD-L1/2, PD-1 ligand 1/2; SLAM, signaling lymphocyte activation molecule; ICOS, inducible costimulator; +, stimulatory; −, inhibitory.

on T-cell activation. Several costimulatory molecules that are constitutively expressed (e.g., CD40L interacting with CD40 on B cells and dendritic cells, and CD28 interacting with CD80 and CD86 on dendritic cells) appear to play some role in interactions of all T cells with antigen-presenting cells, and only CD40L is relatively abundant on Th2-type effector T cells. This made it an attractive target for the inhibition of Th2 cell responses until it was discovered that CD40L is also expressed on platelets and that administration of anti-CD40L monoclonal antibodies causes thromboembolic episodes (Kawai et al. 2000). There is now great interest in the roles of induced costimulatory molecules in T-cell activation. These latter comprise the positive costimulators ICOS, OX40, CD30, 4-1BB, and SLAM and the negative costimulators CTLA-4 and PD-1. While positive costimulatory molecules are possible therapeutic targets in strategies to inhibit inflammation, negative costimulators are less attractive because studies in animals suggest that blockade of these molecules may result in unbridled T-cell proliferation. Of the positive costimulators, ICOS, CD30, OX40, and possibly 4-1BB are particularly involved in the activation of Th2 effector cells. In addition to driving Th2-type T-cell responses (Gonzalo et al. 2001), ICOS substantially contributes to the induction of antibodies, including IgE, by B cells and is absolutely required for the generation of memory B cells (Grimbacher et al. 2003). OX40 is particularly involved in regulating longterm T-cell survival and the generation of T-cell immunologic memory (Croft 2003) (see also discussion above). The roles of CD30 and 4-1BB are less well defined but can result in Th2 cell differentiation (Croft 2003; Tarkowski 2003). The

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relative contribution of the interactions of these molecules with their respective ligands to ongoing Th2 cytokine production is not well understood. Their precise contribution to T-cell activation in human allergy and asthma remains to be defined. Furthermore, the contributions of particular molecules may vary according to the nature of the antigenpresenting cell and its environment. For example, costimulation through CD86 appears to be important in the function of alveolar macrophages in presenting allergens to T cells in atopic asthmatics (Larche et al. 1998), whereas with respiratory tract dendritic cells and whole bronchial biopsy cultures, both CD80 and CD86 appear to be implicated (Jaffar et al. 1999; Faith et al. 2005). There may in some circumstances be redundancy in signaling, requiring simultaneous blockade of several molecules. Blockade of ICOS and OX40 interactions with their ligands are particularly attractive therapeutic strategies, at least in theory, in asthmatic and allergic inflammation, since the expression of these ligands is limited to activated T cells with a bias to Th2 differentiation (Lane 2000). It is conceivable that short-term, accurately targeted blockade of these molecules could not only abolish Th2 effector T-cell activation, with concomitant reduction of help for allergen-specific IgE antibody synthesis by B cells, but also substantially ablate memory cells, creating a sustained effect. These are exciting prospects for the future. A caveat is that T regulatory responses may also depend on costimulatory molecule signaling (Lohning et al. 2003). Animal models of asthma and allergy have shed some further light on the possible roles of costimulatory molecules in allergic inflammation. Mice lacking ICOS show marked

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defects in humoral (especially IgG1 and IgE) responses and Th2 cytokine expression (Dong et al. 2001). ICOS blockade reduced lung inflammation and airway hyperresponsiveness following adoptive transfer of highly polarized Th2 cells into naive mice and was effective up to 21 days following allergen priming of naive animals (Coyle et al. 2000), suggesting that ICOS is important for maintaining Th2 effector function but not differentiation. In contrast, blockade of CD28–CD80/86 interactions after priming had no effect on the Th2 effector response. As suggested in humans, however, ICOS may also be important in the development of allergen-specific, IL-10producing adaptive T regulatory cells (Akbari et al. 2002). In summary, there is still much to be learnt about the influences of costimulatory molecules on T-cell activation. It seems likely that a balance between stimulatory and inhibitory signals is required for effective T-cell responses and for maintaining T-cell tolerance. It is not clear whether these possible interactions provide redundant positive and negative regulatory signals or whether there is some hierarchy in the organization of the signals. CD28 and ICOS synergize to promote T-cell activation, with CD28 having a predominant role in initial T-cell activation and ICOS regulating differentiated T cells. Of the multiple negative inhibitory signals provided through these interactions, CTLA-4 is the predominant one, but it has synergistic nonredundant interactions with those of PD-1 (Oflazoglu et al. 2004; Radhakrishnan et al. 2005).

gd T cells As discussed above, early T-cell differentiation in the thymus gives rise to CD8+ and CD4+ T cells which recognize peptides presented with MHC class I and II molecules respectively. In addition, a subset of T cells arising in the thymus recognizes a limited range of antigens in a non-MHC-restricted fashion. These cells have antigen receptors composed of distinct polypeptide chains called γ and δ, in contrast to the α and β chains used by the majority of MHC-restricted T cells. In humans, γδ T cells comprise 1–5% of circulating T cells. They have been implicated (mostly from animal experiments) in specific primary immune responses, immunoregulation, and tumor surveillance (Hayday & Tigelaar 2003). They are particularly abundant at mucosal surfaces, especially of the skin and gut, and their tissue localization appears to be dependent on the particular γ and δ TCR genes they express (Pennington et al. 2005). γδ T cells do not pass through a “double-positive” CD4/CD8 stage during maturation, but appear to develop from double-negative cells in the thymus. Since physiologic ligands for their receptors have so far been poorly characterized, it is not clear if and how they are positively and negatively selected. Mature γδ T cells are activated in the presence of products of some microorganisms such as Mycoplasma and cell wall components of Mycobacterium, as well as self glycosphingolipids induced in antigen-presenting cells in association with a variety of bacterial, and possibly also viral,


infections (De Libero et al. 2005). They also recognize self molecules produced by cellular “stress,” such as CD1 and F1ATP synthase (Scotet et al. 2005). In addition to antigen receptors composed of γ and δ chains, these cells also express innate cell-surface receptors such as TLRs, and under certain conditions can act as antigen-presenting cells, expressing the lymph node homing receptor CCR7, MHC class II, and costimulatory molecules such as CD40, CD80 and CD86 (Brandes et al. 2005). They also induce dendritic cell maturation (Conti et al. 2005). A recent study (Jin et al. 2005) suggests that a subset of γδ cells expressing the Vγ4 TCR gene can suppress airway hyperresponsiveness in an ovalbuminchallenge murine model of asthma. Since these γδ cells are not antigen-specific, they are perhaps activated by antigenpresenting cells such as dendritic cells.

Natural killer T cells NKT cells are a specialized subset of thymus-derived cells that express conventional αβ TCR chains as well as NK-specific markers such as NK1.1. In humans, a large majority of these cells (termed type I, or iNKT, or classical NKT cells) express an invariant TCR comprising the Vα24/Jα18 TCR α chain paired with a Vβ8 or Vβ2 β chain, hence their name “invariant” NK T cells. These cells are restricted by the nonpolymorphic MHC class I-like protein CD1d. CD1d is widely expressed in the body by mucosal epithelial cells, hepatocytes, T cells, B cells, macrophages, and dendritic cells. Like iNKT cells, type II NKT cells (or noninvariant NKT cells) are CD1d restricted, but have a more diverse TCR repertoire. These cells have been implicated in the pathogenesis of some human diseases such as ulcerative colitis (Fuss 2004). Type III NKT cells express a diverse repertoire of T-cell antigen receptors restricted by conventional MHC class I and II molecules. Evidence suggests that NKT cells develop from committed, double CD4+CD8+ T-cell precursors by a mechanism that is not yet clear. Many inherited diseases that affect T-cell development also affect NKT cells, such as X-linked severe combined immunodeficiency (mutation of CD132, the common γ-chain receptor of IL-2, IL-4, IL-7, and IL-15) (Jordan et al. 2004). Following expression of the TCR, CD1d-restricted cells can be identified before the appearance of NKT cell-specific markers. The most immature cells lack the surface markers DX5 and NK1.1. Mature NKT cells are divided almost equally into DX5+/NK1.1+ and DX5–/NK1.1+ cells (Gadue & Stein 2002). In humans, most iNKT cells also express CD4, while a small proportion expresses CD8 and a more substantial proportion expresses neither CD4 nor CD8. NKT cells are distributed in the same way as conventional T cells. Their most striking effector function is to produce large quantities of the cytokines IL-2, IL-4, IFN-γ, and TNF-α following activation. They are able to do this because they accumulate cytokine-specific mRNA in the resting state (Stetson et al. 2003). The invariant receptor of NKT cells is unique in its interaction with the nonclassical MHC molecule CD1d. This

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molecule has a deep, hydrophobic, antigen-binding pocket that allows it to present lipid and glycolipid antigens rather than conventional peptide antigens. Natural ligands for NKT cell receptors are poorly characterized, although some have been described (Brigl & Brenner 2004; Zhou et al. 2004). A synthetic glycolipid, α-galactosylceramide (α-GalCer), derived from marine sponges has been identified as a surrogate NKT cell ligand in humans and mice and has been useful not only for functional studies but for identifying these cells by their binding to α-GalCer complexes with CD1d tetramers. Stimulation of NKT cells with α-GalCer induces a proliferative response, expression of activation molecules, elevated cytotoxic activity, and production of large amounts of cytokines. As with T cells, NKT cell cytokine production can be skewed by external influences. For example, stimulation through the NK1.1 receptor results in high production of IFN-γ but not IL-4. Exogenous cytokines also exert an influence. For example, exogenous IL-7 encourages IL-4 production, whereas exogenous IL-12 favors IFN-γ (Godfrey et al. 2000). NKT cells can also exhibit cytotoxic activities similar to those of CD8+ T cells. Studies in humans suggest that this may be a particular property of subsets of NKT cells. In vitro, activation of NKT cells by α-GalCer leads to multiple secondary effects on other cells such as NK cells, dendritic cells, and subsets of conventional T and B cells, probably through their production of cytokines. The rapid response of NKT cells to nonpeptide ligands suggests that they play a role in innate rather than adaptive immunity. They do however regulate adaptive immune responses: in mouse models they have been implicated in regulating adaptive immune responses against a number of viral infections, tumor immunity and autoimmune diseases (Mercer et al. 2005). Of particular interest here is a possible role in asthma. CD1d knockout mice, which lack NKT cells, showed markedly attenuated airway hyperresponsiveness and inflammation in a model of allergen sensitization and challenge (Bilenki et al. 2004). Furthermore, activation of NKT cells using α-GalCer exacerbated airways inflammation and bronchial hyperreactivity in this model, an effect that was not seen in mice lacking CD1d. Adoptive transfer of NKT cells from mice deficient in both IL-4 and IL-13 to Jα18-deleted (and therefore iNKT deficient) mice failed to restore the induction of airway hyperresponsiveness following allergen challenge (Akbari et al. 2003), suggesting that production of IL-4 and/or IL-13 by NKT cells is required for the development of airway hyperresponsiveness in this situation. This observation is remarkable since allergen-specific Th2 cells as well as IgE responses develop normally in CD1d or Jα18 knockout mice, but at least in these experiments such normal T-cell responses appear to be insufficient for the development of airway hyperreactivity. Consideration of an additional role for NKT cells in the development of organ-specific disease despite normal Th2 and IgE responses to allergens might throw some light on the

56

age-old question of why only some subjects sensitized to allergens develop allergic diseases such as asthma and rhinitis in the target organs. Using CD1d tetramers loaded with α-GalCer and PCR analysis of the expression of the invariant TCR of NKT cells, it has recently been suggested that, in human asthma, approximately 60% of airway CD4+ cells are not conventional MHC class II restricted T cells, as has hitherto been assumed, but NKT cells expressing the invariant Vα24/Jα18 receptor (Akbari et al. 2006). These cells exhibited a cytokine expression profile similar to that of conventional Th2 cells. This finding was striking considering that NKT cells comprise 105 molecules/cell) of FcεRI (Prussin & Metcalfe 2006). The presence of IgE-secreting plasma cells in the same tissues (KleinJan et al. 2000; Smurthwaite et al. 2001), and/or the transport of IgE into tissues by CD23-bearing cells, may contribute to this high expression, because IgE protects its receptors against proteolytic cleavage at the cell surface (Lee et al. 1987; MacGlashan 2005). For all these reasons IgE is well adapted to its barrier function in tissues. CD23 expressed on B cells functions in IgE antibodydependent antigen presentation (Kehry & Yamashita 1989; Pirron et al. 1990; Heyman et al. 1993; van der Heijden et al. 1993; Squire et al. 1994; Gould et al. 1997), while CD23 expressed on monocytes and eosinophils functions in IgE antibody cell-mediated cytotoxicity against tumor cells (Karagiannis et al. 2007). CD23 also participates in the transport of IgE to

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Immunoglobulin Gene Organization and Expression and Regulation of IgE the mature mRNA. The 5′ exon, encoding the leader sequence of the mature immunoglobulin chain, and an exon of 300 bp, encoding the first 100 amino acids of the rearranged V region, lie downstream of the promoter. Each D segment also possesses a promoter, driving germline transcription, upstream of a single exon of variable length (∼ 15 bp). Each functional J segment comprises a single exon, ending in a 3′ RNA splice signal. Each C germline gene contains an exon corresponding to each immunoglobulin domain or hinge region, followed by one or two exons encoding the membrane and cytoplasmic sequences of the immunoglobulin chains and an RNA chain termination signal. Each CH gene, except δ, also has a promoter, driving germline transcription, upstream of a short germline or intervening (I) exon, followed by a GC-rich switch (S) region 5′ of the CH exons. The organization of the constant regions differs between mouse and human (Fig. 6.4). In the mouse, the four γ genes are clustered upstream of the single ε and α gene, while in the human two γ-γ-ε-α clusters appear to have arisen from a duplication event during evolution. Transcriptional enhancers are characteristically positioned in each of the three loci in the JC intron (intron enhancer or Ei) and downstream of the last membrane exon (3′ enhancers

sites of allergic inflammation (Yu et al. 2001, 2003). We discuss the function of CD23 in the homeostasis of IgE in a separate section.

Immunoglobulin gene organization The genetic loci encoding the κ, λ, and H chains are on chromosomes 2, 22, and 14, respectively, in humans and on chromosomes 6, 16, and 12, respectively, in the mouse. Each has a characteristic arrangement of genetic elements that must be properly recombined in B cells for immunoglobulin expression. The coding sequences, exons, separated by introns, all reside on one strand (the template or “sense” strand) of the DNA. We refer to the unrearranged genes in the immunoglobulin loci as germline genes and those at various stages of somatic recombination in B cells as rearranged genes. In the H-chain and κ-chain loci all the separate elements, V, (D), J and C, are found in discrete clusters (Fig. 6.4). In the λ locus, however, each C gene is juxtaposed with its own J gene segment. Each V segment consists of a promoter upstream from the coding sequence, which drives the production of germline transcripts and, after successful recombination, of l light-chain locus L1 Vl1

L2 Vl2

L Vl-30

Jl1

Cl1

Jl2

Cl2

Jl4

Cl4

(a) k light-chain locus L1 Vk1 L2 Vk2

L3 Vk-3

L Vk-40

Jk1–5

Ck

(b) Heavy-chain locus L1 VH1 L2 VH2 Fig. 6.4 Genomic organization of the mammalian l and k L-chain and H-chain immunoglobulin loci. The immunoglobulin loci contain V, [D], and J gene segments linked to C gene segments. A leader (L) exon lies upstream from each V-gene exon. In the l locus (a) each J gene segment is positioned upstream of one of four separate Cl genes. In the k locus (b) the J gene segments are organized in a tandem array upstream of a single Ck exon. In the H-chain locus (c) the V, D, and J gene segments are organized in tandem arrays upstream of Cm. The JC intron enhancer (Ei) and 3′ enhancers (3′E) are portrayed by ovals (D and E). The CH regions of the human (d) and mouse (e) differ in the number and arrangement of the isotypes. The Ce locus is expanded in (D) to illustrate the relative positions of the I exon promoter, the I exon (Ie), the switch region (Se), the four CH exons (Ce1–4), and the two membrane exons (M1 and M2).

L3 VH-3

LH VH-50

DH1–25

JH1–6

Cm

(c) Human IgH locus Ei (Em) enhancer V genes

D J genes genes

Cm Cd

3’E (Ca1) enhancer Cg3 Cg1

Ye

Ca1

Yg

3’E (Ca2) enhancer Cg2 Cg4 Ce

Ca2

(d)

Germline Ie promoter

Se

Ce1–4 M1,2

Mouse IgH locus Ei (Em) enhancer V genes

D genes

J genes

Cm Cd

3’E (Ca) enhancer Cg3 Cg1 Cg2b Cg2a Ce

Ca

(e)

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Vl


CACAGTG Heptamer GTGTCAC

Vk

VH

GGTTTTTGT CACTGTG Nonamer 12 Heptamer CCAAAAACA GTGACAC

ACAAAAACC Nonamer TGTTTTTGG

23

12

23

23

23

D

12

V(D)J recombination Each V gene segment is followed by a recombination signal sequence (RSS), comprising a conserved heptamer (consensus sequence 5′-CACAGTG-3′) and nonamer (consensus sequence 5′-ACAAAAACC-3′), separated by a relatively nonL2 V2

l chain

Jk

k chain

JH

H chain

12

or 3′Es). In the human, an enhancer is located on the 3′ side of each α chain in the duplicated clusters (Fig. 6.4). The 50 or 51 V-gene segments in the human fall into seven families, which vary widely in size, and generally contribute in roughly corresponding proportions to the antibody repertoire. Notable exceptions occur when B-cell superantigens bind to the common framework regions of particular VL or VH families (see below).

L1 V1

Jl

Ln Vn

Fig. 6.5 Alignment of sequences for V(D)J recombination. The V and J (L chain), or the V, D and J (H chain), sequences are flanked by the required specific recombination signal sequences, separated by spacers of the required length, for V(D)J recombination.

conserved spacer of either 12 or 23 bp. Similarly, each J gene segment is preceded by an RSS, while D gene segments are flanked by RSSs on both sides; the orientation of the RSSs and, crucially, the size of the spacers present at each of the loci are shown in Fig. 6.5. RAG function requires that for recombination to occur between two gene segments, one RSS must have a 12-bp and the other a 23-bp spacer, a restriction referred to as the “12/23 rule” (Fig. 6.5). This rule ensures that one of the V, D, and J segments from each family are correctly recombined to form the H-chain V-region coding sequence, linked to Cμ, and one V and one J segment are recombined to form the L-chain V-region coding sequence, linked to Cκ or Cλ (Fig. 6.6). The intervening sequences are deleted from the chromosome and the ends are linked to Jl1

Cl1

Ln V2

Vn L2

L1 V1

Jl1

Cl1

Ln V2

Vn

L2 L1 V1

Jl1

L1 V1 Jl1

124

Cl1

Cl1

Fig. 6.6 Recombination of V and J gene segments at the l locus. The 23-bp spacer downstream of the ¨V gene segment (→) and 12-bp spacer upstream of the J segment (←) are apposed for synapsis and the intervening sequence is deleted. The ends of the deleted sequence are linked to form a circle, and V and J gene segments are linked to form the expressed gene.

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form circles. This DNA cannot be replicated during cell division and is diluted out during cell proliferation. The RAG protein complex (RAG1/RAG2) introduces DNA double-strand breaks specifically where two coding segments meet their flanking RSSs (Gellert 2002) (Fig. 6.7). Following cleavage of the DNA, the coding ends are splayed out and joined together to form a hairpin. An enzyme called Artemis nicks the hairpin at random positions and the staggered ends are then repaired. This seemingly unnecessary nicking and repair in fact serves an important function, for during their single-stranded state the junctional sequences are diversified. At the VJ and DJ junctions an exonuclease can erode the overhanging sequence. Instead, or in addition, two types of nucleotide insertions, templated and nontemplated, may occur. The enzyme terminal deoxynucleotidyltransferase (TdT) may add nontemplated nucleotides to form tracts of up to 15 nucleotides (so-called N regions). Alternatively, templated addition of nucleotides may occur on the recessed

RAG1/RAG2 binds to the 12 bp spacer

DNA–RAG complex recruits the 23 bp spacer

The DNA is cleaved and the complementary strands are joined to form hairpins

The Ku70/Ku80–DNA PKcs complex binds to the hairpins, and the DNA is cut by Artemis and modified by TdT and an exonuclease

XRCC4 repairs the DNA and DNA ligase IV links the coding and RSSs together

Coding joint

Signal joint

Fig. 6.7 Mechanism of VJ recombination. The RAG1/RAG2 complex binds to the recombination signal sequences (RSSs) associated with the 12-bp spacers 5′ of the J gene segment (shaded) and recruits the RSS associated with the 23-bp spacers 3′ of the V gene segments. DNA is cleaved and broken ends of coding regions are linked to form “hairpins,” which are cleaved at random positions by Artemis. Nucleotides are then added by TdT and subtracted by an endonuclease, leading to junctional diversity in the rearranged DNA. Meanwhile Ku70/80 apposes the coding ends, which are repaired by XRCC4 to provide blunt ends for ligation by DNA ligase IV. See text for definition of other abbreviations.

strand to form short palindromic (P) sequences, complementary to the overhang. Junctional diversity affords a 100-fold amplification of combinatorial diversity (Janeway & Travers 2003). The junctional sequences of the immunoglobulin genes uniquely identify a B-cell clone, comprising all the B cells descended from a common B-cell progenitor and offer a convenient means of tracing the ancestry of a B-cell clone, as exemplified in studies of IgE (Coker et al. 2003). The processed DNA ends are held together (synapsed) prior to recombination through the “nonhomologous endjoining” (NHEJ) pathway, involving the DNA end-binding protein complex, Ku70/Ku80, DNA phosphokinase catalytic subunit (DNA-PKcs) and the general DNA repair proteins, X-ray repair cross-complementing protein 4 (XRCC4) and DNA ligase IV (Fig. 6.7). Synapsis could well be a function of the DNA end-binding Ku70/Ku80 protein complex (Jones et al. 2001). Finally, DNA-PK, XRCC4, and DNA ligase IV repair the ends of the DNA and catalyze formation of the phosphodiester bonds. As mentioned above, rearrangement of the H chain precedes that of the L chain, and that of the κ chain precedes that of the λ chain. In the H-chain locus, DJ recombination occurs on both alleles and precedes V-DJ recombination, which is restricted to the active allele. Transcription from the D-region and V-region promoters must occur before each stage of recombination. These sequential events are regulated through epigenetic mechanisms, as briefly outlined in a later section. The ATG translation initiation codon at the 5′ end of the V exon is fixed within a triplet-reading frame. Since the precise site of recombination varies in the creation of junctional diversity, only one of three recombination events in the L- and H-chain locus leads to the synthesis of a functional immunoglobulin chain. Receptor editing can rescue B cells with out-of-frame rearrangements and also those that express autoantibodies (see below). If this also fails, recombination is allowed to proceed on the inactive allele. If recombination is unsuccessful on both alleles, the B cell undergoes apoptosis. “Allelic exclusion” is the mechanism by which the B cell is prevented from expressing two different immunoglobulins (one from each chromosome), one of which could be an antibody and the other, obviously disadvantageous, an autoantibody. The current state of knowledge about the mechanism and control of V(D)J recombination is surveyed in several excellent reviews (Gellert 2002; Oettinger 2004; Cobb et al. 2006; Jung et al. 2006; Lieber et al. 2006).

Receptor editing and revision V(D)J recombination generates the combinatorial and junctional diversity of the immunoglobulins synthesized in the primary B-cell repertoire, prior to receptor editing or deletion of clones in the bone marrow. Receptor editing (RE) in naive B cells in the bone marrow, or receptor revision (RR) – the term assigned to a similar process in antigen-experienced B cells in the periphery – refers to the process by which

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secondary V(D)J rearrangements in the expressed immunoglobulin gene can replace a nonfunctional or autoreactive V-region exon by a new one, thereby changing antigen specificity (Nemazee 2006). This process is triggered by either insufficient or excessive signaling through the antigen receptor. As we have noted, 40– 60% of the B cells resulting from primary V(D)J rearrangements are thought to express autoantibodies. RR occurs in the germinal centers of lymphoid tissue and also, significantly, in tertiary lymphoid tissue, formed de novo at sites of inflammation such as the target organs of autoimmunity (Armengol et al. 2001; Aloisi & Pujol-Borrell 2006) and allergy (P. Takhar et al. 2007). RE and RR are carried out by RAG proteins, reexpressed in those sites. Editing or revision at L-chain V-region genes occurs more frequently than in H-chain V-region genes, which is readily understood: following VJ recombination, upstream V gene elements and downstream J elements that obey the 12/23 rule remain in the chromosome and can often undergo further RAG-mediated secondary rearrangement, even though all the downstream V gene elements and upstream J gene elements are deleted. However, in the H-chain locus, the recombination of D with V on one side and J on the other eliminates all the other D elements and the RSSs of the remaining one. Nevertheless, a cryptic heptamer, highly conserved in evolution, is present in the bodies of VH gene elements, close to the ends that are joined to the functional D elements (Zhang 2007). These can undergo RAG-mediated recombination between upstream V genes and the preexisting DJ cassette. It is estimated that 5 –10% of the VDJ regions in the normal human B-cell repertoire have resulted from VH replacement events. In both the revised H- and Lchain V regions, the clonotypic CDR3 region, formed in the primary V(D)J rearrangement, is conserved. This allows clones with a history of RE and RR to be identified by cDNA sequencing. RR and RE occur preferentially on the rearranged allele, and so maintain allelic exclusion.

Somatic hypermutation SHM further diversifies the antibody repertoire and leads to affinity maturation, triggered in naive and memory B cells by antigens. Mutations are introduced into the V regions of the expressed immunoglobulin genes at a rate of 10 −3 per bp per cell division, around 100 times the usual frequency in somatic cells (Odegard & Schatz 2006; Di Noia & Neuberger 2007). Follicular B cells also proliferate very rapidly, dividing on average every 6 hours. Rapid proliferation may expose naked DNA or allow remodeling of the chromatin, increasing the accessibility of the DNA to SHM. The mutations introduced by SHM are predominantly point mutations, targeted to the three CDRs in the V regions of the expressed H- and L-chain genes. This is at least partly due to the preference of AID for deaminating cytidines in the sequence (A or T)(A or G)C (abbreviated to WRC, where W denotes A or T and R

126

denotes a purine nucleotide), which is more prevalent in the CDRs than in the framework regions. This sequence motif is nonetheless abundant in other parts of the genome. AID introduces somatic mutations into only a few other genes, including those for BCL-6, FAS, and B29. The mechanism by which AID is targeted to the immunoglobulin genes is not fully understood. The boundaries of the sequence targeted by AID in the V region are well defined, beginning at the 5′-end of the coding region, 100–200 bp from the transcriptional start site, and terminating after 1–2 kb, within the 3′ intervening sequence, excluding the C-region exons (Longerich et al. 2005). These are the regions that are highly transcribed during V(D)J recombination. Promoters are crucially present upstream of each of the elements, since transcription is required for SHM. Transcription may promote SHM by exposing singlestranded DNA during the action of RNA polymerase. AID is a specific single-stranded DNA deaminase, which can act on stretches of DNA as short as nine nucleotides (Yu et al. 2004). Replication protein A (RPA), a single-stranded DNAbinding (Chaudhuri et al. 2004) protein that binds to phosphorylated AID, may guide the enzyme to its substrate in the cell nucleus. AID contains both a nuclear-localization and a nuclear export sequence, but is restricted to the cytoplasm in the absence of cell activation. On the induction of SHM (or CSR) it is phosphorylated by protein kinase A and translocated to the nucleus, where it is sequested by RPA (Basu et al. 2005). AID homolog, ABOBEC1, is an RNA-editing protein that introduces a point mutation in the mRNA for apolipoprotein B. APOBEC1 recruits an accessory protein to locate this target, so AID may require an analogous factor to find its targets in immunoglobulin genes. Mutagenesis of AID itself has allowed the identification of discrete positions in the protein sequence required for SHM or CSR; these lie on either side of the catalytic center (Chaudhuri & Alt 2004) and could be binding sites for accessory proteins implicated in SHM or CSR. AID acts by deaminating cytidine (C) residues in DNA to uridine (U). Uridine occurs of course in RNA, but not DNA, where it is recognized as an error that requires repair. The repair process can proceed along several alternative pathways, each of which results in a characteristic spectrum of mutations or restores the wild-type C (Fig. 6.8). Replication over the U–G lesion leads to C→T transitions; one daughter cell will have a wild-type C–G pair, the other will have a U–A pair (yielding T–A pairs in subsequent generations). Excision of the U residue by uridine nucleotide glycolase (UNG) will give rise to an abasic site, replication over which could yield either transition or transversion mutations at C–G pairs, depending on which nucleotide is inserted opposite the abasic site. The abasic site formed by UNG-mediated excision of a U residue could form a substrate for apyrimidinic/apurinic acid endonuclease, which can nick the DNA strand that contains the abasic site and allow polymerase-mediated repair by

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Transitions at C-G T

T

A

A

C

T

G

A

C

T

G

A AID

DNA replication

U

T

G

A

T

G

A

T

T

A

A

A

T

T

A

G

T

C

A

Mutagenic patch repair

UNG

Transitions and transversions at C-G C

MSH2-MSH6

DNA synthesis

T G

MMR

A

APE

T G

A

Repair

C

T

G

A

Mutation of C-G pairs by deamination of C to U

Mutations mainly at A-T pairs

Fig. 6.8 Pathways in the resolution of AID-mediated lesions in SHM. AIDmediated deamination of C to U generates a lesion comprising a mispairing of U with G. Replication over this U–G lesion leads to C→T transitions; one daughter cell will have a wild-type C–G pair, the other will have a U–A pair (yielding T–A pairs in subsequent generations). Excision of the U residue by UNG will give rise to an abasic site, replication over which could yield either transition or transversion mutations at C–G pairs, depending on which nucleotide is inserted opposite the abasic site. The abasic site formed by

UNG-mediated excision of a U residue could also form a substrate for apyrimidinic/apurinic acid endonuclease (APE), which can nick the DNA strand that contains the abasic site and allow polymerase-mediated repair by the conventional base excision repair pathway (no mutation). Alternatively, U–G lesions can be processed by a form of mutagenic patch repair, involving Exo1, MSH2 and MSH6, and DNA polymerase h; this generates mutations at mainly A–T pairs near the initiating U–G lesion. MMR, mismatch repair. See text for definition of other abbreviations.

the conventional base excision repair (BER) pathway, restoring the original DNA sequence. More recently, the MRE11 (meiotic recombination 11 homolog)/RAD50/Nijmegen breakage syndrome 1 (NBS), or so-called MRN protein complex, has been implicated in cleavage at the abasic site. An errorprone DNA polymerase (e.g., η) is then required to insert any of the four nucleotides in place of the original C. In place of UNG, the U–G mismatch can recruit the mismatch repair machinery, which triggers short-patch mutagenic DNA repair, creating mutations at mainly A–T pairs near the initiating U–G lesion. The proteins known to be involved in this pathway are exonuclease 1 (Exo1), the E coli MutantS homologs, MSH2 and MSH6, and DNA polymerase η. The present state of knowledge on the mechanism of SHM is reviewed in Li et al. (2004a), Odegard and Schatz (2006), and Di Noia and Neuberger (2007).

Class-switch recombination The first immunoglobulin classes to appear in naive B cells in the bone marrow and the circulation are membrane-bound IgM and IgD. These are expressed by the synthesis of a common mRNA precursor, with the VDJ region spliced to either Cμ or Cδ in the mature mRNA. CSR is required for the expression of the other H-chain isotypes (Chaudhuri & Alt 2004). This involves a nonhomologous recombination event between the switch regions of two CH genes, one between the expressed VDJ and CH regions (donor) and the other upstream of the new CH gene (acceptor). This process creates a composite switch junction containing sequences from both donor and acceptor switch regions. The intervening DNA segment is looped out and its two ends are joined to form a switch circle (Fig. 6.9). Linkage of the VDJ region and the intron enhancer (Ei) to the new CH gene cassette drives transcription

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Immunology of the Allergic Response Sm

VDJ

Cm

Cd

Ie

Se

Ce1–Ce4

Poly A M1 M2 Poly A

Sm-Se ligation CSR Cd VDJ

Sm

Se

Ce1–Ce4

Poly A M1 M2 Poly A

+ Ie

Transcription

VDJ

Sm

Se

Ce1–Ce4

M1 M2 Poly A

Splicing VDJ Ce1–Ce4 M1M2Poly A Membrane e-chain mRNA

Cm Se

VDJ

Sm

Se

Sm

Ce1–Ce4

Poly A

Splicing VDJ Ce1–Ce4 Poly A Secreted e-chain mRNA

Fig. 6.9 Class switching from IgM to IgE. The stages of class switching are exemplified by class switching from IgM to IgE. The first stage is CSR, involving nonhomologous recombination between the switch region (Sm) upstream of Cm to the switch region (Se) upstream of Ce, linking the V region, previously expressed with Cm, to Ce. The ends of the deleted sequence are linked to form a “switch circle.” The second stage of class switching is transcription of the recombined gene into one of the e-chain mRNA precursor(s). Transcription of the recombined gene is initiated from the V-region promoter. It can be terminated in the intron between Ce and the membrane exons to yield the precursor

mRNA encoding the secreted form of the e chain. Transcription through this intron and the membrane exons leads to the precursor mRNA encoding the membrane form of the e chain. The third stage of class switching is splicing of the mRNA precursors to yield the mature mRNA. Finally, the mRNAs are exported to the cytoplasm and translated into the corresponding e chains, which are paired off with an L chain and conducted through the cell membrane (not shown). The secreted form of IgE is exported from the cell, whereas the membrane form of IgE is anchored in the cell membrane and associated with its a and b signaling subunits.

of the switched immunoglobulin H-chain gene. Recombination occurs first from IgM to one of the other CH cassettes, after which sequential switching to additional CH cassettes can follow in the downstream direction in the B cell or B-cell clone. Targeting the switch regions of particular isotypes to participate in CSR requires transcription initiated from the promoters located upstream of these sites (germline gene transcription). This is contingent on stimulation by specific cytokines, e.g. interferon (IFN)-γ for IgG1 and IgG3 in the human system, interleukin (IL)-4 or IL-13 for IgG4 (IgG1 in the mouse), and IgE and IL-4 and transforming growth factor (TGF)-β for IgA. We describe the activity of IL-4 and TGF-β in the stimulation and inhibition, respectively, of ε germline gene transcription in the section on IgE regulation. The second signal required for CSR is CD40-ligand (L), expressed on activated T helper 2 (Th2) cells, which binds to CD40 on the cognate B cells. However, alternative ligands secreted by dendritic cells are now known to be able to deliver the second signal to B cells. The interaction of either BAFF (B cellactivating factor of the tumor necrosis factor family) with

BAFF-R (BAFF receptor), or TACI (transmembrane activator and calcium-modulator and cytophilin ligand interactor), or APRIL (a proliferation-inducing ligand) with BCMA (B-cell maturation antigen) or TACI, stimulate CSR to several isotypes, including IgE (Litinskiy et al. 2002; Castigli et al. 2005; Schneider, 2005). The germline gene transcripts are propagated through the intervening sequence exon (I exon), switch region (S), CH and the membrane exons. Transcription is terminated, as in the rearranged gene (and other mRNAs), by cleavage of the RNA engendered by a “termination signal” in the DNA, leading to polyadenylation of the RNA. The primary transcripts are then spliced, so as to join the I exon to the CH exons, eliminating the intervening sequence. The germline gene transcripts cannot encode proteins because they have stop codons in all three reading frames (and are therefore referred to as “sterile transcripts”). The deleted switch circles are also transcribed and spliced to yield “switch circle transcripts.” The switch circles lack replication origins and therefore each circle represents a single recombination event. Their transcripts are unstable and likewise decay rapidly in

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the dividing cell population. These characteristics have been exploited to estimate the frequency of CSR and reveal the pathway of CSR, using DNA and cDNA sequencing (Cameron et al. 2003; Takhar et al. 2005, 2007). CSR appeared later in evolution (in amphibians, birds, and mammals) than VDJ recombination and SHM (which first emerged in cartilaginous fish). It would appear that CSR is an evolutionary adaptation of SHM, since both require AID; these processes display both similarities and differences. Doublestrand DNA breaks are involved in both VDJ and CSR, but those in VDJ occur at specific sites, whereas in CSR they appear anywhere in the switch regions, varying in size from 1 to 12 kb. Imprecise breakpoints can occur in CSR because the switch regions are located in introns, spliced out of the mRNA precursor. In contrast, the DNA breaks in VDJ occur at the edges of coding regions, which need to be precisely stitched together to encode a functional immunoglobulin. The switch regions in mammals are rich in GC pairs, with the transcribed strand being invariably C-rich. The switch regions are primarily composed of tandem repetitive sequences with motifs such as TGGGG, GGGGT, GGGCT, GAGCT, and AGCT, but there are characteristic differences between particular switch region sequences, certain of which also contain much longer repeats. The different switch regions, then, vary in respect of primary sequence, sequence length, and position in the H-chain locus, any of which could influence the relative frequencies of CSR. The prevalence of GC pairs in the switch regions is not in itself essential for CSR, since the switch regions in amphibian immunoglobulin genes are ATrich. A vestigial similarity has nevertheless endured through evolution, in that AID preferentially targets one of the same sequence motifs, namely AGCT, in the amphibian switch regions. The composition of the switch regions in mammals may have evolved to facilitate CSR. As RNA polymerase transcribes the C-rich strand, the G-rich strand of the DNA is transiently displaced. A GC-rich RNA–DNA hybrid is uniquely stable, and therefore persists, leaving the displaced G-rich single strand as an optimal target for AID. The resulting three-stranded structure is called an “R-loop.” Ultimately, the C-rich strand must also undergo scission to allow recombination. It is conjectured that the breaks in the C-rich strand are introduced in the single-stranded segments of the DNA where the RNA–DNA hybrid abuts the DNA duplex. As for SHM, AID initiates CSR by cytidine deamination, and interacts with the single-stranded DNA-binding protein RPA, which may direct it to the unpaired G-rich strands in the R-loops. After deamination of Cs in the switch region, CSR employs the BER pathway that operates in SHM and then NHEJ to repair and join the two end of the DNA, as in V(D)J recombination. In CSR, conversely, the dominant agent in holding the chromosome intact is thought to be a histone variant, phosphorylated H2AX (γH2AX), which plays no part in V(D)J recombination and is of lesser importance in SHM

(Petersen et al. 2001). H2AX is conveyed to the DNA breakpoints and is there converted to its phosphorylated form, γH2AX, by DNA-PKcs. This region of γH2AX-enriched nucleosomes is extended along several million base pairs of DNA, encompassing many switch regions and, importantly, keeps the chromosome intact (Franco et al. 2006a,b).

Epigenetic control of somatic recombination and hypermutation of immunoglobulin genes Principles of gene expression Selective gene expression is regulated by changes in chromatin structure and nuclear location. DNA is first packaged into nucleosomes containing 146 of DNA wrapper around complexes of histone octamers containing two each of the generic core histones H2A, H2B, H3, and H4. These are connected by a variable length of linker DNA, forming “beads on a string”. The fifth histone, H1, binds to the linker regions and induces higher-order chromatin folding. The resulting chromatin is further organized into loops, attached to a scaffold, with each chromosome occupying a designated “territory” in the cell nucleus (Marshall et al. 1996). Specific histone variants and the methylation of CpG sequences in the DNA are associated with the activation or repression (“silencing”) of gene expression. Some of the histone variants arise by posttranslational modifications in situ, while others are expressed from different genes. The status of histone modifications in the genome is deemed to be a “histone code,” which, by analogy to the genetic code, determines the developmental fate of all cells (Jenuwein & Allis 2001). Although the globular octameric histone complex is enveloped by DNA, the N-terminal “tails” of the histones are free to attach themselves to the minor groove of the DNA duplex and block the access of nonhistone proteins. Modifications, including acetylation, methylation, and phosphorylation of the N-terminal histone “tails,” affect the accessibility of regulatory proteins with response elements in the DNA and serve as docking sites for accessory factors. In addition, ATP-dependent remodeling complexes can alter the positioning of nucleosomes along the DNA, thereby exposing the binding sites for transcription factors in linker regions. The DNA itself can be methylated at CpG sequences, an event generally associated with gene repression. The enzymes that modify histones and DNA are histone acetyltransferases (HATs), histone deacetylases (HDACs), and histone and DNA methyltransferases. The variety of available modifications of chromatin allows the use of a limited library of transcription factors in the simultaneous regulation of many genes, exemplifying again the principle of combinatorial diversity, applied here to the regulation of gene expression.

Control of gene expression in the immune system The importance of chromatin accessibility in the immunoglobulin H-chain locus, originally postulated by Alt and coworkers (Yancopoulos & Alt 1985), was demonstrated in a

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Progenitor

Pre-B

Pro-B

Ac

Ac

Ac

Ac Me

Me

Me

Ac

Ac Ac

Me

Ac

Ac

Ac Me

Ac

model system: RAG proteins were introduced into isolated nuclei derived from cells at differing developmental stages (Stanhope-Baker et al. 1996). V(D)J cleavage was detected only at the sites undergoing recombination at the developmental stage of the cells from which the nuclei were derived. It follows that factors tightly bound to DNA, changes in chromatin structure or higher-order chromosomal organization determine RAG access, and that this structural state persists. The operation of epigenetic mechanisms has been studied in relation to V(D)J recombination, SHM and, CSR, mainly in mouse systems. Histone hyperacetylation, which reduces the positive charge on the histone tails, is correlated with VDJ regions poised to undergo recombination in the murine Hchain locus, and inversely correlated with silencing of the same regions in other cell types (Oettinger 2004). Other modifications correlated with VDJ rearrangement include H3-K79 dimethylation, a marker of active chromatin, and reduced H3-K9 methylation, associated with gene silencing. Whereas, the aforementioned modifications are spread evenly throughout the DJ region, another modification, dimethyl H3-K4, peaks sharply at the boundary of the accessible locus, and may signify the presence of a barrier element that prevents the spread of repressive modifications into the active region (Morshead et al. 2003). In pro-B cells, before DJ recombination, the DJ, but not the VH region, is hyperacetylated (Chowdhury & Sen 2001; Maes et al. 2001). Before VH to DJH recombination, the histones in the region of the VH genes are acetylated in three stages, correlated with the initial bias toward recombination of the DJ proximal segments. Johnson et al. (2003) also demonstrated

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Ac

Fig. 6.10 Mechanism of allelic exclusion. Each panel shows the two immunoglobulin alleles packaged into a nucleosome structure. There are at least three stages of activation. In progenitor B cells (left panel), both alleles are methylated (Me) and the histones deacetylated, but one allele replicates earlier (light blue DNA strands) than the other (dark blue DNA strands). In pro-B cells (middle panel), the two alleles are already packaged with hyperacetylated histones (Ac), even though the DNA is still methylated. In pre-B cells (right panel), the early allele (light blue DNA strands) undergoes monoallelic demethylation, making it susceptible to rearrangement.

that histone acetylation is confined to the gene segments, their flanking promoters and RSSs, and that a drop in histone acetylation accompanies the inhibition of VH to DJH rearrangement at a later stage of B-cell ontogeny. An IL-7 signal provokes acetylation of distal VH gene segments undergoing VH to DJH recombination. Deletion of the gene encoding PAX5 protein (or B-cell activation protein, BSAP) leaves transcription and acetylation unchanged, yet blocks rearrangement. This reveals that layers of occlusion still prevail (Hesslein et al. 2003). Further details can be found in recent reviews (Oettinger 2004; Cobb et al. 2006; Jung et al. 2006). In an unusual and enlivening foray into the human immune system, Woo et al. (2003) studied the relation between transcriptional activation, histone acetylation, AID expression, and the association of γH2AX in SHM of VH regions in the BL2 cell line. In this cell line the VH regions are constitutively transcribed, but not hyperacetylated. Induction of SHM leads to hyperacetylation of the VH, but not the Cμ, region and the expression of AID. The increased acetylation of H4, relative to that under nonmutating conditions, was comparable to the changes seen in histone acetylation during the onset of V(D)J recombination (Chowdhury & Sen 2001; Johnson et al. 2003). When AID expression was inhibited, acetylation still occurred, and must thus be downstream of transcription but independent of AID. If deacetylation is inhibited by HDAC inhibitors, Cμ becomes acetylated and mutations are introduced into the Cμ as well as the VH region. Similar results ensued when AID was overexpressed in the BL2 cell line in the absence of immune stimulation. γH2AX is also associated with the VH but not the Cμ region, and may thus

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be linked to the resolution of the DNA breaks required for SHM. Woo et al. concluded that acetylation of histones in the V-region chromatin is used to target SHM. Simliarly, in a mouse system, Odegard et al. (2005) found that induction of SHM is accompanied by changes in histone acetylation in the V region of chromatin and inferred that these changes are instrumental in targeting the mutations (see Odegard & Schatz 2006 for details). Three studies have addressed the possible role of histone acetylation in CSR: Nambu et al. (2003) showed that H3 acetylation of switch regions is linked to CSR. They used lipopolysaccharide (LPS), LPS and IL-4, or LPS, IL-4 and TGF-β1 to direct switching, respectively, to Sγ3, to Sγ1 together with Sε, and to Sα with accompanying inhibition of the switch to Sε in the mouse. They found that H3 acetylation at the corresponding I exon promoters, and at switch regions was correlated with transcription and recombination of the genes targeted by the cytokines. The outcomes of two other studies (Li et al. 2004b; Wang et al. 2006) were broadly consistent with the results and conclusions of Nambu et al. See Chaudhuri et al. (2007) for a comprehensive review. The strategies of nucleosome positioning and the substitution of histone variants have also been used in the regulation of immunoglobulin gene expression. Sikes et al. (2002) observed a dominant role of promoter positioning in determining the accessibility of gene segments for V(D)J recombination. Along the same lines, Kwon et al. (1998) and Baumann et al. (2003) demonstrated the importance of RSS positioning for V(D)J cleavage, and Patenge et al. (2004) revealed the potential role of an ATP-dependent mechanism of chromatin remodeling. Of special interest is the activation of chromatin by the substitution of the histone variant H3.3 for H3 in nucleosomes, in a replication-independent manner (Ahmad & Henikoff 2002).

Nuclear topology An emerging theme is the relationship between epigenetic events (histone modifications and CpG methylation), higherorder chromatin structure, and the packaging of genes in designated subcompartments of the cell nucleus. Each chromosome occupies a specific territory, and different chromatin segments exhibit movement in this territory (Marshall et al. 1996; Abney et al. 1997). Freedom of the DNA to move is restricted by the attachment of chromatin segments to nuclear substructures, such as the nuclear periphery, by “looping” within chromosomes, and by the association of genes with centromeric heterochromatin. The extent of the freedom to move has been mapped by immunohistochemistry and fluorescence in situ hybridization (FISH) confocal microscopy (Skok et al. 2001; Kosak et al. 2002; Fuxa et al. 2004; Roldan et al. 2005; Sayegh et al. 2005), or followed by the method of chromosome conformation capture (3C) (Liu & Garrard 2005). The movements and configuration of murine VH and Vκ

gene segments have been studied in early B cell development from the pro-B cell to the immature B cell (Kosak et al. 2002). Before recombination both immunoglobulin H-chain alleles are observed in the periphery of the nucleus in an extended conformation. Early pro-B cells are characterized by the relocation of both H-chain alleles to a central nuclear position and histone acetylation in response to IL-7 signaling (Chowdhury & Sen 2001). Expression of PAX5 leads to longrange contraction of the locus, resulting in VH to DJ recombination on one allele (Kosak et al. 2002; Fuxa et al. 2004). Upon expression of membrane IgM (with surrogate L chains) on the cell surface, the unrearranged H-chain allele is packaged into centromeric heterochromatin, effecting allelic exclusion. The subsequent Vκ to Jκ rearrangement is accompanied by decontraction of the VH locus (Roldan et al. 2005), evidently precluding further rearrangement. Nuclear topology of κ-chain locus undergoing recombination has also been studied by the 3C methodology (Liu & Garrard 2005; Wuerffel et al. 2007), which demonstrated dynamic looping by association of the three H-chain gene enhancers, implicating activation protein 5 (AP-5) and enhancer 47 (E-47) transcription factors.

DNA replication and allelic exclusion Histone modifications, DNA demethylation, nucleosome repositioning, nuclear location, and chromatin compaction, looping and transcription all contribute in various combinations to the accessibility of immunoglobulin genes for V(D)J recombination, SHM, and CSR. However, these events can only occur at the designated stage of the cell cycle and may require several rounds of replication. A possible reason for this is that chromatin remodeling requires unraveling of the DNA in the mechanism of action of DNA polymerase. Replication timing has been studied in relation to allelic exclusion in V(D)J recombination. Replication origins in the H-chain locus prior to VDJ recombination were mapped at several stages of B-cell development (Norio et al. 2005). Prior to VDJ recombination, replication begins at the 3′ end of the H-chain locus early in S phase, proceeds towards the 5′ region, and terminates at the 5′ end of the locus at the end of the S phase of the cell cycle. In contrast, during the early stages of B-cell development (in pro-B and pre-B cells), the entire locus replicates in early S phase. Late in B-cell development (in immature and mature B cells), replication reverts to the original timing. The changes in replication timing are brought about by changes in the number and position of the replication origins, rather than the speed of replication (1–2 kb/min). Origins are activated independently within several functional domains, corresponding to the D region, the C region, the VD intergenic region, and one or more V-region domains. These changes are correlated with the progressive modifications known to take place in chromatin structure and transcriptional activation (described above), sometimes preceding these changes.

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When there are multiple origins, only a few, e.g., two or three in the 3′ DJCH region (amounting to one per 100 kb, although some are much closer together) are fired in any one cell undergoing division. It may take 1–2 hours before the DNA polymerase reaches the end of each unit of replication. This means that only one or two of the origins are activated during each cycle, and that each allele is likely to begin DNA replication initiation from different origins. Therefore, on each allele, any given portion of the H-chain locus will replicate at a different time in the S phase, which depends on its actual distance from an active origin. Although replication timing following the induction of CSR has not yet been examined, this might well dictate the frequency of CSR to different isotypes, and constitute a mechanism involved in the regulation of IgE. Indeed, it was shown that CSR is coupled to cell division and that the peak of CSR to IgE occurs after five divisions, compared with three for IgG (Tangye et al. 2002). The probability of CSR to different isotypes as a function of cell division cycles also indicates that it occurs in a “stochastic” manner, consistent with the model of Norio et al. for the firing of replication origins. The allele, maternal or paternal, that is first to undergo all the somatic changes in DNA sequence in all B cells is set during embryogenesis and “remembered” during the whole life of an individual. Both alleles have an equal “chance” of being the first, implying that the choice is made at random, and that negative feedback mechanisms operate to silence the second allele unless the first fails to perform its functions. The active allele is marked by hypomethylated CpG sequences compared with the inactive allele (Mostoslavsky et al. 1998) and is always the first to undergo replication (Mostoslavsky et al. 2001). The inactive allele may be unable to compete with the active allele for a limited pool of transcription factors or HATs, and CpG methylation may specify its packaging into centromeric heterochromatin. The methylation status of the DNA is determined during replication by the action of DNA methyltransferase on the nascent strand of DNA. See Bergman et al. (2003) for the hierarchy of epigenetic mechanisms that regulate antigen receptor gene expression and allelic exclusion. In V(D)J recombination, RAG gene expression is switched on and off at the appropriate times to assist the process (Fig. 6.10).

Expression and regulation of IgE Regulation of class switch recombination to IgE The stimulation of germline gene transcription determines the selectivity of CSR to one of the IgGs or IgAs or to IgE (Agresti & Vercelli 1997). Transcription of the ε germline gene, and hence CSR to IgE, is highly specific in its requirement for either IL-4 or IL-13. In the mouse these cytokines also stimulate γ1 germline gene transcription and switching

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to IgG1, but a higher concentration of IL-4 is needed for stimulation of ε than of γ1 germline gene transcription. In human, IL-4 and IL-13 stimulate the transcription of germline genes corresponding to all isotypes (Fujieda et al. 1995; Mills et al. 1995; Fear et al. 2004), but other cytokines, including IFN-γ, also induce transcription of the γ germline genes. In fact, IFN-γ inhibits transcription of the ε germline gene in both species (Xu & Rothman 1994; Pene et al. 1988), affording an example of mutual antagonism that also appears at other levels of IgE regulation (see below). The combination of TGF-β and IL-4 inhibits CSR to IgE, while stimulating CSR to IgA (Islam et al. 1991). Stimulation of B cells by IL-4 (or IL-13) causes binding of the transcription factor STAT6 (signal transducer and activator of transcription 6) to a response element in the ε germline gene promoter (and to the promoters of other germline genes responsive to these cytokines, e.g., AID). But the binding site for STAT6 is only one of several response elements, all of which must be occupied to activate transcription of the ε germline gene (Fig. 6.11). The Iε promoter contains binding sites for STAT6, nuclear factor (NF)-κB, PU-1, enhancer-box (E-box)-binding protein E2A, and PAX5 defined earlier. Of these, only PAX5 is a B cell-specific protein (downregulated in plasma cells, as described above). The other transcription factors are present in all B cells, and (all but PAX5) in most other cell types; it is the combination of factors and the accessibility and strength of their binding sites that underlie the selectivity of transcription, yet another example of combinatorial diversity. As mentioned above, cytokine stimulation is necessary, but not sufficient, for CSR. Recombination occurs only in the presence of a “second signal.” One such is CD40L, expressed by antigen-activated T helper cells and mast cells. CD40L binds to CD40 on B cells in the context of cognate interactions with T cells. Allergen-activated Th2 cells provide both of the signals (IL-4/IL-3 and CD40L) required to stimulate CSR to IgE. The more recently discovered CD40L homologs BAFF and APRIL, secreted by monocytes and dendritic cells, also promote CSR, including CSR to IgE (Litinskiy et al. 2002). The action of these cytokines may be restricted to tertiary lymphoid tissue, formed in response to chronic inflammation, and would thus bear on the pathogenesis of both autoimmune and allergic diseases (see below). Both the IL-4 and CD40L signal transduction pathways have been well explored (Agresti & Vercelli 1997; Chai & Rothman 1997) (Fig. 6.11). The Janus-activated kinases (JAK1 and JAK3) and tyrosine kinase 1 (TYK1) are associated with the IL-4 receptor and JAK1 and TYK2 with the IL-13 receptor; both stimulate phosphorylation of STAT6 on IL-4 or IL-13 binding to their receptors. STAT6 must be phosphorylated if it is to bind to its consensus sites in the DNA (TTCNNN[N]GAA). The cytoplasmic sequence of CD40 associates with, and signals through, three proteins that belong

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T cell IL-4

TGF-b

CD40L

Dendritic cell

Allergen IgE

CTLA4 gC

IL-4Ra

TGF-bR

JAK1

CD40

JAK3

Plasma cell

IgE FceRI

TRAF6

lgE

TYK1 TRAF2,3 B cell Induces transcription of ID2

P P

JNK/p38

1-kB NK-kB

Nuclear membrane

ID2

IgM

CSR to IgE and plasma cell differentiation

IeGLT

B cell

MHC II

STAT6 TCR

1-kB

NK-kB

Ie

IL-4 CD4 T cell

eGLT

CD80/ CD28 CD86

CD40L CD40 IL-4R

Se

IL-4

P P

ID2+E2A

AP1

STAT6

NK-kB ID2+PAX5

AID STAT6

NK-kB

AID

P P

Fig. 6.11 Regulation of e germline gene transcription and AID expression. IL-4 stimulation, in conjunction with cognate B cell–T cell contact, mediated by CD40–CD40L interaction, initiates a signaling cascade within the B cell resulting in the activation of e germline gene transcription and AID expression. IL-4 stimulation results in the phosphorylation of STAT6, by JAK1, JAK3 and TYK1, which allows STAT6 to enter the cell nucleus. The CD40–CD40L interaction results in the dissociation of IkB, which retains NF-kB in the cytoplasm, allowing it also to enter the cell nucleus. Together with the constitutive B-cell transcription factors PAX5, E2A and AP1, STAT6 and NF-kB stimulate e germline gene transcription. This process is inhibited by CTLA-4 signaling and expression of the transcription factor ID2, upregulated by TGF-b. See text for definition of abbreviations.

to the family of tumor necrosis factor receptor-associated factors (TRAFs), TRAFs 2, 3 and 6. Cross-linking of CD40 by membrane-bound trimeric CD40L promotes the association of CD40 with, and signaling through, TRAFs. As a consequence of this interaction IκB dissociates from NF-κB, allowing the translocation of NF-κB to the cell nucleus. NF-κB acts synergistically with STAT6 to activate the Iε promoter, and may also promote CSR by way of binding sites in the ε switch region in mouse (Kenter et al. 2004). The interaction of CD40L with CD40 in the presence of IL-4 leads to upregulation of the co-stimulatory factors CD80, on the B cell, and CD28, on the T cell (Fig. 6.12).

Fig. 6.12 Cell–cell interactions in IgE production. Allergen presentation to Th2 cells leads to the secretion of IL-4 and upregulation of CD40L. Cognate interaction between B cells and T cells, via CD40L on the T cell and CD40 on the B cell, stimulates the expression of costimulatory proteins, CD28 on the T cell and CD80 or CD86 on the B cell. Upregulation of e germline gene transcription and CSR from IgM (IgG or IgA) to IgE is followed by plasma cell differentiation and secretion of IgE. GLT, germine gene transcript; TCR, T-cell receptor. See text for definition of other abbreviations.

Other T-cell cytokines, such as IL-5, IL-6, and IL-10, impinge on later stages of class switching to IgE, but are not specific to IgE. Epstein–Barr virus (EBV) infection of B cells stimulates switching to IgE, due to an EBV-encoded protein, latent membrane protein 1 (LMP1), which mimics the effect of CD40 by recruiting TRAFs (Brodeur et al. 1997) and promoting cell survival (Fries et al. 1999; Kieser et al. 1999). Steroids, while useful in treating allergy, paradoxically stimulate IgE synthesis. This is due to the stimulation of CD40L expression on T and B cells, probably through gluocorticoid response elements in the CD40L promoter (Jabara et al. 2001). IL-4 and CD40 orchestrate the expression of a variety of genes involved in IgE expression and function. IL-4 enhances its own expression in Th2 cells, and expression of MHC class II antigen (involved in T cell-dependent antigen presentation) and that of both IgE receptors (Gould et al. 1997). IL-4 plus CD40L stimulates AID expression through STAT6 and NF-κB (Dedeoglu et al. 2004) (see Fig. 6.10). CD40 in various cell types is implicated in the expression of Lyn, Syk, phosphatidylinositol 3-kinase phospholipase C-γ2, p28, JAK3, STAT3, and STAT5, and thus coordinates many disparate functions in the immune system (Banchereau et al. 1994).

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Several cytokines are implicated in the downregulation of ε germline gene transcription, either directly (see Fig. 6.11) or indirectly (e.g., by their effects on IL-4 or IL-13 expression). TGF-β induces expression of the transcription factor ID2. ID2 is expressed in immature B cells and dendritic cells and appears to have a dual effect on CSR to IgE, first on the Th1/Th2 balance through regulation of dendritic cell populations and, second, by sequestering E2A, preventing its binding to the response element in the ε germline gene promoter and the activation of transcription. The transmembrane phosphatase CD45 dephosphorylates JAK1 and JAK3 to prevent STAT6 activation. Expression of the inhibitory costimulatory molecule CTLA-4 cytotoxic T-lymphocute-associated protein 4) inhibits the activation of NF-κB and STAT6, perhaps by competing with CD28 for its partners, CD80 and CD86, on T helper cells (which participate in costimulation of CSR) (Fig. 6.12). BCL-6 has pleiotropic effects in retarding B-cell differentiation (see above) and it competes with STAT6 for their common binding site in the ε germline gene promoter (Harris et al. 1999). The most important of the regulatory mechanisms that we have discussed are illustrated in Fig. 6.11. See also Geha et al. (2003) for a comprehensive account.

Regulation of IgE-committed B-cell differentiation into plasma cells B cells that have undergone CSR to IgE still cannot secrete IgE at a high rate until they have differentiated into plasma cells. We describe two post-CSR processes that distinguish IgE regulation from that of other antibody classes in this section. First is the choice of RNA chain termination signals in the common mRNA precursor for the secreted and membrane forms of IgE (Karnowski et al. 2006). RNA polymerase transcribes the rearranged ε gene from the transcription start site through the sequence encoding the (untranslated) ribosome binding site in the mRNA, the leader exon, the exons encoding Cε1–4, and the two exons encoding the membrane and cytoplasmic sequences. One RNA chain termination signal falls within the intron separating the Cε4 exon from the membrane exons; three more termination signals are located downstream of the membrane exons. The chain termination signal is a hexamer, but sequence variations influence the efficiency of termination. Most immunoglobulin genes use the consensus sequence AAATAAA, which is the first termination signal, to express the secreted form of the ε chain. However, the termination signals used to express the membrane form all deviate from this sequence, having instead the sequences AGTAAA, AAGAAA, and ATTAAA, and are known to be less efficient (Sheets et al. 1990). The RNA polymerase can bypass these signals without causing termination. When this happens the RNA is generally degraded in the nucleus. Since expression of membrane IgE on the cell surface is required for B-cell survival (Achatz et al. 1997), only a minor proportion of the cells that have undergone CSR to IgE

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survive and go on to differentiate into IgE-secreting plasma cells. It has been suggested that this could limit the expression of IgE, relative to other antibody classes (Karnowski et al. 2006). Possibly related to this are the missing IgE+ memory cells in a T/B monoclonal mouse, following immunization (Erazo et al. 2007). IgE+ B cells, unlike IgG1+ B cells, are rarely seen in germinal centers, and are found instead in the medullary cords and at the boundary of the T-cell areas in the lymph nodes and spleen. The IgE+ cells also have a more plasmacytoid phenotype than the IgG1+ cells (see Fig. 6.1) and express the plasma cell markers BLIMP1 and XBP1, rather than the Bcell markers PAX5, BCL-6, and AID. A population of doublepositive IgG1+/IgE+ cells with the same phenotype and gene expression profile was found and taken to reflect CSR from IgG1 to IgE and the simultaneous synthesis of the γ and ε chains from both mRNAs in the B cells. The IgE+ cells exhibited evidence of SHM, resulting in higher affinity for the antigen. Erazo et al. suggest that affinity maturation of IgG1+ B cells occurs in the germinal center and that some of these cells undergo sequential switching to IgE as they migrate out of the germinal center during terminal differentiation. It was found, in support of this view, that the IgE+ cells, unlike their IgG1+ counterparts, expressed CXCR5, which debars B cells from germinal centers. Erazo et al. further suggest that the inhibition of CSR to IgE in germinal center may result from the secretion of IL-21 by follicular T helper cells. IL-21 inhibits IL-4- and anti-CD40-stimulated CSR to IgE, but not to IgG1, in vitro. This provocative model for the developmental pathway of IgE-secreting plasma cells is illustrated in Fig. 6.13. Its relevance to IgE regulation in human B cells remains to be explored.

Role of CD23 in the homeostasis of IgE A third post-CSR process is implicated in the homeostasis of IgE. It was shown in separate studies that membrane CD23 functions in the downregulation of IgE synthesis in B cells (Sherr et al. 1989; Luo et al. 1991; Saxon et al. 1991; Yu et al. 1994; Payet & Conrad 1999), whereas soluble proteolytic fragments of CD23 lead to the upregulation of IgE synthesis when they bind to CD21 on B cells (Aubry et al. 1992). These opposing activities have been incorporated into a model for the homeostasis of IgE (Sutton & Gould 1993; Gould et al. 1997, 2003) (Fig. 6.14). In this scheme the central factor is the IgE concentration in the range 0.1–10 nmol/L (corresponding to the dissociation constants of the two IgE–receptor complexes). At concentrations below 10 nmol/L , fragments of CD23 fragments are released, which stimulate IgE synthesis. Higher IgE concentrations protect the membrane CD23 against proteolysis, so that no fragments are liberated to stimulate IgE synthesis. Attachment of antigen–IgE complexes to IgE antibody-expressing B cells may additionally displace membrane-bound IgE from CD21 and nullify the upregulating mechanism (Fig. 6.14). Another observation is

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IgD+

IgD– BcI6+ Tfh IL-21

+

IgG1+ BcI6+

IgG1 BcI6

Th2 IL-4

IgG1– IgE+ IgG1+ PB IgG1+ MC

IgE+ PB

IgE+ PC

IgG1+ PC

Fearon & Carter 1995; Dempsey et al. 1996). Although CD23 can evoke a similar response in B cells (Reljic et al. 1997), it is not yet certain that membrane IgE has the same signaling capacity as membrane IgM. The mouse would not have the benefit of this mechanism: murine CD23 does not bind to CD21, probably due to the absence of a CD21-binding site that is only present in human CD23 (Hibbert et al. 2005). Thus, in mouse, membrane CD23 is available for the negative feedback of IgE synthesis, but fragments cannot play a role in homeostasis by exerting a positive effect. CD23, unlike other immunoglobulin receptors, is a C-type lectin (see Chapter 5). C-type lectins (e.g., mannosebinding protein) are more commonly engaged in innate immune responses. Both CD23 and CD21 which functions as a complement receptor as well as a counterreceptor for CD23, link innate to adaptive immunity in humans.

Local IgE regulation in the target organs of allergy

Fig. 6.13 Do IgE memory B cells exist? Follicular T-helper cells (Tfh) provide an IL-21-rich environment in the germinal center. BCL-6 expression and signaling through the IL-21 receptor inhibit class switching to IgE in most germinal center B cells. Class switching to IgE can occur in germinal center cells that downregulate BCL-6 function and interact with Th cells in a high IL-4 and low IL-21 microenvironment. Switching to IgE is therefore linked to a pathway of exit from the germinal center and differentiation of IgG1+ plasmablasts. MC, memory B cell; PB, plasmablast; PC, plasma cell.

that oligomeric CD23 fragments stimulate, and monomeric fragments inhibit, IgE synthesis (McCloskey et al. 2007). One may therefore conjecture that the oligomeric fragments act by coligating membrane IgE and CD21 in IgE-committed B cells (Hibbert et al. 2005; McCloskey et al. 2007). This scheme is based on analogy with the action of C3dg–antigen complexes in stimulating the immune response through the coligation of membrane IgM and CD21 (Carter & Fearon 1992;

Primary and secondary immune responses in the germinal centers of lymphoid organs have long been studied, but only recently has evidence come to light of a third stage in the immune response. This can take place in the “tertiary lymphoid organs” that develop at sites of chronic inflammation in response to persistent local antigen challenge. Germinal center-like reactions are well documented in the target organs of autoimmune diseases (Armengol et al. 2001; Aloisi & Pujol-Borrell 2006), and the evidence now is that they also prevail in the respiratory tract mucosa in allergic rhinitis and asthma (Gould et al. 2006). The classical germinal center reactions, defined by the sequence of events in secondary lymphoid tissue, are RR, SHM, and CSR (as outlined above). It was long assumed that these are the source of the memory B cells and plasma cells found locally in inflamed tissues, especially at sites of allergic inflammation. The observation of local IgE synthesis (Smurthwaite et al. 2001) is consistent with an external origin of these cells. On the other hand, it has been established that both SHM (Coker et al. 2003) and CSR (Snow et al. 1997;

Negative feedback Fig. 6.14 Homeostasis of IgE. IgE synthesis is regulated by a homeostatic mechanism involving CD23 and CD21. In the presence of excess IgE, the cleavage of CD23 from the cell surface is prevented, causing cross-linking of CD23 and membrane IgE by allergen and resulting in the downregulation of IgE synthesis. However, in the absence of IgE, CD23 is readily cleaved from the cell surface by the metalloproteinase ADAM10, which results in the coligation of membrane IgE and CD21, causing upregulation of IgE synthesis.

Positive feedback

Allergen

CD23

IgE

+

IgE synthesis

CD21

+

IgE synthesis

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Coker et al. 2003; Takhar et al. 2005, 2007), and probably RR (P. Takhar et al., unpublished results 2007), occur locally in the nasal mucosa in allergic rhinitis and the bronchial mucosa in asthma. In autoimmune disease, especially of the thyroid, fully developed germinal center-like structures appear in the target organ (Armengol et al. 2001; Aloisi & Pujol-Borrell 2006). Yet the germinal center reactions occur in the absence of these structures, which evolve gradually from loose cell aggregates to the mature state, including the presence of FDCs, with progress of the disease, and according to its severity. It was also observed that local B cells in the respiratory tract mucosa in rhinitis and asthma exhibit a greater abundance of B cells expressing the minor VH5 family, compared with those in the circulation or in normal tissues (Snow et al. 1999; Coker et al. 2005) The 50 or 51 functional VH genes in the human repertoire can be grouped into seven families (VH1 – VH7) on the basis of amino acid sequence homologies. The most abundant family is VH3 (30 members), whereas the VH5 family comprises only one member in 75% and two in 25% of the population. The superabundance of VH5 hints at action of a B-cell superantigen, which could bind to the VH5 framework regions in membrane-associated immunoglobulins, and preferentially expand this population of B cells (Gould et al. 2007). The nasal mucosa in 20% of the population is permanently, and in a much higher proportion occasionally, colonized by Staphylococcus aureus. This microbe secretes a plethora of enterotoxins, superantigenic in both T and B cells, and so there may well be a connection between this infection and the observed VH5 bias. The local inflammation caused by S aureus superantigens is likely to exert a wider range of effects in the tissue, such as T-cell and B-cell proliferation, stimulation of germinal center reactions, and resistance to steroid therapy in patients with asthma and related diseases (Gould et al. 2007).

Th2 cell polarization We have remarked on the importance of IL-4 and IL-13 in CSR to IgE in B cells. These and other cytokines are produced mainly by one of two types of T helper cell, Th1 and Th2 cells, which stem from a common CD4+ Th0 cell precursor. Th1 cells secrete IL-2 and IFN-γ, inducing CSR to IgGs, whereas Th2 cells secrete IL-4 and IL-13, which induce CSR to both IgGs and IgE. Thus, the mechanism of T helper cell lineage determination bears on the regulation of IgE. The origin and function of many T-cell subtypes, including T regulatory cells for example, is the subject of other chapters in this book. Indeed, the mechanisms involved in the transcription of the IFN-γ and IL-4 locus alone in the two T-cell subsets is a complex story. It involves, like that governing the immunoglobulin locus in B cells, mutual antagonism, which is inseparable from the polarization of T helper cells (Ansel et al. 2006). Yet T-cell polarization is only one step back in a hierarchy of

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events that ultimately decide the fate of B cells: “to E or not to E” (Vercelli 1999). The predominance of Th2 cells in the lung, skin, and gut (target organs of allergy) stems from the presence of epithelial, smooth muscle, and activated mast cells all expressing thymic stromal lymphopoietin (TSLP) (Liu et al. 2007). TSLP induces dendritic cells to express OX40L, which in turn initiates an inflammatory Th2 cell program in naive T and memory T cells in the absence of IL-12. IL-12 is noted for stimulating the Th1 cell program, including expression of IFN-γ, in uncommitted T helper (Th0) cells. IL-12 production by dendritic cells is stimulated by PAMPS, expressed by pathogens, and recognized by PRRs, such as Toll-like receptors on host cells (Kapsenberg 2003). Thus the development of inflammatory Th2 cells is determined by both positive (TSLP via OX40L) and negative or “default” (IL-12) signaling mechanisms. OX40L-stimulated Th2 cells express a modified set of cytokines. Like CD40L, OX40L stimulates production of IL-4, IL-5, and IL-13 by Th2 cells. CD40L-stimulated cells also express IL-10, whereas TSLP stimulation leads to expression of tumor necrosis factor (TNF)-α. TSLP can act directly on mouse but not human CD4+ Th0 cells, as only the former expresses the TSLP receptor (Liu et al. 2007). Tissue-specific expression of TSLP in transgenic mice predisposed them to localized manifestations of allergy (Yoo et al. 2005). Although this hints at a possible basis for tissuespecific manifestations of allergy, it begs the question of what determines the levels of TSLP in allergic as against normal tissues, and indeed the relative contributions of local TSLP expression and other predisposing states of different tissues or individuals. Other chapters deal with genetic and environmental factors associated with susceptibility to allergic disease, and the window of vulnerability in early life. We have long passed the stage of belief that any single agent engenders the development of allergy or asthma. There may, however, be many good ways of blocking the activities of the many essential agents, with side effects of varying seriousness. The magic bullets await discovery.

References Abney, J.R., Cutler, B., Fillbach, M.L. et al. (1997) Chromatin dynamics in interphase nuclei and its implications for nuclear structure. J Cell Biol 137, 1459–68. Achatz, G., Nitschke, L. & Lamers, M.C. (1997) Effect of transmembrane and cytoplasmic domains of IgE on the IgE response. Science 276, 409–11. Agresti, A. & Vercelli, D. (1997) Regulation of ε germline gene transcription: Q & A. In: Vercelli, D., ed. IgE Regulation: Molecular Mechanisms. John Wiley & and Sons, Chichester, pp. 179–90. Ahmad, K. & Henikoff, S. (2002) The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol Cell 9, 1191–200.

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7

Environmental Factors in IgE Production Anne Tsicopoulos, Catherine Duez and Andrew Saxon

While genetics have long been appreciated to play a major role in both IgE production and the development of allergic disease, equally important are environmental factors as they are what regulate the expression of this genetic potential. Environmental factors likely explain the recent increase in the prevalence of allergic disorders. Among these factors, allergens are a major contributor in the development and expression of IgE-mediated allergic diseases. Changes in allergen exposure can be seen to be related to issues such as early age of allergen exposure, changes in lifestyle favoring exposure to indoor versus outdoor allergens, dose of allergen, increased environmental levels, and geographic spread of allergens linked to changes in climate and in ecology. Airborne pollutants, both particulate and gaseous, represent a major environmental factor promoting allergic sensitization and disease expression. These adverse effects of particulate matter are highly dependent on the nature and size of the particles, their content of chemicals and metals, the age of exposure of the subjects, and the subject genetic makeup. Diesel exhaust and gases, in particular ozone, have been shown to exacerbate cellular inflammation and to act as mucosal adjuvants to skew the immune response to inhaled antigens toward a Th2-like phenotype. Levels of action include increased allergen presentation and increased IgE production through indirect and direct effects on B cells. Growing evidence suggests that mechanisms of pollutant-induced IgE production depend on hierarchical oxidative stress that is under the control of susceptibility genes. Environmental tobacco smoke appears to affect both the primary and secondary allergic responses, much like diesel exhaust. Exposures to microbes and their products have a more complex role on allergic sensitization and disease. Thus, exposure to bacterial endotoxin was found to protect, have no effect, or exacerbate IgE production depending on the time and dose of exposure. Polymorphisms in the receptors involved in endotoxin recognition may explain some of these discrepancies. Similarly, some viruses, such as hepatitis A, decrease allergic sensitization, whereas respiratory syncytial virus infection increases it. Mechanisms may involve modulatory effects on

Th1 and Th2 cytokine production through Toll-like receptors. Helminth infections have a paradoxical effect and have been linked to decreased allergic responses, probably through induction of T-regulatory responses, even though they increase the production of Th2 cytokines and IgE. Given the marked increase in both allergic airways disease and food allergy that has occurred over the past century, with the more recent further upturn in the last several decades, there can be no question that environmental factors are driving these changes. Interventions spanning those directed to individuals all the way to those directed at global change, are going to be required to address these environmental factors.

Introduction The role of IgE in allergic disorders is well established. Stratification of serum total IgE levels is a good predictor of the risk of asthma (Fig. 7.1) (Burrows et al. 1989; Owen 2007), and removal of total IgE with anti-IgE treatment is effective in allergic diseases (Holgate et al. 2005). While genetics have IgE and asthma relationship in adults

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Serum lgE (IU/mL) Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Fig. 7.1 Relationship between serum IgE concentrations and likelihood of asthma or allergic rhinitis. (From Owen 2007, with permission.) (See CD-ROM for color version.)

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long been appreciated to a major effect on both IgE production and expression of allergic disease, equally important are environmental factors in the expression of this genetic potential. Atopic individuals bear a complex genetic constitution that includes factors facilitating the increased production of IgE towards common environmental allergens (see Chapter 55). By way of gene–environment interactions, this inherited ability leads to alteration of the immune response and susceptibility to allergic disorders. The prevalence of allergic diseases has doubled in the past 20 years, such that 10–30% of the population in developed countries are likely to suffer from allergy (ISAAC 1998). The factors underlying this phenomenon are complex, and the rapid increase in allergic burden has occurred in parallel with urbanization and industrialization. A variety of environmental factors have and will affect the development and severity of allergic diseases, by enhancing induction of allergen-specific IgE (sensitization) and increasing IgE production, respectively. Among the most important are allergens, endotoxin, viruses, parasites, anthropomorphic pollutants, and tobacco smoke.

Allergens Allergens are obviously central to allergic disease: “no allergen – no allergy.” The airborne allergens responsible for the bulk of allergic disease worldwide are both indoor allergens, particularly those derived from arthropods [house-dust mite (HDM) and cockroach], animals (dog and cat), and outdoor allergens, particularly seasonal pollens and some molds. Issues such as age of allergen exposure, indoor versus outdoor allergens, dose of allergen, increased environmental levels, and geographic spread of allergens can play an important role in the development of IgE responses. Although IgE-dependent sensitization is a strong risk factor for the development of allergic diseases, increases in allergen exposure as discussed in this section are not enough per se to explain the marked rise in the prevalence of these diseases. The nonallergen factors involved are discussed in the later sections.

Age of exposure: timing of initial exposure to allergens and the development of IgE-dependent sensitization Although sensitization to allergens not previously encountered can occur throughout life, the high-risk period appears to be in early childhood when most important allergens are initially encountered. The seminal observations linking allergen exposure in early childhood to sensitization were related to allergy to seasonal allergens in northern Europe, and particularly the association of sensitization to birch pollen with birth during the pollen season (Bjorksten et al. 1980). Many studies suggest that infancy represents the prime time for

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initial sensitization (Wahn et al. 1997; Lau et al. 2000). Studies on cord blood cells showed that a generalized Th2 skewing of immune function was present during fetal and neonatal life (Prescott et al. 1999). The principal determinant of the sensitization risk seems to be the maturational state of the immune system in relation with the regulation of the Th2 response at the time aeroallergens are first encountered (Holt & Thomas 2005). As IgE production starts in week 11 of gestation, several groups have tried to establish whether priming of the fetus by allergens can occur during pregnancy. Although the presence of aeroallergen-reactive T cells has been reported in cord blood (Jones et al. 1996), it seems that effective priming of aeroallergen-specific memory T cells rarely occurs before birth (Smillie et al. 2001) and, instead, is initiated during infancy (Finn et al. 2000) and consolidated by the end of the preschool years, i.e., prior to age 5. The continued persistence in childhood of a Th2-polarized pattern has been linked to a deficient upregulation of the Th1 activity, which may involve a delayed maturation of associated antigen-presenting cells or regulatory cells (Holt & Thomas 2005). In support of this hypothesis, protective responses within the respiratory tract in very early life can be induced in children following high endotoxin exposure (see Endotoxin section), and in mice by both endotoxin and allergen exposure through induction of regulatory T cells and Th1 cells, respectively (Wang & McCusker 2006).

Indoor versus outdoor allergen exposure and changes in lifestyle According to the allergen involved, the induction of the IgE response is associated with different endorgan responses. Several studies have shown that IgE-dependent allergy to indoor allergens is mostly associated with asthma whereas outdoor seasonal allergen allergy is linked to allergic rhinitis (Omenaas et al. 1996; Duffy et al. 1998; Magnan et al. 1998). A recent longitudinal general population survey that followed over 600 children from the onset of asthma to age 26 years showed that sensitization to HDM was one of the strongest risk factors for persistence of asthma (Sears et al. 2003). These data led to the hypothesis that increases in the prevalence of asthma may be linked to changes in lifestyle such as housing. Thus, the decrease in the air exchange rate in homes, increases in indoor temperature, and soft furnishings, as well as increased time spent indoors, have increased exposures to indoor perennial allergens that are most likely to induce IgE production and lung inflammation. The importance of other allergens associated with urban lifestyle has also become clear. Cockroach allergens not only increase the risk of asthma attack but also may increase a child’s risk of developing asthma (Litonjua et al. 2001). Cat allergen can be found at levels associated with symptoms in 50% of homes in the USA where no cat is “in residence,” and is well known to be widely disseminated in public buildings including schools (Arbes et al. 2004). More recently, mouse allergen has been

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shown to contribute to the burden of asthma, and has been detected in approximately 75% of middle-class suburban homes of asthmatic children. In these children, 13% were sensitized to mouse allergen (Matsui et al. 2004).

Allergen dose exposure Studies in the 1990s demonstrated a simple linear dose– response relationship between indoor allergen exposure and specific sensitization and allergic disease (Wahn et al. 1997). The threshold concentration for developing mite sensitivity in atopic children was determined to be 2 μg Der p1 (a major allergen of HDM) per gram of dust, and 80 μg/g for nonatopic children (Kuehr et al. 1994). In both children and adults the severity of asthma symptoms was found to vary with the level of exposure to indoor allergens. Sporik et al. (1990) showed that exposure to >10 μg Der p1 per gram of dust at 1 year of age was associated with a fivefold greater likelihood of asthma at age 11. Similar results were reported in adults (Custovic et al. 1996). Accordingly, reduction in HDM exposure in young adults with asthma resulted in an eightfold improvement in bronchial hyperresponsiveness (Platts-Mills et al. 1982). However, the Multicentre Allergy Study, a prospective study of 1318 infants, showed that if IgE-dependent sensitization was associated with asthma, early indoor allergen exposure was not (Lau et al. 2000). Later studies with cat allergen reported a protective effect, i.e., high levels of allergen exposure were associated with decreased development of allergic diseases. It was shown that the prevalence of respiratory allergy and allergen sensitization was significantly lower in children who were exposed to pets during the first year of life (Hesselmar et al. 1999), suggesting that early exposure to high levels of pet allergens could induce a form of immune tolerance. This concept was supported by a study showing that children exposed to high levels of cat allergen made a modified Th2 response with production of IgG4 antibodies without IgE (Platts-Mills et al. 2001), and of interleukin (IL)-10 after allergen-specific T-cell stimulation in vitro (Reefer et al. 2004). This finding has been extended to adults (Custovic et al. 2001) and rodents (Jeal et al. 2006). A study using a model of primary allergen sensitization in allergic subjects confirmed that initial high-dose exposure to a neoallergen prevented allergic sensitization to this allergen, with a concomitant increase in IgG levels, whereas the opposite was observed in initial low-dose exposure (Riedl et al. 2005). Altogether, these studies demonstrate apparent dose–response relationships that fit a biphasic pattern in which sensitization risk increases with exposure concentration until a plateau is reached, above which risk decreases with further increases in exposure (Cullinan et al. 2004; Holt & Thomas 2005). However, this pattern does not seem to apply to HDM (Erwin et al. 2005) for which a more linear relationship is observed. Due to physical properties, environmental exposure to HDM is 100-fold lower than for mammalian allergen (Custis et al. 2003), and may therefore not

Environmental Factors in IgE Production

have achieved the allergen levels where allergic tolerance occurs. Another explanation is that immune response to allergens differs according to their nature. A direct effect on the IgE response has been suggested for Der p1, which is a potent cysteine protease that can enhance the IgE response by cleaving the low-affinity FcεRII/CD23 (Hewitt et al. 1995), by inhibiting interferon (IFN)-γ and Th1 responses in mice (Comoy et al. 1998), and by inducing IL-4 in human T cells (Ghaemmaghami et al. 2001). Other contaminants of HDM allergens also include endotoxin and unmethylated bacterial and mite DNA, which can stimulate the innate immune response through Toll-like receptors (TLRs) and then potentially modify the IgE response (Platts-Mills 2007).

Changes in allergen levels and geographic spread of allergens Changes in climate and in ecologic environment have led to an increase in the load of allergen exposure and in the rate of IgE-dependent sensitization.

Changes in climate Climate warming that has occurred over recent decades (about 0.6°C thus far), mainly related to air pollution, has dramatically advanced budburst in spring (Fitter & Fitter 2002), therefore bringing forward the allergenic pollen season for spring-flowering taxa (Rasmussen 2002; Gilmour et al. 2006) (Fig. 7.2). The rate of this advance is estimated at 0.84 –0.9 days/year. Increased temperatures prolong the duration of the pollen season and the abundance of pollens, in particular through direct stimulatory effect of CO2 (Ziska et al. 2003). Controlled environmental studies with simulated changes in the timing of spring, at both current and future predicted CO2 levels showed that high CO2 exposure increased ragweed pollen production by 55% (Rogers et al. 2006). Similarly, in a 6-year study, CO2 enrichment led to an average annual growth increase of 149% of poison ivy compared with ambient CO2 (Mohan et al. 2006). These climatic changes are also contributing to the geographic spread of allergens, exemplified by the march of ragweed into Europe and Asia. 26 April 30 April 4 May 8 May 12 May 16 May 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 Fig. 7.2 Changes in peak of airborne birch (Betula) pollen concentrations in Copenhagen, Denmark 1978–1999. (From Gilmour et al. 2006 and Rasmussen 2002, with permission.)

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The spread of ragweed and birch throughout Italy provides an example of new exposures (Asero 2004).

Changes in ecologic environment Ecologic changes introduced by humans have also contributed to the development of more widespread sensitization and symptoms as exemplified by Japanese cedar pollen (Cryptomeria japonica) allergy. Japan carried out a huge reforestation effort in the 1950s. By the time it was completed, cedar trees covered 12% of the Japanese archipelago. The first cases of allergy were reported in the early 1960s and the prevalence of Japanese cedar pollinosis has increased 2.6-fold between 1980 and 2000. The estimated prevalence in 2004 was 28.7% in metropolitan areas and 24.5% in the general population in urban areas (Kaneko et al. 2005). Similarly, the Asian lady beetle Harmonia axyridis, introduced into the USA over the period 1918–1990 as a biological control for aphids. In the fall, they seek shelter from the cold and often shelter in homes. In 1999, the two first cases of ladybug allergic rhinitis were published (Yarbrough et al. 1999). Since then, many case series have reported sensitization, being found commonly in endemic areas (Goetz 2007).

Pollutants Airborne particulates and gases related to human activities are important issues that affect allergic diseases; among others the combustion of fossil fuels produces a number of unhealthy substances such as carbon monoxide, nitrogen oxides, benzene, sulfur dioxides (SO2), and particulate matter (PM). Refinement in epidemiologic tools and air exposure assessment has provided more conclusive associative studies, leaving little doubt that air quality affects allergic airway diseases (Kramer et al. 2000; Janssen et al. 2003), and may contribute to new-onset asthma, allergic rhinitis, and atopy (Penard-Morand et al. 2005; Gordian et al. 2006; McConnell et al. 2006). In particular, more accurate methods for characterizing exposure to traffic pollution have been developed, including spatially resolved exposure biomarkers (Kulkarni et al. 2006; Oudinet et al. 2006).

Air particulate matter Effect of particle size Ambient PM consists of very heterogeneous groups of components, arbitrarily divided into different size fractions: coarse (PM ≤ 10 μm), fine (PM ≤ 2.5 μm), and ultrafine (PM ≤ 0.1 μm). Particle size is very important as size determines, to a great extent, inhalation, deposition, and elimination of particles. Ultrafine particles have substantially higher deposition efficiency. Moreover, ultrafine particles may penetrate through the epithelium and vascular walls and be transported to the blood to distal organs where proinflammatory events may occur (Elder & Oberdorster 2006).

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Studies on ultrafine particles have shown that the allergic pulmonary effects were related to the total surface area and number of particles rather than to the instilled mass (Nygaard et al. 2004). Mouse footpad injections of coarse and fine PM sampled from different European cities showed that the fine particles had a stronger adjuvant effect on ovalbumin (OVA)-induced IgE (Dybing et al. 2004). When comparing fine with ultrafine PM, only the ultrafine PM was able to increase Th2 cytokine and allergen-specific IgE production to administration of the antigen in sensitized mice (Alessandrini 2006; De Haar et al. 2006). Short-term exposure to PM 2.5 has also been associated with increased allergic inflammation in nasal lavage from asthmatics, but not healthy controls (Nikasinovic et al. 2006).

Effect of age The age of the subjects is also an important consideration in PM exposure. Children have greater physical activities, spend more time outdoors, and are likely to be more exposed than adults. In exposure studies, childrens’ exposure was twice that of adults exposed to the same outdoor concentrations of PM 10 (Janssen et al. 1997, 1998). Children are also more vulnerable to the effects of pollution, because their lungs are not fully developed when exposure begins. In a prospective 8-year study, adverse effect on lung development, assessed by decreased flow expiratory volume, was associated with pollution by PM 2.5 and NO2 (Gauderman et al. 2004).

Effect of particle-associated metals Metals have been scrutinized as important constituents of PM. In the Utah valley, closure of a steel mill factory releasing metal-rich PM in the 1980s was associated with decreased mortality and morbidity (Ghio 2004). Metal-rich particles have been linked to allergic diseases. Evidence was provided from regional differences in industrial air pollution that were shown to account for differences in prevalence rates of allergic sensitization in children living in different cities in eastern Germany (Heinrich et al. 1999). These differences correlated with the presence in samples of PM 2.5 of severalfold higher levels of zinc, magnesium, lead, copper, and cadmium (Gavett et al. 2003). There is evidence that metals induce IgE production (Prouvost-Danon et al. 1981; Murdoch et al. 1986) and stimulate Th2-driven immune responses in different species (Heo et al. 1997). A correlation between in vitro biological effects of residual oil fly ash (ROFA), a fine PM sample, and its content in transition metals has been reported (Dreher et al. 1997), leading to its use as a surrogate of metals. In animals, ROFA and metal-rich particles can enhance allergic responses to OVA and HDM in models of asthma (Lambert et al. 1999, 2000; Gavett et al. 2003), including increased release of pro-Th2 cytokines, increase in allergenspecific IgE levels, eosinophil recruitment, and airways hyperresponsiveness (Gavett et al. 1999). Moreover, metal ions such as aluminum, cadmium, nickel, and strontium can directly

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Exacerbation of inflammation Many studies have shown that both particulate and gaseous pollutants can initiate and exacerbate cellular inflammation, by interacting with the immune system. Ozone, nitrogen dioxide (NO2), and DEPs can all initiate the recruitment of inflammatory cells such as neutrophils, lymphocytes, and macrophages (Sandstrom et al. 1990; Schelegle et al. 1991; Salvi et al. 1999), but also, through their direct interaction with the airway epithelium, can lead to the generation of increased amounts of proinflammatory cytokines, chemokines, and adhesion molecules. DEPs can induce release of soluble intercellular adhesion molecule (ICAM)-1, and cytokines like granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-8 from bronchial epithelial cells (Bayram et al. 1998; Takizawa et al. 2000). DEP extracts can also directly stimulate macrophages leading to the production

Pioneering studies in Japan, where diesel vehicles are most common, showed that respiratory allergy was more prevalent in subjects living near busy highways than in subjects living in less busy areas with equivalent atmospheric concentrations of cedar pollen allergens (Ishizaki et al. 1987). As discussed below, it is now appreciated that DEPs are able to potentiate the effect of the allergen on the immune response at several steps (Fig. 7.3). Macrophage

Airway epithelium IL-10 CD80

CD28

TH1

–

Proinflammatory IL-B, Rantes GM-CSF, others

TH0 APC

TCR

IL-4 IL-13

B cell Antigen DEP

YY

Y

YY

DEP + ROS

Y Specific lgE

YY

TH2

Y

MHC-II

Y

Diesel exhaust consists of a complex mixture of DEPs and gases. Diesel engines emit 30–100 times more PM than gasoline engines, making DEPs one of the major components of airborne PM in urban and industrialized areas, and the best-studied particulate pollutant. The 3.3 million diesel trucks and buses in the USA contribute 58% of respirable particulates, although they comprise less than 2% of the total vehicles (National Air Pollutant Emission Trends 1997). The same proportion is observed in France (55.6% of PM) (Kunzli et al. 2000), whereas in other countries such as Japan, diesel vehicles contribute almost 100% of PM emissions (Kagawa 2002). After combustion of diesel fuel, the exhaust components tend to aggregate into discrete spherical respirable particles approximately 0.1– 0.5 μm in diameter, which consist of an elemental carbon core with a large surface area to which hundreds of chemicals and transition metals are attached. The major particle-associated chemical compounds are PAHs, which contain three to five benzene rings that can easily diffuse through cell membranes. Of the gaseous pollutants, the main focus has been on ozone. The principal source of lower atmosphere ozone is automobile exhaust as a consequence of photochemical reactions of nitric oxides, volatile organic compounds, and ultraviolet light.

Interactions with allergen Diesel exhaust

Y

Diesel exhaust and gases

of chemokines able to recruit neutrophils and eosinophils (Fahy et al. 1999). Beside these interactions with the innate immune system leading to bronchial inflammation, interesting results have shown that pollutants are able to modify the IgE response by interacting with allergens.

YY

affect mast cells by enhancing their IL-4 release and degranulation (Walczak-Drzewiecka et al. 2003). Recently, aerosolized ROFA in pregnant mice was shown to favor IgE-dependent asthma susceptibility in their offspring (Hamada et al. 2007). Taken together, these studies suggest that soluble metal from PM has an influence on subsequent allergic responses. Identifying the specific causative agents in PM is difficult because of its multiple constituents. Beside metals, biological contaminants such as endotoxin (see Endotoxin section) and polyaromatic hydrocarbons (PAH), contained in diesel exhaust particles (DEPs) and cigarette smoke, have been shown to contribute to their adjuvant effect on IgE production.


Y

Y Mast cell

Plasma cell

Fig. 7.3 How diesel exhaust particles (DEPs) modify the immune response to allergen. DEPs impact on, and are internalized by, the airway epithelium and macrophages. The ensuing generation of oxidative stress causes the release of proinflammatory cytokines from both cell types. One outcome is increased antigen presentation because of decreased IL-10 plus upregulation of costimulatory molecules such as CD80 on antigenpresenting cells (APC). In this setting, APCs seem to favor the development of a Th2 cytokine milieu, which has a positive feedback loop on IL-4 production. DEPs have indirect effects on B-cell development and isotype switching through the secretion of IL-4 and IL-13 from Th2 cells. DEPs also have a direct effect on B cells that can drive enhanced IgE isotype switch and production. The IgE produced by the resulting antigen-specific plasma cells leads to the priming of mast cells that after antigen exposure degranulate and release cytokines such as IL-4 that produce positive feedback on both B and T cells. Finally, DEPs themselves can enhance mast cell and basophil degranulation, and cytokine release. MHC II, major histocompatibility class II; TCR, T-cell receptor. See text for definition of other abbreviations. (From Saxon & Diaz-Sanchez 2005, with permission.) (See CD-ROM for color version.)

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Effect on allergen presentation One level of action of pollutants is the modification of the immune system’s handling of the allergen. DEPs and their derived chemicals affect allergen presentation by upregulating costimulatory molecules such as CD80 and CD86 (Nel et al. 1998), and HLA-DR major histocompatibility complex class II molecule on human macrophages (Don Porto Carero et al. 2002) and nasal epithelial cells (Takizawa et al. 2007). In particular, pollutant-induced increases in allergen-induced macrophage-derived chemokine (MDC) production from human peripheral blood mononuclear cells (PBMCs) can be completely blocked by inhibition of the B7–CD28 pathway (Fahy et al. 2002). Furthermore, because DEPs and their resident chemicals may inhibit the tolerogenic cytokines IL-10 and transforming growth factor (TGF)-β while increasing IL-1 and soluble CD23 in lipopolysaccharide (LPS)stimulated PBMC/macrophages, exposure to DEPs has been suggested to be important in increasing allergenicity during the early phase of allergen presentation (Nel et al. 1998; Pacheco et al. 2001). In vivo human data support this concept by showing that primary human mucosal sensitization (IgE), as well as increased IL-4 levels, can be driven by a neoantigen (keyhole limpet hemocyanin, KLH) administered with DEPs, whereas the antigen alone leads only to a protective IgG response in allergic subjects (Diaz-Sanchez et al. 1999). Indirect effects on IgE production DEPs also augment the effect of the allergen on the immune system, by both inducing and exacerbating in vivo allergic responses in the human upper respiratory tract. In subjects challenged intranasally with DEPs, increased IgE isotype switching results in increased total IgE levels (Diaz-Sanchez et al. 1994, 1997; Fujieda et al. 1998). In atopic patients, in contrast to the two- to three-fold increase in allergen-specific IgE produced with allergen alone, combined challenge with DEPs plus allergen enhances local specific IgE production 16-fold (Diaz-Sanchez et al. 1997). Similar deviations of the IgE response have been seen in mice and rats repeatedly challenged with DEPs and ovalbumin (Muranaka et al. 1986; Fujimaki et al. 1994; Al-Humadi et al. 2002) and with PAH, in particular phenantrene, anthracene, and quinone derivatives (Heo et al. 2001; Hiyoshi et al. 2005). This potentiation of the IgE response is accompanied by an increased IL-4 and IL-13 Th2 cytokine profile evidenced both in mice (Takano et al. 1997; Yanagisawa et al. 2006; Inoue et al. 2007a) and humans, as well as a decrease in IFN-γ Th1 cytokine (Diaz-Sanchez et al. 1997). Along the same lines, in allergic subjects, diesel extracts also induce increased production of Th2-attracting chemokines (such as MDC and I-309) (Fahy et al. 2002; Senechal et al. 2003) and eosinophil-attracting chemokines (such as RANTES) (Diaz-Sanchez et al. 2000a; Fahy et al. 2000), and decreased production of Th1-attracting chemokines (such as IP-10) (Fahy et al. 2002). It is of interest that parts of these effects on the Th1/Th2 polarization of the immune

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response have also been shown in nonatopic subjects (Pourazar et al. 2004; Ohtani et al. 2005; Chang et al. 2006) or nonsensitized mice (Finkelman et al. 2004; Chan et al. 2006; Inoue et al. 2007b), suggesting that DEPs might also have a role in the genesis of allergic reactions. Moreover, DEPs and PAHs, such as benzo(a)pyrene and quinines, enhance IgEdependent histamine release and IL-4 production from human mast cells and basophils (Wang et al. 1999; Diaz-Sanchez et al. 2000b; Devouassoux et al. 2002; Kepley et al. 2003). Direct effects on B cells and IgE production In vitro studies have shown that human B cells and cell lines cultured with IL-4 and CD40 monoclonal antibodies in the presence of DEP-derived chemicals demonstrate up to a 360% increase in IgE production (Takenaka et al. 1995; Tsien et al. 1997). Moreover, DEP exposure in humans leads to the attraction of B cells in airway lavage, showing that a direct interaction can occur in the lung (Salvi et al. 1999). Thus, DEPs appear to have direct effects on B cells as well.

Gases Although not as well studied, gaseous pollutants may also modify responses to allergen. In animal models, ozone, SO2, and NO2 can all augment allergic antibody production and pulmonary inflammation after allergen challenge (Gilmour 1995; Park et al. 2001; Gershwin 2003). In particular for ozone, increases in Th2 cytokines and in IgE production have been observed after antigen challenge in mice (NeuhausSteinmetz et al. 2000), as well as in nonhuman primates (Schelegle et al. 2003). However, results of controlled human exposure studies have been more equivocal. Whereas some studies have reported enhanced airway responses to inhaled allergen after exposure to ozone (Molfino et al. 1991; Jorres et al. 1996), the combination of NO2 and SO2 (Devalia et al. 1994), or NO2 (Tunnicliffe et al. 1994; Strand et al. 1997), others have found enhanced late inflammatory or early bronchoconstrictor responses to inhaled antigen in only a subgroup of allergic asthmatic patients (Vagaggini et al. 2002; Chen et al. 2004). Overall, these data suggest that many airborne pollutants function as mucosal adjuvants and, in interacting with both innate and adaptive immune cells, skew the immune response to inhaled antigens toward a Th2-like phenotype (Saxon & Diaz-Sanchez 2005).

Mechanisms Despite our knowledge that air pollutants alter immunophysiologic outcomes, the mechanisms that underlie these outcomes are only now being elucidated. The most plausible model to explain these effects involves hierarchical oxidative stress (Xiao et al. 2003). This model postulates that with low exposure the formation of reactive oxygen species (ROS) leads to the activation of antioxidant response elements, followed by transcription of enzymes important in detoxifica-

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tion, cytoprotective, and antioxidant responses, such as phase II enzymes. At higher exposure, the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 response elements are activated, leading to NF-κB and mitogenactivated protein kinase (MAPK) signaling, and resulting in increased expression of proinflammatory and allergy-related genes. Enhanced inflammation leads to additional generation of ROS, which are normally curtailed by antioxidant defenses. With greater exposure, defenses are overwhelmed and inflammation and cytotoxic effects may occur. Experimental evidence in support of this model come from increases in markers of oxidative stress reported in both human and animal models after exposure to DEPs (Lim et al. 1998; Hiura et al. 1999; Li et al. 2002). Ultrafine particles, because of their greater relative surface area, contain significantly more organic carbons and PAHs than coarse or fine particles, and are more active in their redox cycling capacity (Li et al. 2003a), and may well have greater proinflammatory effects than larger particles. It is believed that particleassociated transition metals, such as iron and copper, generate formation of ROS through the Fenton reaction and also synergize with organic PM components in this generation (Gilmour et al. 1996; Barchowsky & O’Hara 2003). DEPs associated with PAHs and their quinone oxygenated derivatives directly induce oxidative stress. In animal models, antioxidant treatment can inhibit DEP-enhanced IgE production and its proinflammatory effects (Whitekus et al. 2002). PAHs and quinone derivatives have been shown to modify IL-10, RANTES, and macrophage inflammatory protein (MIP)-1α chemokine production (Shi et al. 1996; Nel et al. 1998), all able to modulate IgE production (Kimata et al. 1996; Jeannin et al. 1998). Moreover, DEPs and their resident chemicals can activate the redox-sensitive transcription factors N-terminal Jun kinase and p38 MAPK (Ng et al. 1998), and NF-κB pathways, leading to expression of proinflammatory cytokines, chemokines, and adhesion molecules (Ng et al. 1998; Takizawa et al. 1999; Fahy et al. 2000; Wang et al. 2005). DEPs and their associated PAHs can also activate phase II drug metabolizing enzymes (Li et al. 2000, 2004). Recently, it has been shown that B cells can make a cytoprotective response to diesel extracts by increasing such an enzyme, the NADPH quinone oxidoreductase (NQO1), and that when this response was sufficiently elevated it could block the IgE potentiation effect of DEP extracts (Wan & Diaz-Sanchez 2006). Therefore, oxidative stress can also be partly protective (Fig. 7.4). This hierarchical oxidative stress model can probably be extended to gaseous pollutants and thus underlies the adjuvant effects of air pollution in general. Ozone is a potent oxidant that produces free radicals and ROS. The epithelial surface of the respiratory tract is rich in antioxidants, such as glutathione and ascorbate. In controlled exposure experiments, ozone induces considerable depletion of this antioxidant storage (Mudway et al. 1999). Similarly, SO2 inhalation


Reactive oxygen species/ oxidative stress

Sensor/detector ASK1?

Keap 1 ?

MAPKs, e.g. JNK

NK-kB kinases

Nrf2

AP-1 RE

NK-kB RE

ARE

Transcription of: (a) Cytokine (b) Chemokines (c) Adhesion molecules

Transcription of: (a) Heme oxygenase-1 (b) Phase II enzymes, e.g. GST, NQO1

Fig. 7.4 Cellular sensors and signaling pathways involved in oxidative stress. Reactive oxygen species (ROS) generation and/or oxidative stress is detected by cellular sensors. The possible candidates for the sensors include ASK1 for the AP-1 pathway and Keap1 for the ARE pathway. These afferent components activate the mitogen-activated protein kinase (MAPK) cascade and, in the case of Keap1, lead to the release of the transcription factor, Nrf2, to the nucleus. The sensor for the NF-kB cascade is unknown, but ultimately leads to the phosphorylation and degradation of IkBa, thereby releasing the attached Rel protein to the nucleus. Binding of AP-1 and/or NF-kB transcription factors to their respective DNA-binding sites eventually leads to the production of cytokines, chemokines, and adhesion molecules. These products exert proinflammatory effects. The binding of Nrf2 to ARE results in the expression of heme oxygenase 1 (antioxidative) and phase II (detoxifying) enzymes. These products are cytoprotective. GST, glutathione S-transferase; NQO1, NADPH quinone oxidoreductase. (From Li et al. 2003b, with permission.) (See CD-ROM for color version.)

affects the intracellular glutathione redox state in airway epithelial cells (Todokoro et al. 2004), as well as NO2 (Kelly et al. 1996). In conclusion, there is growing evidence that pollutantinduced oxidative stress may be responsible for generating IgE production. Moreover, there is the realization that not all oxidative stresses are injurious; there is also a protective level of oxidative stress that could form the basis of disease susceptibility. However, the complex gene–environment interactions involved need to be further deciphered to understand the subtleties involved in pollution effects.

Genetics The results discussed above showing the underlying role of oxidative stress in the proinflammatory and proallergic effects of pollutants suggest that the key to protection from such pollutants is to mount an effective cytoprotective

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response. It follows that people with diminished ability to detoxify xenobiotics and metabolize ROS are at increased risk for adverse outcomes from pollutant exposure. Controlled human exposure studies have shown a large interindividual variation in responses to pollutants such as SO2, ozone, and DEPs (Horstman et al. 1986; Holz et al. 1999; Bastain et al. 2003), leading to the idea of pollutant “susceptibility genes.” Members of the glutathione S-transferase (GST) superfamily of phase II xenobiotic metabolizing enzymes, GSTP1 and GSTM1 are proving to be ideal candidate genes. Their products are present in the respiratory tract and they have common variant alleles with decreased or abolished enzyme function, which are associated with asthma (Fryer et al. 2000; Gilliland et al. 2002). The frequency of the homozygous GSTM1-null and of the GSTP1-Ile/Ile genotype is very high, around 40% (London et al. 1995). Taken together, these data suggest that a decreased ability to mount an effective cytoprotective response to pollutants in relation with a GST polymorphism may increase the allergic airway response. This hypothesis was confirmed in allergen-sensitive patients by nasal provocation challenges with allergen alone, or plus DEPs. Individuals with low responsive genotypes (GSTM1null or GSTP1-Ile/Ile) showed enhanced susceptibility to the adjuvant effect of DEPs, but not to allergen alone (Gilliland et al. 2004). These studies have been extended by showing that these genes are also involved in susceptibility to ozone in children who live in high-ozone areas in Mexico (Romieu et al. 2004, 2006). Asthmatic children with the nonfunctional variant GSTM1-null had greater ozone-related decreases in forced expiratory flow than children who received antioxidant supplementation with vitamins C and E. Interestingly, this population of GSTM1-null children with high ozone exposure were at significantly reduced risk of asthma when they also carried the protective NQO1 Pro-Ser polymorphism, whereas children with GSTM1 carrying the serine allele did not show modified risk of asthma (David et al. 2003). These data suggest a hierarchy of genes determining susceptibility, but also the complexity of the gene–environment interactions. In mice, other susceptibility genes involved in lung hyperpermeability to ozone have been located on chromosome 4, which contains the TLR4 gene, a regulator of innate immune responses (Kleeberger et al. 2000). It is of note that TLR4-deficient mice do not exhibit airway hyperreactivity after subchronic ozone exposure as do wild-type mice (Hollingsworth et al. 2004). Susceptibility to ROFA may similarly be associated with TLR4 (Cho et al. 2005). Tumor necrosis factor (TNF)-α has also been identified as a candidate gene in ozone susceptibility in mice (Kleeberger et al. 1997); however, in humans it is not clear if the –308G polymorphism of this gene protects (Li et al. 2006) or not (Yang et al. 2005) from ozone exposure.

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Tobacco Particulates are also a significant problem in indoor environments, with environmental tobacco smoke (ETS) being perhaps the most remediable source of indoor PM. Tobacco smoke is a complex mixture of more than 5000 components including SO2, NO2, particles, and PAH (Leikauf et al. 1995). An epidemiologic survey in the 1990s in the USA showed that 87.9% of nontobacco users had detectable levels of cotinine, a surrogate of ETS exposure (Pirkle et al. 1996). Epidemiologic studies have shown a link between tobacco and asthma, in particular in children. Metaanalyses found a dose-dependent increase in children’s rates of asthma related to increasing number of household smokers (Cook & Strachan 1997). Recent studies have addressed the role of prenatal versus postnatal exposure. In utero exposure to maternal smoking without subsequent ETS exposure was associated with asthma in school-age children (Gilliland et al. 2001), and the risk of developing asthma among children 7 years of age was increased with the mother’s smoking rates in pregnancy (Jaakkola & Gissler 2004). The finding that maternal smoking during pregnancy has a stronger relationship to asthma onset than later ETS exposures is supported by other studies that separated postnatal exposures from maternal in utero exposures (Cunningham et al. 1996). Recently, an increased risk of adult-onset asthma has been reported in relation to ETS exposure (Jaakkola et al. 2003; Piipari et al. 2004).

Effect on allergic primary and secondary responses Tobacco smoke is considered a cofactor in promoting IgE production in humans. Serum IgE, IL-4, and IL-13 are higher in smokers than in nonsmokers (Byron et al. 1994; Sherrill et al. 1994; Oryszczyn et al. 2000; Cozen et al. 2004), and are possibly higher in ETS-exposed subjects (Ronchetti et al. 1990; Oryszczyn et al. 2000; Feleszko et al. 2006). A recent experimental study has definitely shown that in humans ETS exacerbates allergic reactions by evidencing, in nasal lavages from allergic patients challenged with both allergen and ETS, enhanced allergen-specific IgE and Th2 cytokine milieu as compared with allergen alone (Diaz-Sanchez et al. 2006). However, few animal studies have addressed the mechanistic effects of tobacco on allergic responses. These few studies have shown that ETS exposure enhances secondary allergic responses to inhaled allergen in mice, in promoting IgE antibody, Th2 cytokines, peripheral eosinophilia (Seymour et al. 1997), and airway hyperresponsiveness (Moerloose et al. 2005). These results may be explained in part by PAHs contained in ETS and the generation of oxidative stress (see Pollutant section). Indeed, cigarette smoke is an incredibly potent oxidant mixture (Church & Pryor 1985), which is expected to induce ROS leading to the activation of a Th2

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pattern response, although this hypothesis awaits experimental evidence. The question arises whether environmental noxious agents, such as cigarette smoke, could be one of the mechanisms responsible for the suppression of the normal tolerogenic status to harmless allergens (Holt & Mcmenamin 1989). Animal studies have evaluated the effect of ETS on primary allergic sensitization and found increased IgE responses to harmless allergens in rats (Zetterstrom et al. 1985) and in mice, and induced airway eosinophilia on restimulation with allergen in mice (Rumold et al. 2001). More recently, ETS effect on primary sensitization in mice was found to be associated with increases in lung tissue dendritic cells, suggesting that the mechanism involved might involve altered antigen presentation (Moerloose et al. 2006).

Genetics Asthma gene linkage analysis has been performed, including ETS as a potential risk factor for asthma. Genes were found in three chromosomal regions (1p, 5q, 9q) that might interact with ETS to confer risk of asthma (Colilla et al. 2003). Chromosome 1p contains the GSTM1 gene. In the GST gene family, GSTP1 and GSTM1 variant alleles have been associated with asthma in smokers. In utero exposure to secondhand smoke has been associated with increased prevalence of early-onset asthma in GSTM1-null children (Gilliland et al. 2002), as well as passive smoking (≥ 20 cigarettes per day) (Kabesch et al. 2004). In ETS preexposed allergic subjects challenged with allergen, GSTM1-null individuals had a larger increase in allergen-specific IgE than nonaffected individuals (Gilliland et al. 2006). The 5q region was confirmed to interact with ETS exposure in infancy and asthma development (Meyers et al. 2005). This region contains genes coding for IL-13 and CD14, both of which have been linked to ETS and serum IgE levels by candidate gene approaches. Maternal smoking was found to increase the effects of –1055C→T and Arg130Gln IL-13 polymorphisms on total serum IgE level (Liu et al. 2003). Cigarette smoke also contains endotoxin, and passive smoking results in exposure levels of endotoxin that are 120 times higher than in smoke-free air. The CD14 gene encodes part of the endotoxin receptor and the CD14 −159 polymorphism has been associated with decreased IgE levels in ETS-exposed subjects (Choudhry et al. 2005). It is of interest that opposite effects of this polymorphism have been observed on IgE levels in children recruited from the same population but raised in environments with different levels of microbial exposure (see discussion on endotoxin above), suggesting that the environment may elicit different, and even opposite, phenotypes. One possible explanation is the involvement of epigenetic mechanisms able to affect gene expression, as exemplified by the effect of different amounts of folic acid in diet (required for DNA


methylation) in early life which change the coat color of agouti mice (Vercelli 2004). The extent to which environmental factors may provoke epigenetic responses represents a very exciting area of future research.

Endotoxin The “hygiene hypothesis” (see Chapter 2) proposes an association between the decreased exposure to microbes in early life and the increased incidence of atopic diseases in the recent decades. Exposure to bacteria and their relationship with atopy has thus attracted much scientific interest. Special attention has been focused on endotoxin, which comprises the outer LPS component of the cell wall of Gram-negative bacteria. Although the word “endotoxin” is used in epidemiologic literature, the effect of “LPS” is analyzed in experimental studies. Epidemiologic studies in humans have found that exposure to endotoxin can protect against, have no effect on, or exacerbate asthma. Similarly, LPS has variable effects on allergic pulmonary inflammation in the mouse depending on exposure time points, doses, and administration procedures of LPS, as well as the model of allergic sensitization. Clearly, this is a complex issue.

Endotoxin: beneficial or detrimental? Children raised on farms have been shown to be at lower risk for allergic sensitization to inhalant allergens. Studies analyzing rural communities in Switzerland and Austria found that farmers’ children who had regular contact with livestock were significantly less likely to demonstrate an elevated level of allergen-specific IgE (Braun-Fahrlander et al. 1999; Riedler et al. 2000). In a search for the protective factor in livestock exposure, endotoxin levels were measured in households and found to be associated with farming and nonfarming lifestyle (Von Mutius et al. 2000). A cross-sectional study in rural areas of Austria, Germany, and Switzerland showed that exposure to stables and farm milk in the first year of life was protectively associated with serum allergenspecific IgE compared with children with no such exposure in their first year. This suggests that the protective factor mediated its effect early in life (Riedler et al. 2001; Stern et al. 2007). Protection from allergic sensitization by early exposure to endotoxin was also found in nonfarming children (Gereda et al. 2000a; Gehring et al. 2002). However, no relationship was found between high endotoxin levels and sensitization to food allergens nor atopic dermatitis, but stratification by parental atopy showed a positive association of endotoxin exposure with sensitization to inhalant allergens in infants with parental atopy (Bolte et al. 2003). Finally, many studies have shown that endotoxin increases asthma severity in adults, but did not report their IgE levels (Michel et al. 1991).

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The dual effect of endotoxin was further analyzed using animal models. Timing of LPS exposure determines the outcome on IgE production, early exposure being associated with protection. Prenatal and neonatal LPS exposure diminished OVA-specific IgE, and suppressed IL-5 and IL-13 production while strongly enhancing IL-10 and IFN-γ production by spleen cells of OVA-sensitized mice (Gerhold et al. 2006). In a rat model, timing of LPS exposure modifies its effect on IgE production: early aerosol LPS inhibited the increase in OVAspecific IgE, while late exposure did not alter the serum antibody levels in sensitized animals (Tulic et al. 2000). The site of LPS injection differentially affects Th1 cytokine secretion, as both systemic and local LPS administration before OVA sensitization reduced serum OVA-specific IgE, and diminished Th2 cytokine production by splenic mononuclear cells, but only local LPS administration induced IFN-γ production by peribronchial lymph nodes (Gerhold et al. 2002). Finally, the effect of LPS exposure is also dependent on the dose of endotoxin. Indeed, mice sensitized by intranasal exposure to OVA plus a high or low dose of LPS showed a bimodal immune response. In contrast to low levels of LPS, which induce Th2 responses, inhalation of high levels of LPS with antigen induces Th1 responses (Eisenbarth et al. 2002). However, both doses of LPS increased serum IgE levels (Kim et al. 2007).

Association with expression of receptors for endotoxin The expression of, and polymorphisms in, the genes encoding endotoxin receptors have been linked to allergic diseases. Two TLRs were found to be differentially involved in endotoxin recognition: many LPS species (from Gram-negative bacteria) signal through TLR4 (Poltorak et al. 1998), while a few LPS species signal through TLR2 (Table 7.1) (Werts et al. 2001; Girard et al. 2003). CD14 is an important cofactor in binding endotoxin and initiating the immune response (Wright et al. 1990). Expression of mRNA encoding TLR2, TLR4, and CD14 was significantly higher among farm children than reference children, and was associated with endotoxin maternal exposure during pregnancy, which was found to be protective on atopic sensitization of the child (Ege et al. 2006). Similarly, prenatal and postnatal LPS exposure, which prevents allergic sensitization, inflammation, and hyperresponsiveness, increased soluble CD14 (sCD14) serum levels and TLR2 and TLR4 mRNA levels in lung tissues from OVA-sensitized mice (Gerhold et al. 2006). In contrast, low sCD14 levels in amniotic fluid or breast milk were found to be associated with an increased risk of developing atopic sensitization (Jones et al. 2002).

Table 7.1 Toll-like receptors (TLR) involved in the recognition of bacteria, viruses, and helminths. (From Duez et al. 2006, with permission.) Receptors

Microbial ligands

TLR2/TLR1 or TLR6

Diacyl lipopeptide (Pam2CSK4) (TLR2/TLR6), triacyl lipopeptide (Pam3CSK4) (TLR1/TLR2) Lipoteichoic acid (TLR2/TLR6) Lipoarabinomannan (Mycobacterium tuberculosis) (TLR2/TLR6) Porins (Neisseria meningitidis) (TLR2/TLR6) Macrophage-activating lipopeptide 2 (MALP2) (TLR2/TLR6) Glycophosphatidylinositol (Trypanosoma cruzi) Viral protein (herpes simplex virus) Phospholipomannan (Candida albicans)

TLR3

Double-stranded RNA from viruses or helminths (Schistosoma mansoni)

TLR4

Lipopolysaccharide (also involves CD14, MD2 molecules) F protein (from the respiratory syncytial virus) Phosphatidylinositol mannosides (Mycobacterium tuberculosis) Glycoinositol phospholipid ceramides (Trypanosoma cruzi)

TLR5

Flagellin

TLR7, TLR8

Guanosine and uridine-rich single-stranded RNA

TLR9

Bacterial and viral DNA

TLR11

Profilin (Toxoplasma gondii )

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Functional mutations in the genes encoding these receptors were found to be associated with risk of atopy. The CD14 gene was identified as the nearest gene to the loci most closely associated with total serum IgE (D5S399/D5S393 on chromosome 5q31–33) (Gao et al. 1999). A C→T transition at position –159 in the promoter of the gene encoding CD14 was associated with increased levels of serum circulating sCD14, which were positively and negatively related to IFN-γ and IL-4, respectively (Baldini et al. 1999). Several studies have found consistent associations between the CD14 –159TT genotype and lower levels of total IgE (Baldini et al. 1999; Koppelman et al. 2001; Buckova et al. 2003; Leung et al. 2003; O’Donnell et al. 2004), whereas others have not (Sengler et al. 2003; Kedda et al. 2005). Environment (endotoxin or allergen exposure) may greatly differ between the populations considered in these studies and gene–environment interactions might explain these discrepancies (Eder et al. 2005). Other studies suggest that the −159C→T CD14 promoter polymorphism might have a differential effect according to the dose and the timing of LPS exposure, and might influence the prevalence of atopy at different ages, as well as the degree of atopy among those already sensitized (Leynaert et al. 2006; Simpson et al. 2006). Results obtained with TLR4 polymorphisms are just as complex: 29 single-nucleotide polymorphisms identified in the TLR4 locus were analyzed in two different cohorts, but none were found to be associated with asthma or total serum IgE (Raby et al. 2002; Werner et al. 2003), whereas asthmatic people with the D299G polymorphism exhibit an increased severity of atopy (Yang et al. 2004). Analysis of polymorphisms in the TLR2 gene has shown that adult patients with atopic dermatitis with the TLR2 R753Q polymorphism had significantly higher total IgE levels (Ahmad-Nejad et al. 2004). However, in children with asthma, no association was found between polymorphisms in TLR2 and total IgE levels (Eder et al. 2004; Noguchi et al. 2004). In conclusion, the protective response to endotoxin exposure appears to be dependent in part on the time of exposure and on the receptors involved in endotoxin recognition. However, one might still elucidate why, paradoxically, a mutation that is thought to diminish responsiveness to LPS also appears to decrease the risk for atopy in highly exposed individuals. More studies are needed to clarify the gene– environment interaction.

Mechanisms of endotoxin effects Endotoxin exposure clearly modulates the profile of the immune response. Increased house-dust endotoxin concentration correlated with increased proportion of IFN-γproducing CD4 T cells, suggesting that indoor endotoxin exposure early in life may protect against allergen sensitization by enhancing type 1 immunity (Gereda et al. 2000b). In mice, an inhibitory effect of LPS was found to be dependent


on IL-12 (Gerhold et al. 2002) or nitric oxide synthase 2 activity (Rodriguez et al. 2003). Costimulation of explanted nasal mucosa from atopic children with allergen and LPS abrogated Th2 cytokine expression and increased Th1 cytokine production, as well as the number of IL-10+TLR4+ cells and CD4+CD25+ cells, when compared with allergen stimulation alone. LPS-induced Th1 cytokine production was dependent on IL-10, IL-12, and IFN-γ. LPS effect appeared diminished in adults compared with children (Tulic et al. 2004). In a mouse model, protection by LPS administration paralleled the generation of CD25+IL-10+ T cells (Wang & McCusker, 2006). Finally, a marked downregulation of immune responses was found in exposed children: the production of TNF-α, IFN-γ, IL-10, and IL-12 by peripheral blood leukocytes stimulated with LPS was shown to be inversely related to the endotoxin level in the bedding (Braun-Fahrlander et al. 2002). These findings suggest that LPS may downregulate allergic response through Th1 skewing and/or the expansion of regulatory T cells, but that these effects were more prominent in children, whose developing immune system is still susceptive. Different cytokines have been found to be involved in the IgE-enhancing effects of LPS, depending on the LPS dose. Thus, OVA-specific IgE and IL-4 production enhanced by low-dose LPS was dependent on TNF-α, whereas production enhanced by high-dose LPS was dependent on IL-4 and IFN-γ. IL-12p40 levels in bronchoalveolar lavage fluid were significantly higher in mice sensitized with OVA plus highdose LPS than in those sensitized with OVA alone or OVA plus low-dose LPS (Feleszko et al. 2007). TLR4 and dendritic cells play crucial roles in LPS effects. tlr4-deficient mice subjected to sensitization and pulmonary challenge with OVA had reduction in allergen-specific IgE levels and Th2 cytokine production compared with wildtype mice. The reduced response was attributable, at least in part, to decreased dendritic cell function, as dendritic cells from tlr4-deficient mice expressed lower levels of CD86 and induced less Th2 cytokine production by naive CD4 T cells in vitro (Dabbagh et al. 2002). Similarly, Th2 response induced by low-level inhaled LPS was shown to be mediated by signaling through TLR4, and activation of dendritic cells (Eisenbarth et al. 2002). Inhibition of Th2 responses by LPS was also found to be dendritic cell dependent, but IL-12 independent (Kuipers et al. 2003). Moreover, stimulation of TLR4 on dendritic cells was described to remove the suppressive effect of CD4+CD25+ regulatory T cells on effector T cells, suggesting that ineffective TLR4 signaling may result in unopposed inhibition of both Th1 and Th2 expression by regulatory T cells (Pasare & Medzhitov 2003). MyD88 is a critical common adaptor molecule shared by TLR. The MyD88-independent LPS signaling pathway induces Th2 cell development and is responsive to LPS at low concentration, whereas the MyD88-dependent pathway dominates at

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LPS

Viruses TLR4 EC Mast cell

NKT

Treg CD4+CD25+

BEC

Cell recruitment

mDC

Low High dose of LPS Th1

IL-13

IL-4 (early)

Th2

Increased suppressive function

B cell/plasmacyte Increased IgE Fig. 7.5 Effect of lipopolysaccharide (LPS) on IgE production. Differences in timing, dose, and compartment targeted (local/systemic) by LPS result in the stimulation of different cell types through TLR4, which leads to activation or suppression of IgE production. Plain lines indicate activation, dotted lines inhibition EC: Endothelial cells, BEC: bronchial epithelial cells, MDC: myeloid dendritic cells. (See CD-ROM for color version.)

high LPS concentration and favors Th1 cell development (Kaisho et al. 2002; Piggott et al. 2005). Finally, long-term allergen exposures of tlr4-deficient mice dramatically increased serum IgE and Th2 cytokines compared with similarly challenged wild-type mice, suggesting that tlr4 functions in an antiinflammatory pathway that limits the extent of allergic responses to continued allergen challenge (Hollingsworth et al. 2006). The effect of LPS on allergic sensitization is likely to be the result of the activation of multiple cells expressing LPS receptors (Fig. 7.5). Mast cells, which were found to be involved in Th2 cytokine production induced by low-dose LPS, express TLR4 and on LPS stimulation produced IL-5, IL-13, TNF-α, and IL-6 (the later cytokines being involved in dendritic cell maturation and participating in Th2 polarization, respectively) (Nigo et al. 2006). TLR4 is expressed on CD4+CD25+ T regulatory cells, and LPS activation of CD4+CD25+ cells increases cell survival/proliferation and suppressor efficiency (Caramalho et al. 2003). Functional TLR4 is expressed on invariant Vα14+Jα18+ natural killer (NK)T cells, and in vivo and in vitro LPS stimulation induced rapid production of IL-4 in hepatic iNKT (Askenase et al. 2005). Endothelium and epithelium also express TLR4 and, on LPS stimulation, upregulate the expression of adhesion molecules and increase their production of chemokines and proinflammatory cytokines (Monick et al. 2003; Dauphinee & Karsan 2006). All these mechanisms may contribute to the regulation of the IgE production and explain the apparently conflicting data of the literature.

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Clinical observations and experimental data clearly suggest that viruses may inhibit or exacerbate atopic disorders depending on the virus and the conditions of infection. Seropositivity for hepatitis A was associated with a lower prevalence of atopy (Matricardi et al. 1997; Linneberg et al. 2003), and this has now been linked to the TIM1 gene. TIM-1 functions as a receptor for hepatitis A and is expressed on Th2 cells (McIntire et al. 2001, 2004). Recent studies have characterized several TIM1 polymorphisms in human subjects and also demonstrated that hepatitis A seropositivity protects against atopy, but only in individuals with a specific insertional mutation in the sequence corresponding to amino acid position 157 of TIM1. In contrast, measles vaccination or infection was associated with a higher frequency of atopy (Shaheen et al. 1996; Paunio et al. 2000). Respiratory viruses (respiratory syncytial virus or RSV, rhinoviruses, influenza, and parainfluenza viruses) can also exacerbate atopy and the symptoms of asthma (Sigurs et al. 1995, 2005; Mallia & Johnston, 2002; Umetsu 2004). The role of RSV has been extensively studied and found to have either no effect or accelerate the rate of sensitization depending on the severity of infection and the timing of atopy assessment (Murray et al. 1992; Sigurs et al. 1995; Noble et al. 1997; Schauer et al.2002).

Regulation of IgE production by viruses Viral infections generally induce a Th1 cytokine pattern. It is therefore not surprising that children who had hepatitis A or B virus infection had lower levels of allergen-specific IgE than controls (Kocabas et al. 2006). However, increases in systemic IgE levels have been found following infection with several other viruses like the Epstein–Barr virus, cytomegalovirus, and the measles virus (Griffin et al. 1985), and following vaccination with influenza in nonallergic subjects (Davidsson et al. 2005). In allergic rhinitis subjects, total serum IgE levels have been noted to be increased following experimental rhinovirus infection (Skoner et al. 1995). Increases in IgE levels could be viral- or allergen-specific or could simply represent a polyclonal upregulation of IgE production. In some patients viral infections seem to induce allergic reactions directly. The most prominent example is the induction of acute urticaria shortly after viral infections, especially in children, suggesting the presence of virus-specific IgE (Mortureux et al. 1998). RSV-specific IgE has been detected after infection, the magnitude of the response correlating with degree of wheezing, and children with an atopic predisposition developed an IgE response more readily (Welliver et al. 1981). RSV-specific IgE was also detected in some studies in nasopharyngeal secretions and was significantly higher in infants with wheezing (Welliver et al. 1981), whereas others failed to measure RSV-IgE antibodies in nasal washes

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from children with wheezing (Toms et al. 1996; De Alarcon et al. 2001). Higher virus load associated with more severe illness may be needed to detect the presence of RSV-IgE (Russi et al. 1993; Rabatic et al. 1997). Specific IgE antibodies to parainfluenza virus have also been demonstrated (Welliver et al. 1982). Virus-specific IgE has been reproduced in mouse models, as mice infected with influenza A virus produce virus-specific IgE antibodies and develop active and passive cutaneous anaphylaxis with the influenza A virus antigen (Grunewald et al. 2002). Similarly, infection with RSV induces serum RSV-IgE (Dakhama et al. 2004). The role of IgE in the course of viral infection is unknown and has still to be elucidated. Viruses have also been shown to increase allergen-specific IgE. In a small study of 12 children with asthma attacks precipitated by influenza, the levels of HDM-specific IgE increased during the acute phase, although total serum IgE levels remained unchanged (Lin et al. 1988). Similarly, increased allergen-specific IgE has also been observed after infection of OVA-sensitized mice with influenza virus (Suzuki et al. 1998). Prospective study of a cohort of children hospitalized with RSV bronchiolitis in infancy found that serum IgE antibodies to common allergens were higher in this group compared with the control group, up to age 13 (Sigurs et al. 2000, 2005). The timing of RSV infection is critical for RSV effects on IgE production. Indeed, prior inoculation with RSV decreased the concentration of both total and OVA-specific IgE in serum of OVA/RSV-treated mice, compared with OVA/RSV-treated mice that were not previously inoculated with RSV (Barends et al. 2004). However, RSV infection before OVA sensitization did not enhance the production of OVA-specific IgE (Schwarze et al. 1997). Finally, in vitro, a human B-cell line treated with IL-4 and infected with measles virus increased its IgE class switching in comparison with control cells treated only with IL-4, suggesting that measles virus may increase the production of IgE during infection in allergic patients (Imani et al. 1999). RSV activation of the antiviral protein kinase in B cells was also shown to induce isotype switching to IgE in vitro (Rager et al. 1998). Therefore, virus effect on IgE production appears to depend on the virus, the severity of infection, and the timing of exposure and assessment. Moreover, it is likely that exposure to other environmental factors (as was shown with allergen) and genetic predisposition might affect the outcome of virus infection on allergic diseases. This latter is suggested by a study comparing the effect of influenza A viral infection on OVA-induced responses in C57BL/6 mice and BALB/c mice. Indeed, prior influenza A viral infection of C57BL/6 mice, which have a Th1 bias compared with BALB/c mice, prevented Th2-type allergic responses (Wohlleben et al. 2003).

Virus-induced mechanisms Most studies devoted to understanding the effect of viral infection on IgE production have focused on respiratory


viruses and their regulation of the production of Th1- and/or Th2-type cytokines. Virus infection alone was found to lead to Th1 cytokine production, both in respiratory secretions and PBMCs (Van Schaik et al. 1999; Gern et al. 2006). In mice, influenza A respiratory infection leads to high local IFN-γ production by CD4+ and CD8+ T cells, and strong Th1 responses in lymph nodes, while a low to undetectable amount of ex vivo Th2 cytokine expression is detected (Doyle et al. 1999; Grunewald et al. 2002; Roman et al. 2002). However, the initial induction of RSV-IgE antibody production is likely to be dependent on the Th2 response. Consistent data from clinical studies described a significant local and systemic Th2 response that was associated with RSV bronchiolitis, influenza, and parainfluenza infections in infants (Roman et al. 1997; Bendelja et al. 2000; Pala et al. 2002; Tripp et al. 2002; Legg et al. 2003; Kristjansson et al. 2005; Murai et al. 2007). RSV protein G was found to induce a Th2-like immune response both in humans and mice (Alwan et al. 1993; Jackson & Scott 1996). In mice, lung infection with RSV resulted in significant increases in mRNA expression for IL-4, IL-13, CD40, and IgE, and for both of the high- and low-affinity receptors in the lungs (Johnson & Graham 1999; Varga et al. 2000; Dakhama et al. 2004). IL-13 production was elicited upon reinfection only if RSV first infection occurred early in life with live viruses, emphasizing the requirement for active lung infection (Dakhama et al. 2005). Both these Th1 and Th2 responses induced by respiratory viruses might play a role in exacerbating asthma. KLH sensitization of BALB/c mice after influenza A viral infection and clearance promoted dual allergen-specific Th1 and Th2 responses and enhanced later allergen-specific asthma. This effect was dependent on IFN-γ secreted during acute viral infection and on lung dendritic cells (Dahl et al. 2004). Viruses have been found to upregulate some chemokines involved in IgE production, as well as Th1 and Th2 cytokines. Mononuclear cells from infants with severe respiratory tract illness stimulated in vitro with RSV produce RANTES and MIP-1α (Tripp et al. 2002). Significantly higher levels of RANTES and MIP-1α have been detected in nasopharyngeal secretions of RSV-, influenza virus- or parainfluenza virusinfected infants (Kristjansson et al. 2005; Murai et al. 2007). Moreover, RSV alone, or in combination with IFN-γ, increases the secretion of RANTES from respiratory epithelial cells (Saito et al. 1997; Olszewska-Pazdrak et al. 1998). All this may contribute to the exacerbation of allergic diseases. Molecular events involved in the regulation of Th1 and Th2 cytokine production induced by viruses have still to be identified, but some TLRs will certainly play a role. Indeed, rhinovirus and RSV are partly recognized by TLR3 and TLR4 (associated with CD14) (see Table 7.1), and were also found to increase the expression of these TLRs on airway epithelial cells (Kurt-Jones et al. 2000; Hewson et al. 2005; Groskreutz et al. 2006).

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Therefore, respiratory viruses may exacerbate asthma and IgE production not only by increasing the production of Th1 and Th2 cytokines and several chemokines, but also by modifying the response to subsequent exposure of other environmental factors.

Parasites Underdeveloped nations are relatively protected from the allergy epidemic, but are more chronically infected with various parasite worms than industrialized nations. A hallmark of a helminth infection is the large amount of IgE detected in the serum of infected animals and humans (Yazdanbakhsh et al. 2001). Helminths induce the production of large amounts of polyclonal IgE and very low levels of helminth antigen-specific IgE, the latter providing an important component of the immune protection (Lynch et al. 1993; Capron & Capron 1994; Allen & Maizels 1996).

Parasites and allergen-specific IgE Despite this enhanced total IgE production associated with helminths, a consistent negative relation between helminth infections and the prevalence of atopic responses has been seen in various developing countries (Van Den Biggelaar et al. 2000; Nyan et al. 2001; Yazdanbakhsh et al. 2001, 2002; Huang et al. 2002; Cooper et al. 2003). Along the same lines, long-term antihelminthic chemotherapy increased HDM reactivity in chronically infected children (Van Den Biggelaar et al. 2004). Experimental animal models also support a dual effect of helminths on allergen-specific and total IgE production. In mice, infection with Strongyloides stercoralis or Nippostrongylus brasiliensis leads to a reduction of allergenspecific IgE in the bronchoalveolar lavage fluid, whereas serum total IgE is increased (Wang et al. 2001; Wohlleben et al. 2004). In a model of food allergy, infection with the enteric helminth Heligmosomoides polygyrus resulted in a decrease of allergen-specific IgE production in the serum (Bashir et al. 2002). However, the role of helminths in protecting against allergies is not universally accepted, as several studies do not show a protective role (Cooper et al. 2006; Karadag et al. 2006). One explanation for contradictory observations may be provided by a paradigm in which lowlevel or acute helminth infections enhance allergic reactivity and high-level or chronic infections suppress allergic inflammation (Cooper 2002).

Mechanisms involved in IgE regulation by parasites The role of Th2 responses in protection against helminth infection has been extensively documented (Lynch et al. 1993; Capron & Capron 1994; Allen & Maizels 1996). An absolute requirement for IL-4 receptor-dependent mechanisms in the clearance of worms has been demonstrated

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(Urban et al. 1998), and thus it is not surprising that such infections lead toward a Th2 polarization. Indeed, Ascaris lumbricoides infections are associated with a highly polarized type 2 cytokine response as indicated by increased IL-4 and IL-5 production by mononuclear cells from infected patients (Cooper et al. 2000). Schistosoma mansoni egg-laying male and female worms and, more particularly, schistosome eggs are potent inducers of Th2 immune responses when injected into mice (Vella & Pearce 1992; Mangan et al. 2006). Mechanisms may partly involve recognition of schistosome N-glycans by CD1d, which induces Th2 polarization in vitro (Faveeuw et al. 2002, 2003). Helminth infections have also been associated with suppressing the development of allergic responses and appear to be, at least in part, associated with the induction of T regulatory responses (Kamradt et al. 2005; Maizels 2005). The regulatory cytokines IL-10 and TGF-β are produced after helminth infection (Mahanty & Nutman 1995; Doetze et al. 2000; Van Den Biggelaar et al. 2000; Cooper 2002). Protection against S. mansoni worms was suggested to involve compartmentalization of immunity, with a helminth-modified type 2 pulmonary immune response characterized by elevated IL10, specific IgG4 antibodies, and reduced IL-5 in the lungs (Mangan et al. 2006). Heligmosomoides polygyrus, S. mansoni and N. brasiliensis have been shown to prevent allergen-induced responses in an IL-10-dependent mechanism (Bashir et al. 2002; Mangan et al. 2004; Wohlleben et al. 2004; Kitagaki et al. 2006). Finally, increased natural regulatory CD4+CD25+Foxp3+ cells were found in thoracic lymph nodes of H. polygyrusinfected/OVA-exposed mice (Kitagaki et al. 2006). In contrast, the suppressive effect of N. brasiliensis excretory/secretory products on the development of allergic responses was found to be independent of the presence of TLR2 and TLR4, IFN-γ, and IL-10 (Trujillo-Vargas et al. 2007). However, these TLRs and others may potentially participate in the regulation of the T-cell response and IgE production, as other helminthderived products, such as helminth RNA, glycolipids, and profilin signal through TLR3, TLR2 and TLR4, and TLR11, respectively (see Table 7.1) (Campos et al. 2001; Oliveira et al. 2004; Aksoy et al. 2005; Yarovinsky et al. 2005). Another mechanism for reduced IgE in certain helminth infections may involve chemokine inhibition, as S. mansoni, S. haematobium and S. japonicum eggs were found to produce a chemokine-binding protein able to block the interaction of MIP-1α and RANTES with host chemokine receptors and their biological activity (Smith et al. 2005). Recent data suggest an evolutionary balance between allergy and helminth infection. Human polymorphisms originally described as predisposing toward asthma have now been linked to resistance to helminth infection. A polymorphism in the 3′ UTR of STAT6 was linked to differential resistance to Ascaris in a Chinese population, as well as to asthma in Japan (Peisong et al. 2004). Similarly, an IL-13

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promoter allele (1055T) first identified as increasing gene transcription, and thereby asthma risk, has subsequently been shown to confer greater resistance to schistosome infection in Mali (Kouriba et al. 2005). These findings suggest that one mechanism for increased susceptibility to allergy may be human evolutionary adaptation to ubiquitous helminth infection.

Conclusion Our expanding knowledge of the effects of environmental factors on IgE production (Fig. 7.6) has broad implications for unraveling the complex factors leading to both allergic disease pathogenesis and the ongoing marked increase in the prevalence of IgE-mediated disease. It is anticipated that an understanding of the genes and gene products that control inflammatory responses to environmental factors, and conversely of environmental factors that may regulate gene function, will clarify the molecular/cellular mechanisms of host response to the exposure, and may lead to new prevention and treatment approaches.

LPS Viruses

DEP LPS Viruses ETS

DEP DEP Metals Ozone ETS Viruses Parasites

–

+

+ CD80 + CD28 Th0

Th1 +

–

LPS Parasites

– IL-4 IL-13

IL-10

YY

Y

Y

Y

YY

LPS

+

YY

–

+

B cell

Th2

MHC-II + TCR Treg

Y

APC

YY

Y

Y +

+

Mast cell

Plasma cell Antigen Y Specific lgE

DEP Viruses

DEP Metals LPS Viruses

Fig. 7.6 Environmental factors and their sites of interaction with IgE production. Environmental factors can act at different stages of the immune allergic reaction, by affecting antigen-presenting cells (APC), the polarized T-cell subsets Th1, Th2, and regulatory T cells (Treg), B-cell switch, and mast cell activation. See text for definition of abbreviations. (See CD-ROM for color version.)


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Antigen-presenting Dendritic Cells and Macrophages Bart N. Lambrecht and Hamida Hammad

Summary

Introduction

The lung contains many subsets of dendritic cells (DCs) that are distributed in various anatomic compartments. These cells possess all the machinery to take up and process antigen into the major histocompatibility complex class I and II presentation pathways. In homeostatic conditions, a fine-tuned balance exists between plasmacytoid and myeloid DCs necessary for maintaining tolerance to inhaled antigen and avoiding overt inflammation. Tolerance occurs when immature DCs present antigen to T cells. Several lung protective pathways therefore suppress the function of DCs in homeostatic conditions. One such pathway involves alveolar macrophages that actively suppress T cell and DC activation in the lung. The same subsets of DCs are also implicated in the process of allergic sensitization. Allergens can directly or indirectly activate the myeloid DC network of the lungs and this can lead to Th2 immunity. Wellknown adjuvant factors like endotoxin, cigarette smoke, diesel particles, ozone, and viral infection also lead to Th2 immunity by altering DC function. It is increasingly clear that DCs also play important roles in establishment of eosinophilic airway inflammation seen in ongoing asthma. DCs are important for local activation of memory Th2 cells, for attracting lymphocytes and inflammatory cells to the lungs, and for regulating the function of antiinflammatory Treg cells. In the future, it will be important to study how DCs communicate with the various structural cells of the airways, how they are recruited to the lungs and draining lymph nodes, and how their removal might affect antimicrobial defense mechanisms in the lungs. Critical cytokines produced by epithelial cells could perpetuate allergic inflammation through a DC intermediate mechanism. Based on these new insights on airway DC biology, several approaches that interfere with DC function show potential as new intervention strategies for these ever-increasing diseases.

The prevalence of sensitization to allergens and allergic diseases has reached epidemic proportions in Western societies. Allergic sensitization is the presence of IgE to common environmental allergens, and is controlled by Th2 cells that provide help for IgE synthesis by B cells. In addition, many of the inflammatory cell types found within sites of allergic inflammation, such as eosinophils and mast cells, depend on Th2 cells for their development and function. Th2 cells will only react to allergen when it is presented in the context of major histocompatibility complex (MHC) molecules by professional antigen-presenting cells such as dendritic cells, macrophages, and B cells. DCs are the most important antigen-presenting cells found throughout the body and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to (aero)allergens. Increasingly, these cells are also recognized for their potential to maintain ongoing effector responses and therefore they might be crucial in maintaining allergic inflammation. B cells present allergen to T cells mainly in the context of immunoglobulin synthesis, for which they need T-cell help. Macrophages are seen as scavenger cells that can also regulate the function of DCs. They are equally important in controlling pathogen clearance and tissue remodeling.


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Dendritic cell terminology and heterogeneity DCs were originally described by their capacity to efficiently process and present antigens and to prime naive T cells (Steinman & Cohn 1973). Over the last three decades, multiple DC subtypes have been defined, differing in phenotype, localization, and immune function (Shortman & Liu 2002). Myeloid DCs, Langerhans cells (LC) as well as natural type I interferon (IFN)-producing cells (IPCs, also called plasmacytoid DCs, pDCs) are part of the hematopoietic system and have a relatively short half-life in tissues. To maintain DC numbers in the tissues, there is continuous renewal of DCs from hematopoietic precursors residing in the bone marrow or within the skin (for LC in steady-state conditions; Merad

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Antigen-presenting Dendritic Cells and Macrophages

CD34+

GM-CSF TNF-a

CD14+ CD1a–

M-CSF IL-6

CD14– CD1a+

Monocyte

TGF-b IL-15

Interstitial Langerhans cell “dermal” DCs Fig. 8.1 MHCII-positive mucosal dendritic cell network visualized by MHCII staining on a murine tracheal wholemount. Trachea was taken from a naive unimmunized mouse. (See CD-ROM for color version.)

et al. 2002). A universal feature of DCs in tissues is their typical morphology with long dendrite-like extensions (hence their name) that can be demonstrated by staining for MHC class II (Fig. 8.1). Myeloid DCs in humans express markers shared with monocytes/macrophages such as CD33, CD4, and CD11c, whereas in the mouse they typically express CD11c and CD11b. In humans, pDCs were described in the bloodstream, lungs, and lymph nodes as lineageneg CD11clo CD123+ BDCA2+ cells (Shortman & Liu 2002). In the mouse, pDCs express specific markers (120G8, PDCA-1) as well as cell markers shared with myeloid DCs (MHC class I and II, CD11c) but also with granulocytes (Gr1) and B cells (B220) (Shortman & Liu 2002). LC express CD1a, langerin, and intracellularly demonstrate so-called Birbeck granules (BG), “tennis racket”shaped organelles. Recently, another DC subset sharing marker expression with natural killer (NK) cells was identified as “natural killer DC.” These DCs originate as cells with NK function, which are capable of taking up killed material for presentation to T cells (Taieb et al. 2006). DCs originate in the bone marrow from a CD34+ precursor and circulate in the bloodstream as a monocyte-like precursor before entering peripheral tissues (Geissmann et al. 2003; Bonasio & von Andrian 2006). The exact nature of the precursor DC cell type is currently unknown (Fig. 8.2), and could vary for myeloid versus plasmacytoid DC, inflammation versus steady state, or for lymphoid organ versus peripheral tissues (del Hoyo et al. 2002; Naik et al. 2006).

Antigen uptake There are various ways by which antigen-presenting cells can acquire foreign antigen. A first mechanism is via receptor-

CD11c+ CD1a– CD9 CD68 CD2 FXIIIa

CD11c+ CD1a+ Birbeck E-cadherin Langerin

GM-CSF IL-4

FIt3L

CD11c– IL-3Ra IL-3 CD40L

Monocyte-derived DC Plasmacytoid DC CD11c+ CD1a+/– CD83 DC-SIGN

CD11c– CD1a– CD4+ IL-3Ra (CD123) BDCA2/4

Fig. 8.2 Different origins and fate of dendritic cell (DC) subsets. All DCs originate from a CD34+ precursor in the bone marrow. These cells then further differentiate under the influence of various cytokines into Langerhans cells of the skin, interstitial DCs of tissues, monocyte-derived DCs, or plasmacytoid DCs, each expressing specific markers. Certain cytokines like IL-6 and M-CSF inhibit DC development when precursors are continuously exposed to them. (See CD-ROM for color version.)

mediated endocytosis involving clathrin-coated pits. Immature DCs express a plethora of specialized cell receptors for patterns associated with foreign antigens, such as the C-type lectin carbohydrate receptors (langerin, DC-SIGN, dectin, BDCA-2, macrophage mannose receptor, and the unique carbohydrate receptor DEC-205) (Figdor et al. 2002). Lectin-receptor mediated uptake by DCs results in about 100-fold more efficient presentation to T cells as compared with antigens internalized via fluid phase (Mahnke et al. 2000). Interestingly, langerin is a C-type lectin displaying mannose-binding specificity and is exclusively expressed by DCs that display BG, such as lung DCs and skin LC, but seems to be functionally irrelevant (Kissenpfennig et al. 2005). Pollen starch granules were shown to bind to C-type lectin receptors on alveolar macrophages (AMs) and DCs, although internalization occurred only in macrophages (Currie et al. 2000). Also, Pestel demonstrated that Der p1 uptake into cultured DCs involves mannose receptor-mediated endocytosis, and that process is more efficient in DCs obtained from allergic donors (Deslee Gt et al. 2002). In allergic individuals, DCs are furthermore armed with allergen-specific IgE bound to the high-affinity IgE receptor (FcεRI), thus enabling efficient receptor-mediated endocytosis of the allergen (Novak et al. 2003). A second mechanism of antigen uptake is constitutive macropinocytosis that involves the actin skeleton-driven engulfment of large amounts of fluid and solutes (about

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one cell volume/hour) by the ruffling membrane of the DC followed by concentration of soluble antigen in the endocytic compartment (de Baey & Lanzavecchia 2000). Macropinocytosis seems to be a dominant mechanism involved in the uptake of recombinant Bet v1 and Phl p1 pollen allergens by LCs and of Der p1 by cultured DCs, and can be inhibited by cytochalasin D and amiloride (Noirey et al. 2000). Thirdly, immature LCs, cultured DCs, pDCs, and macrophages have been shown to phagocytose particulate antigens such as latex beads and even whole bacteria, as well as apoptotic cells, and this could be the dominant mechanism of uptake of particulate allergens (Inaba et al. 1993; Ochando et al. 2006). The extracellular antigens that are taken up by any of these mechanisms accumulate in the endocytic compartment, where they are loaded on newly synthesized and recycling MHC class II molecules but may also be transported into the cytosol, where they become accessible to the class I antigen presentation pathway, a process called “cross-presentation.”

Antigen presentation

Foreign antigen

MHCI peptide

Allergens are extracellular antigens, and like most extracellular antigens they are processed for presentation onto MHC class II molecules. The T-cell receptor of CD4 T lymphocytes will respond only to processed antigen in the context of MHC class II, a process called “MHC restriction.” In contrast to MHC class I, which is expressed on all nucleated cells types, MHC class II is mainly expressed by professional antigenpresenting cells, but also to a lower extent by epithelial cells, mast cells, and eosinophils. MHC class II molecules, which consist of an αβ heterodimer, assemble in the endoplasmic reticulum with the chaperone invariant chain (Ii) (Fig. 8.3) (Chapman 1998). The cytoplasmic tail of Ii contains a motif that targets the Ii-MHC class II complexes to the endosomal pathway. After entry into endosomal/lysosomal compartments, Ii undergoes stepwise degradation by lysosomal proteases, leaving a small fragment of Ii known as the class II-associated invariant chain peptide (CLIP) associated with the MHC class II peptidebinding groove (Riese et al. 1996). This event stabilizes and protects the peptide-binding site from interacting with other polypeptides. To bind the antigenic peptides that have been generated in acidic endosomal and lysosomal compartments, the binding site of the MHC class II αβ dimers must be free of its previous occupant, CLIP. Displacement of CLIP is facilitated by several factors: (i) low pH, which favors an “open” conformation in the MHC class II molecule and peptide exchange; (ii) the activity of the chaperone H-2DM (HLADM), which stabilizes the open conformation (Denzin & Cresswell 1995); and (iii) by proteolytic elimination of the regions of Ii that flank the CLIP peptide (Kropshofer et al.

168

Recycling

Endocytosis Endosome

MIIC Cathepsin S MHC-CLIP

HLA-DM CLIP

TGN

ER

Cytosolic antigen

MHCI-b2

Proteasome

Presentation of exogenous antigens on MHC class II to CD4+ T cells

MHCII peptide

TAP

MHCII-Ii

ab heterodimer

hsp

Fig. 8.3 Antigen processing in DCs. The classical pathway of MHCI presentation is initiated by the cytosolic degradation of protein antigen by the proteasome, a complex multicatalytic protease. This ubiquitindependent protein degradation generates peptide fragments of defined length (“molecular ruler” function). These fragments bind to heat-shock proteins and target an ATPase-dependent TAP (transporter associated with antigen presentation) on the endoplasmic reticulum (ER) membrane. TAP transports peptides (8–12 amino acids) into the lumen of the ER. Inside the ER, peptides are loaded onto newly synthesized MHCI (a chain and b2-microglobulin) molecules. Chaperone molecules (tapasin, calnexin) prevent immature MHCI molecules from being loaded with irrelevant endogenous ER proteins and disengage after high-affinity binding of cytosolic peptides. Peptide-loaded complexes are transported to the cell membrane after passing through the Golgi and trans-Golgi (TGN) network. For successful expression of MHCII–peptide complexes on the cell surface, endocytosed and partly digested antigen needs to be efficiently mixed with newly synthesized or recycling MHCII molecules in a specialized subcellular compartment called MIIC. The highly polymorphic MHCII molecules consist of an ab heterodimer which assembles in the ER with a third molecule, the invariant chain (Ii). After transport to the TGN, the MHCII–Ii complex is targeted to the endocytic MIIC pathway, via a signal sequence on the Ii chain. In addition, some abIi complexes are directly targeted to the cell membrane, followed by recycling to the endocytic compartment. In the endocytic pathway, invariant chain proteolysis by cathepsin S generates the CLIP (class II-associated invariant chain peptide) fragment, which binds to the peptide-loading groove of the ab heterodimer. Binding of CLIP protects the peptide-binding groove from interacting with irrelevant ER peptides (generated for MHCI loading). The CLIP fragment is then exchanged for immunogenic peptides, generated in the endocytic/lysosomal pathway by proteolysis of intact protein antigen into peptides 12–20 amino acids long. The exchange is catalyzed by HLA-DM. The binding of high-affinity antigenic peptide stabilizes the ab heterodimer, HLA-DM binds the released CLIP fragment and physically disengages. Due to the loss of association with the intact invariant chain, stabilized ab–peptide complexes are targeted to the cell membrane and are transiently expressed before being recycled via the endocytic pathway.

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1995). CLIP removal and binding of the antigenic peptides direct the MHC class II/peptide complexes to the cell surface for presentation to CD4+ T cells. Within the endocytic compartment, antigen is cleaved into short immunogenic peptides by proteolytic enzymes of the cathepsin family. Antigen loading on MHC class II molecules occurs in an acidic cellular compartment rich in newly synthesized MHC class II molecules, called the “MIIC compartment” (Nijman et al. 1995). This multivesicular complex is located at the intersection of the biosynthetic [endoplasmic reticulum (ER), Golgi complex, secretory granules] and endocytic pathway of vesicle transport within the cell and contains the MHC II-related HLA-DM peptide exchanger, which is essential for loading high-affinity antigenic peptides on MHC II (Denzin & Cresswell 1995). Alternatively, there is a pathway of peptide loading onto preformed MHC class II molecules that have been internalized into mildly acidic endosomal vesicles after being expressed on the cell surface (Koppelman et al. 1997). Surprisingly, proteolysis of antigen by immature DCs can also occur extracellularly through secreted proteases, generating peptides that can be loaded onto empty cell surface-expressed MHC class II (De Bruijn et al. 1992). It is at present unclear how allergens are loaded onto MHC class II molecules by DCs. In sensitized individuals, internalization of allergens via receptor-mediated endocytosis by multivalent cross-linking of the high-affinity IgE receptor (FcεRI) on immature DCs targets the antigen to the MIIC compartment (Maurer et al. 1998; Holloway et al. 2001; Novak et al. 2003). In contrast, the generation of peptide–MHC complexes derived from macropinocytosis of Bet v1 and Phl p1 pollen allergens was shown to be inhibited only partly when the pH of the endosomes was altered, suggesting that parts of the molecules were not metabolized in the lysosomal MIIC compartment (Riese et al. 1996).

Presentation of endogenous antigen on MHC class I to CD8+ T cells After a virus enters a host and infects cells, the major adaptive immune response that clears the infection is mediated by CD8+ cytotoxic T lymphocytes (CTLs). These cells also provide the major defense against cancers. CD8+ lymphocytes recognize infected cells that display on their surface MHC class I molecules presenting antigenic peptides derived from viral proteins or tumor antigens expressed in the cytoplasm. All nucleated cells have the capacity to present peptides derived from the cytoplasm onto MHC class I molecules. After recognition of peptide–MHCI, the CTL kills the infected cells and thereby eliminates the source of viral replication or the abnormal/cancerous cells. In the classical view, bystander cells that have endocytosed viral debris cannot process such antigens to form MHC class I-restricted complexes and are therefore not targeted. Recently, however, this view has been challenged (see next section).


The classical pathway of MHC class I presentation is initiated by the cytosolic degradation of the protein antigen by the proteasome, a complex multicatalytic protease (Fig. 8.3). This ubiquitin-dependent protein degradation generates peptides of a defined length. A fraction of these peptides (> seven residues) is transported into the ER through the transporter associated with antigen processing (TAP). Inside the ER, peptides are loaded onto newly synthesized MHCI, and the accompanying chaperone proteins that keep MHCI stabilized in the absence of peptide are released. Peptide-loaded complexes are transported to the cell surface for display.

Presentation of exogenous antigen on MHC class I to CD8+ T cells A second, less well-defined, approach to load peptides on MHCI molecules is for the DCs to capture extracellular antigens and to process these captured exogenous antigens into the MHC class I pathway. This form of presentation is referred to as “cross-presentation.” In the field of experimental allergy, evidence is present for this cross-priming. Aerosolization of ovalbumin (OVA) in OVA-sensitized mice leads to the generation of MHC class I-restricted CD8+ T cells that can regulate the magnitude and duration of IgE responses (MacAry et al. 1997) and suppress airway inflammation (Wells et al. 2007). The mechanisms underlying cross-presentation are not very well defined. Particulate exogenous antigens are phagocytosed and there is gradual accumulation of ER proteins in a form of fused ER/phagosome organelle where MHCI can be loaded (TAP-independent cross-presentation). Under some conditions, the phagocytosed antigen is transferred into the cytosol (Rodriguez et al. 1999), in a process involving Sec61, a transporter normally involved in ER import (Desjardins 2003). The protein can then be degraded by closely membraneassociated proteasome and subsequently transported back into the phagosome/ER by TAP in order to bind and be presented by MHC class I molecules (TAP-dependent crosspresentation) (York & Rock 1996). DCs are particularly efficient in generating antiviral or antitumoral MHCI-restricted CD8+ T cell responses in vitro and in vivo. Indeed, when DCs were purified from mice injected with protein antigens or viruses, they cross-presented the antigen on their MHC class I molecules. Moreover, in many of these experiments, antigen cross-presentation was found only in DCs (Heath & Carbone 2001). Consistent with this finding, Jung et al. (2002) generated transgenic mice expressing diphtheria toxin receptor under the control of CD11c promoter, and found that when CD11c+ DCs were depleted by treatment with diphtheria toxin, mice failed to generate a CD8+ T cell response to cell-associated antigen and intracellular pathogens. DCs are heterogeneous and can be divided into pDCs and conventional myeloid DCs (mDCs). The latter subset can be subdivided further according to their expression of CD8α.

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Which subsets are able to cross-present antigens to CD8+ T cells is not fully understood. In experiments in which mice were immunized with cell-associated antigen (den Haan et al. 2000) or infected with virus (Allan et al. 2003, 2006) and DC subsets isolated, the CD8α+ DC was identified as the primary APC that stimulated naive CD8+ T-cell responses. In viral infection models, however, it is less clear whether crosspriming is an exclusive function of CD8α+ DCs. The nature of the antigen also influences what type of DC participates in cross-priming. For example, cell-associated OVA is crosspresented by CD8α+ DCs, while OVA/anti-OVA immune complexes are cross-presented by both CD8α+ and CD8α− DCs. It is generally believed that pDCs do not cross-prime exogenous antigens. However, type I IFN has been suggested to promote cross-priming (Le Bon et al. 2003). As pDCs produce type I IFN during viral infections, they may enhance the cross-priming process to viral antigen during viral infections (Liu 2005).

Table 8.1 Expression of chemokine receptors by DC subsets.

mDCs

Integrated function of dendritic cells in the immune response Dendritic cell activation DCs originate in the bone marrow and circulate in the bloodstream as monocyte-like precursors before entering peripheral tissues (Geissmann et al. 2003; Bonasio & von Andrian 2006). DC migration is a tightly regulated process in which many chemokines and other factors are involved (Table 8.1 and Fig. 8.4) mDCs are attracted to peripheral tissues by a specific set of chemokines such as macrophage inflammatory protein (MIP)-3α (CCL20) and epithelial β defensins acting on CCR6 (Yang et al. 1999; Cook et al. 2000; Biragyn et al. 2002; Bonasio & von Andrian 2006). pDCs respond preferentially to SDF1 (CXCL12) and CXCL9-11 and the newly described chemerin, a ligand for ChemR23 (Vermi et al. 2005). Once DCs extravasate, they form a network in the upper layers of the epithelium and lamina propria of the airways (see Fig. 8.1), gut, and skin. Here, DCs are said to be in an immature state, specialized for internalizing foreign antigens but not yet able to activate naive T cells (Banchereau & Steinman 1998; Vermaelen et al. 2001). The DC network serves a patrolling function, continuously scanning the environment for foreign antigens. The DC is endowed with numerous ancient receptors for foreign antigenic signature molecules such as bacterial cell walls, viral and bacterial DNA, and foreign sugar molecules (Fig. 8.5). These so-called pathogen-associated molecular patterns (PAMPs) are recognized by Toll-like receptors (TLR1–10) and C-type lectin receptors, which are abundantly expressed on the surface of DCs (Figdor et al. 2002). The expression of various TLRs varies between DC subsets, particularly in human DCs. In humans and mice, pDCs preferentially express TLR7 and TLR9, and thus respond to the corresponding ligands (imidazoquinolines and single-stranded

170

pDCs

Chemokine receptors/ molecules controlling migration

Ligands

CCR1 CCR2 CCR4 CCR5 CCR6 CCR7 CCR8 CXCR3 CXCR4

CCL5, CCL3, CCL7 CCL2, CCL7, CCL8, CCL13 CCL17, CCL22 CCL3, CCL4, CCL5, CCL8 CCL20 CCL19, CCL21 CCL1 CXCL9, CXCL10, CXCL11 CXCL12

PAFR S1PR EP4 CD38 CystLT1 ChemR23

PAF Sphingosine 1-phosphate Prostaglandin E2 CD31 Cysteinyl leukotrienes Chemerin

DP1 IP ChemR23 FPRL1

Prostaglandin D2 Prostaglandin I2 Resolvin E1 Lipoxin A4

CCR7 CXCR3 CXCR4 ChemR23

CCL19, CCL21 CXCL9, CXCL10, CXCL11 CXCL12 Chemerin

RNA versus CpG motif bacterial DNA) but not to ligands for TLR2, TLR3, TLR4, or TLR5. In contrast, in vitro generated conventional monocyte-derived DCs or ex vivo isolated mDCs express all TLRs except TLR9. In addition to the direct molecular recognition of foreign antigenic structures, exposure to foreign antigens or necrotic cell death leads to tissue damage and this by itself can activate the DC system. DCs express a plethora of receptors for these so-called damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1) protein, heatshock proteins, uric acid, adenosine triphosphate (ATP), complement cascade fragments, neuropeptides, prostaglandins, etc. (Fig. 8.5). Many of these compounds not only activate the already residing DCs but also attract new waves of cells to the periphery (Lambrecht 2001, 2006a; Lotze & Tracey 2005). DC activation and maturation in the periphery can occur directly by ligation of DAMP or PAMP receptors and can occur indirectly through activation of the same receptors on the surrounding structural cells such as keratinocytes, epithelial cells, or fibroblasts (Lambrecht & Hammad 2003a). Keratinocytes and lung epithelial cells make granulocyte–

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Epithelium

Allergen


Microbes

4. “Danger” LPS, peptidoglycan Mannan CpG DNA, ds RNA PAMPs

3. MIP3a/CCL20 b-defensin 1.

2. TNF-a GMCSF O2 radicals DAMPs

DC

CCR+

CCR7+

5.

Migration via afferent lymph Immature DC High Ag uptake Low signal 2

MIP3b/CCL19

T cell area

Mature DC Low Ag uptake High signal 2

DC

HEV IL2 7. Selection Activation

IL2R CD8

CD4

8.

CXCR5 9.

Clonal expansion

CD44+ L-sel–

Th1/2

Efferent lymph

L-sel+ CD44–

6. Naive T cell

IL-4 Th2

B cell

CD40L germinal centre Bone marrow plasma cell

CD8

Recirculation to tissues

Fig. 8.4 Induction of the primary immune response by dendritic cells (DCs). 1 Under baseline conditions and on exposure to foreign antigens, epithelia produce macrophage inflammatory protein (MIP)-3a (CCL20) and b-defensin to attract CCR6+ immature DCs from the bloodstream. 2 Resident cell types produce inflammatory mediators and growth factors that attract and activate the recently recruited DC. 3 DCs capture allergens and other foreign antigens such as bacteria and viruses. 4 DCs can discriminate between “dangerous” antigens, and nonpathogenic antigens such as self antigens and probably most allergens, by recognizing certain viral and bacterial patterns. 5 The recognition of infection and tissue damage upregulates the CCR7 and CXCR4 and DCs migrate to the T-cell area of draining lymph nodes where the ligand MIP-3b and SDF-1 is constitutively expressed. During this migration, DCs lose the capacity to take up antigen, but become strong stimulators of naive T cells by their strong expression of costimulatory molecules (signal 2). 6 In the T-cell area, DCs produce chemokines to attract naive T cells that continuously leave the

bloodstream via the high endothelial venules (HEV). 7 Naive T cells are first arrested and then selected for antigen specificity. The recognition of the correct peptide–MHC induces the activation of naive T cells, which will lead to further terminal differentiation of DC function. 8 The activation of T cells leads to autocrine production of IL-2 and to clonal expansion of antigenspecific CD4+ and CD8+ T cells. These cells differentiate into effector cells that leave the lymph node via the efferent lymphatic. These effector cells are poised to migrate to peripheral tissues, especially to inflamed areas. 9 On contact with DCs, some antigen-specific CD4+ T cells upregulate CXCR5 receptor and migrate to the B-cell follicles of the draining lymph node. Here they further interact with germinal center DCs to induce CD40L-dependent B-cell immunoglobulin switching and affinity maturation (germinal center reaction). Most high-affinity B cells go to the bone marrow to become immunoglobulin-producing plasma cells. See text for definition of other abbbreviations. (See CD-ROM for color version.)

macrophage colony-stimulating factor (GM-CSF) and thymic stromal lymphopoietin (TSLP) that activate the underlying DC network. These cytokines are regarded as the principal maturation-inducing factors that can also be used to mature DCs in vitro.

Dendritic cell migration to the draining lymph nodes The recognition of danger (PAMPs or DAMPs) by peripheral dendritic cells dramatically alters the migration behavior of DCs and thus induces the surface expression of CCR7 on

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Toll-like receptors 1–10

Protease-activated receptors Complement receptors

Intracellular receptors NOD1/2 TLR7,9 PKB C-type lectin receptors Dectin Macrophage mannose receptor DEC205 BDCA-2

Prostanoid receptors DP1, EP4, IP Neuropeptide receptors NK1, CGRPR Purinergic receptors P2X, P2Y Receptors for Uric acid, HMGB1 Heat shock proteins

Fig. 8.5 Expression of “danger” receptors by dendritic cells. Dendritic cells express the ancient receptors of the innate immune system also expressed by macrophages, such as the Toll-like receptors (TLRs) and C-type lectin receptors. These receptors react to foreign pathogen-associated molecular patterns (PAMPs). In addition, DCs express numerous receptors for inflammatory mediators and necrotic cell debris, the so-called damageassociated molecular patterns (DAMPs). The exact receptors for uric acid, high mobility group box 1 (HMGB1) protein, and heat-shock proteins are not yet known. (See CD-ROM for color version.)

peripheral DCs (Fig. 8.4) (Bonasio & von Andrian 2006). The ligands for CCR7 are secondary lymphoid chemokine (SLC, now known as CCL21) and MIP-3β (CCL19), which are expressed at the luminal side of afferent lymph vessels and by the T-cell area of draining lymph nodes (Sallusto et al. 1998). Another factor attracting DCs to the lymph node is the lipid mediator sphingosine-1-phosphate (S1P). Blocking the S1P-type receptor dramatically reduces the migration of lung DCs to the mediastinal lymph nodes (Idzko et al. 2006). The responsiveness of CCR7 to CCL19 and CCL21 and the consequent lymph node migration of DCs are controlled by lipid mediators such as the leukotrienes and prostaglandins. Prostaglandin (PG)D2 acts on the DP1 receptor expressed by lung DCs and suppresses the migration of lung DCs (Hammad et al. 2003a). The downstream metabolite 15-deoxy(Δ)1214PGJ2 could also slow down the migration of DCs, by acting on the nuclear PPARγ receptor (Hammad et al. 2004a). Leukotriene (LT)B4 is exported from the cytoplasm of the DC by the multidrug resistance (MDR) protein, where it is metabolized into LTC4, which regulates the CCR7 responsiveness (Robbiani et al. 2000). In contrast to skin DCs, it was recently shown that lung DC migration is less dependent on the export of LTB4 by MDR (Jakubzick et al. 2006). For emigration of DCs from the skin, the CCR8 receptor for the chemokine CCL-1 (also known as I-309 in humans and TCA-3 in mice) acts in concert with CCR7 (Qu et al. 2004). Whether this is also true for lung DC migration remains to be shown. It is clear that the regulation of DC migration by arachidonic acid metabolites is very amenable to modification by various drugs already being developed for allergy treatment.

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Just as in the gut, airway DCs extend long dendrites to the lumen of the airways, forming bud-like extensions at the border of the air interface (Brokaw et al. 1998). Within a few hours after inhalation, airway mDCs and pDCs have taken up fluorescently labeled antigen within the draining mediastinal lymph nodes (Vermaelen et al. 2001; Hammad et al. 2003a; De Heer et al. 2004). After 24 hours, both mDCs and pDCs in the mediastinal lymph nodes contain antigen inside vesicles of the cytoplasm. What is unclear at present is whether pDCs take up antigen in the periphery of the lung and subsequently migrate to the nodes, or whether antigen is being transported to them by migratory mDCs or even a specific subset of CD8α− CD11b− migratory DCs recently described by Belz et al. (2004). Transport of immunogenic material from one nonmigratory DC to another is certainly a possibility, as CD8α+ DCs injected into the lung induce an immune response in the mediastinal node without migrating into it (Hammad et al. 2004b). Under steady-state conditions, mDCs continuously migrate to draining lymph nodes and present either (self-)autoantigens or harmless antigen in a tolerogenic form (Steinman & Nussenzweig 2002). Once they have reached the draining lymph nodes, mDCs express intermediate levels of costimulatory molecules and MHC class II.

T-cell activation by dendritic cells By upregulating the lymph node-homing chemokine receptors, DCs that have seen foreign antigen thus direct their interest to the regional draining lymph node T-cell area where they interact with recirculating T cells and B cells (Fig. 8.6) (Stoll et al. 2002). DCs that have arrived in the lymph node undergo short-lived interactions with T cells in the paracortical region and, during this initial antigen independent event, individual T cells are scanned for specificity for antigen. When antigen is being recognized, there is formation of a more long-term immunologic synapse, leading to full-blown T-cell activation, after which the T cell detaches, divides, and differentiates into an effector and possibly memory T cell. DCs also transport antigen without degrading it and thus offer intact protein to B cells at the interface between the paracortex and B-cell follicle (Castellino et al. 2006; Qi et al. 2006). DCs that have reached the T-cell area have lost the capacity to take up antigen, and now express a plethora of cell adhesion and surface molecules interacting with T cells not previously expressed on peripheral-based DCs. This phenotype is called “mature dendritic cell” implying that functionally these cells are now fully adapted to induce naive T-cell responses. DCs express the antigen on MHC molecules, and provide so-called costimulatory molecules [CD80/CD86 family; tumor necrosis factor (TNF)/TNFR family] (Fig. 8.7) together with cytokines to optimally expand and differentiate T cells for the particular task that needs to be carried out to clear the foreign antigen. Initially, T cells are stimulated in the draining lymph node, but after a

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Uptake of Ag in mucosa Local presentation to Teff

DC network takes up antigen

Teff

Lung effector site Day 4 87654 0

Tcm DC migration DC maturation

Draining node Day 0 Tcm

Naive T

naive

Tcm

Other nodes/spleen Day 4 87654 0

Day 4 876543210

KJ1-26

Clonal selection Proliferation Differentiation (Tcm or Teff) CFSE Ovalbumin

T cell

Epithelial cell

Dendritic cell

Eosinophil

Goblet cell

Teff Tcm Tnaive

Fig. 8.6 Integrated overview of DCs and CD4+ T-cell migration during primary and secondary immune responses. Antigen (Ag) is taken up by DCs across the mucosal impermeable barrier. Mucosal DCs continuously migrate from the lungs to the T-cell area of mediastinal lymph nodes (MLNs). In the presence of inflammation, this process is amplified, increasing the possibility that pathogenic substances will be presented to recirculating naive T cells (Tnaive) or central memory T cells (Tcm). At the same time, DC maturation will be fully induced. When mature DCs arrive in the MLNs, they select specific T cells from the polyclonal repertoire of cells that migrates through the high endothelial venules and T-cell area. Within 4 days, this will lead to clonal expansion of antigen-specific T cells. This is illustrated in the FACS plot where antigen-specific T cells, identified by staining with a specific KJ1-26 antibody for the ovalbumin T-cell receptor, dilute the CFSE signal. When a T cell has acquired a certain threshold number of divisions (usually four or more), it will leave the MLN to become either a Tcm cell or an effector

T cell (Teff). This is where migration pathways separate and consequently the anatomic requirements for reactivation diverge. The Tcm cells will extravasate in other nondraining nodes and spleen, and will eventually accumulate in the spleen over time (see FACS plot for nonlung-draining lymphoid tissue where both divided Tcm and naive T cells can be found). Reactivation of these cells will therefore only occur in central lymphoid organs. In contrast, Teff cells will extravasate in peripheral sites of inflammation (see FACS plot for lungs, where only divided cells can be found), including the lung when the original inflammation is still present. In contrast to naive T cells, which are excluded from lung tissues, these Teff cells can be stimulated by local airway DCs to mediate their effector function. In this scenario, alternative antigen-presenting cells might be eosinophils or even epithelial cells, expressing MHC molecules. (See CD-ROM for color version.)

few cell divisions they acquire effector potential (Lambrecht et al. 2000a), start expressing chemokine receptors for inflammatory chemokines expressed at sites of pathogen entry, and lose the expression of CD69, thus rendering them insensitive to the lymph node-retention signal S1P (Shiow et al. 2006).

Th polarization by dendritic cells DCs are crucial in regulating the immune response by bridging innate and adaptive immunity. Signals from the type of antigen and the response of the innate immune system are translated by DCs into a signal that can be read by the cells of the adaptive immune response, leading to an optimal

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Surface ligand

MHC/peptide-TCR Signal 1

Ag uptake

Ag processing

Signal 2 DCSIGN-ICAM-3 CD80/86-CD28 B7RP-ICOS PDL1/PDL2-PD1 CD40-CD40L OX40L-OX40

T cell

T cell response Activation Differentiation

Fig. 8.7 Formation of the immunologic synapse. Dendritic cells internalize antigen and present it into the groove of MHCI and MHCII molecules to, respectively, CD8 and CD4 T lymphocytes (signal 1). In the process of recognizing foreign antigen, they are induced to express some costimulatory molecules for naive T cells (signal 2). The T cell bearing a specific receptor upregulates CD40L, which induces the terminal differentiation of dendritic cells (DCs) through CD40 signaling, inducing the full expression of all costimulatory molecules like CD80, CD86, ICOSL, etc., that further polarize the immune response. In addition, DCs produce cytokines to expand and differentiate the T-cell response. TCR, T-cell receptor. (See CD-ROM for color version.)

response for a particular insult (Fig. 8.8). Together, these signals consist of provision of a particular density of peptide– MHC, the expression of costimulatory or Th-polarizing cell surface molecules, and the expression of soluble cytokines and chemokines that polarize T cells or enhance their survival. At the same time, DCs also control the function and expansion of regulatory T (Treg) cells that tightly control overzealous inflammatory T-cell responses. Although controversial, it has been suggested over recent years that particular functions of DCs such as tolerance or immunity or Th1/Th2 differentiation might be a specialized function of defined subtypes of DCs (Maldonado-Lopez et al. 1999; Rissoan et al. 1999). Others have refuted this idea and have claimed that DCs are very versatile cells, and can virtually induce any type of response depending on the need of the moment (Kapsenberg 2003). Recent studies have suggested that lung mDCs mediate protective immunity to inhaled antigens only when properly activated by innate immune system-activating immune signals, acting through TLRs or other recognition receptors. Under inflammatory conditions such as those provided by lipopolysacharide (LPS) or virus infection, the expansion of T cells induced by mDCs leads to the generation of Th1 or Th2 effector cells in the mediastinal nodes (Eisenbarth et al. 2002; Brimnes et al. 2003). The signals that determine the type of response after encountering a pathogen in the lung are delivered by DCs in the lymph node. Sporri and Reis e

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High IL-6 Low IL-12 T1/ST2L/IL-33 CCL2?

OX40L CD86 Jagged

DC

+

Secreted

ICOSL dim CD86 dim CD80 Jagged?

Th0

Low IL-6 Low IL-12 IL-10 TGFb PGD2

IL-4 IL-5 IL-13 TNF-a

Treg

IL-10 TGF-b

Th17

IL-17

Th1

IFN-g TNF-a

IL-2

High IL-6 High TGF-b CD80 Delta ICAM-1

Th2

IL-23 High IL-6 High IL-12 High IL-18 CCL3 High IFNg?

Fig. 8.8 T-helper cell polarization by dendritic cells (DCs). Depending on the type of antigen, the dose, the genetic background, and the tissue environment where antigen is first introduced, DCs can induce various types of Th response, tailormade to protect the host, while avoiding autoimmunity. Often the response is extremely well balanced, to avoid tissue damage, while allowing clearance of the threat. The various cytokines and costimulatory molecules that favor a particular direction are indicated. See text for further explanation and for definition of abbreviations. (See CD-ROM for color version.)

Sousa (2005) recently suggested that DC maturation and provision of peptide–MHC to T cells is not sufficient to generate effector cells. Cytokines are dominant signals that determine the quality and quantity of an effector immune response. During generation of an efficient effector immune response, DCs also have to overcome suppression by Treg cells, and the dominant way by which they seem to do this is by production of interleukin (IL)-6 that releases the suppression by naturally occurring Tregs (Pasare & Medzhitov 2003). Certain pathogens or pathogen-derived products induce the direct secretion of Th1-polarizing cytokines by DCs and thus instruct the type of immune response generated. Alternatively, it was shown that tissue environment can also determine Th differentiation. Stumbles et al. (1998) and Dodge et al. (2003) have shown that resting respiratory tract DCs mainly induced Th2 responses. As a direct proof that mDCs can induce Th2 sensitization in the lung, it was shown that intratracheal injection of bone marrow-derived mDCs pulsed with OVA induced a Th2 response to OVA and subsequently led to severe features of asthma when mice were rechallenged with OVA aerosol (Lambrecht et al. 2000b). Recently, much information has been gathered on how exactly Th2 polarization is controlled by DCs. Mice that

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conditionally overexpress TSLP in the lungs mount vigorous Th2 responses in the airways, in a process driven by DCs (AlShami et al. 2005; Zhou et al. 2005). TSLP is increased in the airways of asthmatic patients (Ying et al. 2005) and it can activate mDCs to prime naive CD4+ T cells to differentiate into proinflammatory Th2 cells (Watanabe et al. 2005). The Th2 skewing effect induced by TSLP-activated DCs was found to be dependent on OX40L, a costimulatory molecule shown to play a critical role in the development of allergic lung inflammation (Ito et al. 2005). As TSLP is such an important factor in the sensitization process, it will be very important to study how its release by epithelial cells and other inflammatory cells is regulated in response to natural allergen exposure. The type of immune response induced by mDCs also depends on the strength of the activating innate immune system stimulus. Elegant studies by Eisenbarth et al. (2002) showed that low-level TLR4 agonists prime mDCs to induce a Th2 response, by inducing their full maturation, but not their production of IL-12. High-level LPS administration induced high level IL-12. These findings might help to explain the effects of environmental exposure to LPS on the reduced incidence of allergic sensitization. IL-12 seems to be a dominant cytokine for Th1 responses in the lung, yet the LPS-induced Th1 response induced by mDCs in the lung was not dependent on IL-12 (Kuipers et al. 2003). Although IL-12 may be redundant for some Th1-inducing stimuli, it is certainly sufficient as retroviral overexpression of this cytokine in mDCs in the lung induced strongly polarized Th1 responses (Kuipers et al. 2004). The transcription factor Tbet is a master controller of Th1 development and was recently found to be expressed in DCs in addition to T cells. Tbet –/– DCs were less potent at inducing Th1 responses and produced less proinflammatory cytokines (Wang et al. 2006). The exact role of IL-23 and IL-27, as well as surface expression of the Notch ligands Delta/Jagged, in DC-driven Th1 development in the lung remains to be studied (Amsen et al. 2004). Cells of the innate immune system, such as NK cells, are recruited to the draining nodes by DCs and could also be an early source of Th1-polarizing cytokines (Martin-Fontecha et al. 2004). Recently, Th17 cells producing IL-17 that regulate autoimmune inflammation have been identified. They are induced by a cytokine cocktail of transforming growth factor (TGF)-β, IL-6 and their numbers are expanded by IL-23 (Veldhoen et al. 2006). DCs can produce all these factors, and have been shown to induce Th17 cells in a model of experimental allergic encephalomyelitis (Veldhoen et al. 2006). In view of the fact that IL-17 also plays a crucial role in regulating allergic airway inflammation, the involvement of the DC– Th17 axis in allergy will have to be ascertained (SchnyderCandrian et al. 2006).

Tolerance induction by dendritic cells Immature DCs are distributed throughout the lung and are at the focal control point determining the induction of


pulmonary immunity or tolerance (Akbari et al. 2001, 2002; Lambrecht & Hammad 2003b). Airway DCs form a dense network in the lung, ideally placed to sample inhaled antigens, and these cells migrate to draining lymph nodes to stimulate naive T cells (Banchereau & Steinman 1998; Lambrecht et al. 1998; Vermaelen et al. 2001). As most allergens are immunologically inert proteins, the usual outcome of their inhalation is tolerance and inflammation does not develop on chronic exposure (De Heer et al. 2004; Ostroukhova et al. 2004). This is shown best for the model antigen OVA. When given to the airways of naive mice via aerosolization, nasal droplet aspiration, or intratracheal injection, it renders mice tolerant to a subsequent immunization with OVA in adjuvant, and effectively inhibits the development of airway inflammation, a feature of true immunologic tolerance (De Heer et al. 2004; Ostroukhova et al. 2004). It was, therefore, long enigmatic how sensitization to natural allergens occurred. An important discovery was the fact that most clinically important allergens, such as the major Der p1 allergen from house-dust mite (HDM), are proteolytic enzymes that can directly activate DCs or epithelial cells to break the process of tolerance and promote Th2 responses (Hammad et al. 2001; Kheradmand et al. 2002). However, other allergens, such as the experimental allergen OVA, do not have any intrinsic activating properties. For these antigens, contaminating molecules or environmental exposures (respiratory viruses, air pollution) might pull the trigger on DC activation (Dahl et al. 2004). Eisenbarth et al. (2004) showed that low-level TLR4 agonists admixed with harmless OVA prime DCs to induce a Th2 response, by inducing their full maturation, yet not their production of IL-12. This process has been recently described as being dependent on the activation of the adaptor molecule MyD88 in pulmonary DCs (Piggott et al. 2005). This is clinically important information as most natural allergens such as HDM, cockroach, and animal dander contain endotoxin, and undoubtedly other TLR agonists (Braun-Fahrlander et al. 2002). From the above, it seems that the decision between tolerance or immunity (in the lungs) is controlled by the degree of maturity of mDCs interacting with naive T cells, a process driven by signals from the innate immune system (Herrick & Bottomly 2003; De Heer et al. 2005). It has indeed been shown that immature mDCs induce abortive T-cell proliferation in responding T cells and induce Tregs (Akbari et al. 2001; Brimnes et al. 2003; Ostroukhova et al. 2004). Another level of complexity arose when it was shown that (respiratory) tolerance might be a function of a subset of pDCs (De Heer et al. 2004; Oriss et al. 2005). Removal of pDCs from mice using depleting antibodies led to a break in inhalational tolerance to OVA and to development of asthmatic inflammation (De Heer et al. 2004). The precise mechanisms by which pDCs promote tolerance are unknown but, in the absence of pDCs, mDCs become more immunogenic and induce the formation of effector cytokines from dividing T cells (De Heer

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et al. 2005). The negative signal that is delivered by pDCs has not been elucidated, but could be the high-level expression of programmed death ligand (PDL)-1, delivering a negative signal to T cells or to mDCs directly (De Heer et al. 2004; Kohl et al. 2006). Additionally, pDCs can produce the tryptophanmetabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which has strong inhibitory activity on T-cell proliferation (Fallarino et al. 2004) and inhibits inflammatory airway disease (Hayashi et al. 2004). Interestingly, IDO expression has been demonstrated recently in pulmonary CD11c+ cells (Swanson et al. 2004), although the exact cell type involved has not clearly been identified. Another explanation to the tolerogenic properties of pDCs is related to their immature phenotype, as it has been demonstrated that immature DCs can induce Tregs (Dhodapkar & Steinman 2002). Ex vivo at least, lung-derived pDCs promoted formation of Treg cells specific for OVA (De Heer et al. 2004). If pDCs promote tolerance and mDCs immunity, it is logical to assume that the balance between both subsets is tightly controlled. In support of this, the administration of Flt-3 ligand, a cytokine that induces the differentiation of pDCs, to sensitized mice reduced all the features of asthma (Edwan et al. 2004), whereas administration of GM-CSF expanded mDCs and strongly enhanced sensitization and inflammation (Stampfli et al. 1998). Kohl and colleagues recently demonstrated that blockade of the anaphylatoxin C5a receptor during priming reactions to HDMs or harmless OVA was able to strongly enhance Th2 priming, through effects on the selective recruitment of immunogenic mDCs over tolerogenic pDCs (Lambrecht 2006a; Kohl et al. 2006).

in the number of alveolar mDCs, displaying a mature phenotype with increased expression of MHCII, OX40L, and CCR7 (Fainaru et al. 2005), and demonstrating an increased immunostimulatory capacity. Moreover, RunX3–/– DCs are able to mount inflammatory responses to otherwise harmless inhaled antigens, possibly through their lack of responsiveness to locally secreted TGF-β (Fainaru et al. 2004). In mice normally resistant to HDM-induced asthma and airway hyperresponsiveness (AHR) (C3H mice), Treg cell depletion using the CD25-depleting antibody similarly led to increased numbers of pulmonary mDCs with elevated expression of MHCII, CD80, and CD86, and an increased capacity to stimulate T-cell proliferation and Th2 cytokine production. In normally susceptible A/J mice, Tregs did not suppress inflammation and AHR. These data suggest, therefore, that resistance to allergen-driven AHR is mediated in part by CD4+CD25+ Treg cell suppression of DC activation and that the absence of this regulatory pathway contributes to susceptibility (Lewkowich et al. 2005). In the rat, it was shown that Tregs also control the level of CD86 expression on lung DCs and are responsible for the tolerance to inhaled allergen that occurs upon repeated exposure to allergens (Strickland et al. 2006). In humans with allergy, there is a reduction in the number and possibly function of Tregs (Kuipers & Lambrecht 2004), but it is unclear at present whether this would also lead to altered function of DCs in these patients.

Control of lung dendritic cell function by regulatory T cells

Although it has not been proven directly in humans that DCs are responsible for the Th2 sensitization process, some in vitro findings strongly imply these cells. The way in which allergens are handled by DCs is fundamentally different between atopic and nonatopic individuals (Bellinghausen et al. 2000; De Wit et al. 2000; Hammad et al. 2002; Lambrecht & Hammad 2002). When DCs obtained from HDM-sensitive asthmatics were exposed to the endotoxin-free major allergen component Der p1 in vitro, they mainly produced IL-10, but little IL-12. They expressed the costimulatory molecules CD86 and PDL-1 (Hammad et al. 2001, 2003b). When monocytederived DCs from non-HDM-allergic donors or nonallergic donors were exposed to Der p1, they mainly produced IL-12, expressed CD80, and produced the Th1-cell-specific chemokine CXCL10. Not surprisingly, monocyte-derived DCs from allergic patients induced Th2 cell responses of naive alloreactive T cells in vitro, whereas those DCs from nonallergic individuals induced Th1 responses. Therefore, the way HDM is handled by DCs is crucial to the generation of Th2 cell sensitization, and is clearly different in patients with allergy to HDM. The cysteine protease activity of Der p1 induced these changes in the DCs of allergic individuals, indicating that the activation of a protease-activated receptor on DCs leads to

The signals that determine the type of response after encountering a pathogen in the lung are delivered by DCs in the lymph node. Induction of DC maturation and provision of peptide–MHC to T cells is not sufficient to generate effector cells (Sporri & Reis e Sousa 2005). During generation of an efficient effector immune response DCs have to overcome suppression by Tregs, and the dominant way by which they seem to do this is by producing the cytokine IL-6, which counteracts the suppression by naturally occurring CD4+CD25+ Tregs (Doganci et al. 2005). Established airway inflammation seems to be regulated by Tregs expressing membrane TGF-β or secreting bioactive TGF-β and possibly IL-10 (Kearley et al. 2005). This is a pleiotropic cytokine with significant antiinflammatory and immunosuppressive properties in the lungs, as reduced expression of this cytokine exacerbates airway pathology in an asthma model (Ostroukhova et al. 2004). Several papers now support the concept that Tregs alter airway DC function. Mice lacking the transcription factor RunX3, involved in downstream TGF-β signaling, spontaneously develop asthma features (Fainaru et al. 2004). In the lungs of these mice, there is a strong increase

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aberrant cellular activation in patients with asthma (Hammad et al. 2001). The enzyme activity of Der p1 could also indirectly facilitate antigen presentation by DCs in vivo, by allowing access to intraepithelial DCs through cleavage of epithelial tight junctions and by locally activating the release of epithelial GM-CSF (Wan et al. 1999; Lordan et al. 2002). In this way, the epithelial response to allergens might also determine the type of adaptive immune response induced by DCs. Supporting this idea, DCs treated with lipase (an industrial allergen displaying an enzymatic activity) have been reported to induce a strong recall CD4+ T-cell response associated with a high production of IL-4 and IL-13, and a low production of IFN-γ (Lindstedt et al. 2005). However, allergens without enzymatic activity can also directly activate DCs to induce Th2 priming. For instance, phytoprostane lipids contained in pollen allergens can induce DC maturation and inhibit IL-12 production by LPS-activated DCs. When cocultured with allogeneic naive T cells, pollen-treated DCs polarized the immune response toward Th2 (Traidl-Hoffmann et al. 2005).

Dendritic cells in allergic asthma Not only do DCs play a role in the primary immune response to inhaled allergens, but they are also crucial for the outcome of the effector phase in asthma. Indeed, the number of mDCs is increased in the airways of sensitized and challenged mice during the acute phase of the response (van Rijt et al. 2002). However, during the chronic phase of the pulmonary response, induced by prolonged exposure to a large number of aerosols, respiratory tolerance develops through unclear mechanisms. During this regulatory phase, the number of mDCs in the lungs steadily decreased, and this was associated with a reduction of bronchial hyperreactivity. Inflammation, however, reappeared when mDCs were given (Koya et al. 2006). The role of mDCs in the secondary immune response was further supported by the fact that their depletion at the time of allergen challenge abrogated all the features of asthma, including airway inflammation, goblet cell hyperplasia, and bronchial hyperresponsiveness (Lambrecht et al. 1998; van Rijt et al. 2005). Again, the defect was restored by intratracheal injection of mDCs. It therefore seems that mDCs are both necessary and sufficient for secondary immune responses to allergen. Costimulatory molecules expressed by DCs could play a crucial role in established asthma. Pulmonary DCs upregulate the expression of CD40, CD80, CD86, ICOS-L, PD-L1, and PD-L2 during eosinophilic airway inflammation, particularly on contact with Th2 cells (De Heer et al. 2004; van Rijt et al. 2004, 2005). Costimulatory molecules might be involved in activation of effector T cells in the tissues. In allergen-challenged mice, mDCs might also be a prominent source of the chemokines CCL17 and CCL22, involved in attracting CCR4+ Th2 cells to the airways (Vermaelen et al.


2003; Kohl et al. 2006). The proallergic cytokine TSLP induces the production of large amounts of CCL17 by mDCs, thus contributing to the recruitment of a large number of Th2 cells to the airways, explaining how it may act to enhance inflammation (Zhou et al. 2005). In humans, allergen challenge leads to an accumulation of myeloid, but not plasmacytoid, DCs in the airways of asthmatics, concomitantly with a reduction in circulating CD11c+ cells, showing that these cells are recruited from the bloodstream in response to allergen challenge (Jahnsen et al. 2001; Upham et al. 2002). In stable asthma, the number of CD1a+ DCs is increased in the airway epithelium and lamina propria, and these numbers are reduced by treatment with inhaled corticosteroids (Moller et al. 1996). Based on the above data from mouse studies of asthma, it is very likely that part of the efficacy of inhaled steroids might be due to their effects in dampening airway DC function. According to current thinking, epithelial dysfunction, either intrinsic to asthma or caused by persistent inflammation, leads to epithelial release of profibrotic cytokines, such as epidermal growth factor and TGF-β, acting on fibroblasts and smooth muscle cells, disturbing the equilibrium between epithelial destruction, and growth and repair. Moreover, asthmatic epithelium might release factors such as GM-CSF, TSLP or chemokines that profoundly influence DC survival and/or function (Fig. 8.9). The exact consequences of this epithelial remodeling on the functioning of the airway DCs are currently unknown. Finally, many inflammatory cell types such as mast cells, basophils, and eosinophils are recruited to the airways in chronic asthma. These cells release many mediators such as cytokines, neuropeptides, enzymes, and lipid mediators that may also profoundly influence DC function and, in this way, might perpetuate ongoing inflammation (Lambrecht & Hammad 2003b). As only one example, it is known that histamine and PGD2, both released by mast cells upon crosslinking, reduce the potential of DCs to produce bioactive IL-12, and in this way contribute to Th2 polarization (Idzko et al. 2002; Hammad et al. 2003b). The exact role of pDCs in ongoing allergen-specific responses in asthma is currently unknown. It was shown that pDCs accumulate in the nose, but not lungs, of allergenchallenged atopics (Jahnsen et al. 2000). This is not strictly correct: the same author has shown that deliberate challenge of HDM-sensitized atopics with HDM leads to rapid recruitment of mDCs (but not pDCs) to the airway mucosa within the time-frame of the LPR and, moreover, that this response is attenuated by steroid treatment (Jahnsen et al. 2001). When pDCs were pulsed with pollen allergens, they were as efficient as mDCs in inducing Th2 proliferation and effector function (Farkas et al. 2004). Others have suggested that pDCs might also confer protection against allergic responses, as in the mouse. In children at high risk of developing atopic disease, the number of circulating pDCs was reduced (Hagendorens et al. 2003).

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Allergens

Allergens break down epithelial tight juntions

TSLP PGE2 IL-10 CCL5, attracts Eos, Th2, DCs

GM-CSF enhances DC survival

CCL20, attracts DCs

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Ag loaded DCs migrate to draining lymph nodes

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Stimulation of recirculating memory Th2 cells and naive T cells

IgE synthesis

Dendritic cells in atopic dermatitis Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by eczematous lesions and is associated with elevated serum IgE levels, and tissue and blood eosinophilia. AD is characterized by the infiltration of Th2 cells and the increased secretion of Th2-related cytokines (IL-4 and IL-5) and chemokines (TARC) in early lesions. However, Th1 cells also emerge during the chronic phase of the disease (Fiset et al. 2006). Recent evidence suggests that DCs in the skin and the blood of patients with AD play a pivotal role in the generation and/or control of inflammation. In patients with AD, DCs highly express FcεRI, the highaffinity receptor for IgE (Stary et al. 2005). Two FcεRI + subsets of mDCs have been identified in skin lesions of AD patients: (i) LC expressing CD1a and BG found in the epidermis; and (ii) inflammatory dendritic epidermal cells (IDEC) only found in inflamed skin (Wollenberg et al. 1995, 1996). In AD, FcεRI+ LCs bearing the antigen migrate from the skin to the draining lymph nodes where they activate FcεRI-mediated

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Fig. 8.9 Interaction between epithelial cells and dendritic cells (DCs) during established inflammation. Allergens stimulate epithelial cells to release chemokines and growth factors for DCs, Th2 cells, and eosinophils. Thymic stromal lymphopoietin (TSLP) and granulocyte–macrophage colony-stimulating factor (GM-CSF) are instrumental in inducing a Th2 prone phenotype in lung DCs. Epithelial cell tight junctions are opened up by protease activity of certain allergens, such as Der p1 from house-dust mite. In this way, allergens gain access to the DC extensions. The recruited DCs are also stimulated directly by allergen and produce even more chemokines for Th2 cells (TARC and MDC). Locally attracted Th2 cells interact with DCs in the airways, leading to local DC maturation and T-cell costimulation of effector cytokine production. These activated Th2 cells eventually control the inflammatory process by activating eosinophils and mast cells, and by feeding back on the epithelium and DCs. At the same time, DCs also migrate to the draining lymph nodes where they restimulate recirculating memory Th2 cells to become effector cells, and they recruit new cells into the response. In this way, effector cells are continuously replenished. DCs are also crucial for maintaining IgE synthesis, through their stimulation of IL-4-producing Th2 cells. See text for definition of abbreviations. (See CD-ROM for color version.)

Th2 immune responses. At the same time, LCs also present allergen-derived peptides locally to transiting T cells and induce a classic secondary immune response. Moreover, the aggregation of FcεRI on LCs stimulates them to release chemokines such as IL-16, TARC, MDC, and monocyte-attracting chemokines (Novak et al. 2004a). All these molecules contribute to the recruitment of FcεRI hi IDEC into the skin. IDECs are only found under inflammatory conditions, display high stimulatory capacities toward T cells, and serve as amplifiers of the allergic-inflammatory immune response. The stimulation of FcεRI on IDEC induces the release of IL-12 and IL-18 leading to the priming of Th1 cells, probably contributing to the Th1 response observed in the chronic phase of AD. In the mouse, overexpression of TSLP under the control of a keratinocytespecific promoter led to an AD-like phenotype. In these mice, skin DCs were likely activated to induce Th2 responses to some self or environmental antigen (Yoo et al. 2005). In addition to mDCs, pDCs have been found in increased numbers in the blood of AD patients and express FcεRI (Novak et al. 2004b; Stary et al. 2005). pDCs can process allergens by FcεRI-IgE and promote Th2-type immune responses.

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However, in contrast to LCs or IDEC, pDCs fail to accumulate in skin lesions of AD patients and seem to be retained in the bloodstream. Whether this is due to a lack of recruitment from the blood to the skin or to the high sensitivity of pDCs to proapoptotic signals present in AD skin, remains unclear.

Role of dendritic cells in allergic rhinitis In allergic rhinitis (AR), CD4+ Th2 cells control inflammation by secreting Th2 cytokines, but little is known about how these cells are activated to cause disease. Elevated numbers of CD1a+ LC are present in the nasal mucosa of symptomatic grass pollen-sensitive AR patients, and these numbers further increase upon relevant allergen challenge to the nose (Fokkens et al. 1989; Godthelp et al. 1996; Fokkens 1999). In symptomatic AR patients, DCs bearing allergenspecific IgE in the nasal mucosa are present (Kleinjan et al. 1997). In HDM-allergic perennial AR patients, the number of CD1a+ and CD11c+ MHCII+ DCs was higher in the epithelium and lamina propria of the nasal mucosa compared with healthy control subjects. In AR, DCs had a more mature phenotype and were found in close approximation with T cells. Similarly, in a mouse model of OVA-induced AR, CD11c+ DCs accumulated in areas of nasal eosinophilic inflammation and clustered with CD4+ T cells. To address the functional role of DCs in maintaining inflammation, CD11c+ DCs were conditionally depleted during allergen challenge by systemic administration of diphtheria toxin (DT) to CD11cDT-Receptor-Tg mice. In the absence of CD11c+ DCs, nasal OVA challenge in OVA-sensitized mice did not induce nasal eosinophilia, and did not boost OVA-specific IgE levels or Th2 cytokine production in the cervical lymph nodes. Conversely, when OVA pulsed DCs were administered intranasally to sensitized mice, they strongly enhanced OVAinduced nasal eosinophilia and Th2 cytokine production. These data in humans and mice suggest an essential role for nasal DCs in activation of effector Th2 function leading to allergic rhinitis, and identify DCs as a novel target for therapeutic intervention (Kleinjan et al. 2006). In support, treatment of AR patients with intranasal corticosteroid therapy reduced dramatically the numbers of DCs in the nasal mucosa (Holm et al. 1999).

Dendritic cells as drug targets in allergic diseases If DCs are so crucial in mounting immune responses during ongoing inflammation in the lung, nose, and skin, then interfering with their function could constitute a novel form of treatment for allergic diseases. Additionally, pharmacologic modification of DCs might fundamentally reset the balance


of the allergic immune response in favor of regulatory T cells, and thus lead to a more long-lasting effect on the natural course of allergic disease. Steroids are currently the cornerstone of antiinflammatory treatment in allergic disease. Inhaled steroids reduce the number of lung and nose DCs in patients with atopic asthma (AA) and AD, whereas local application of steroids to the skin of AD patients reduces the influx of IDECs (Holm et al. 1995; Moller et al. 1996). The immunosuppressant drug tacrolimus is currently in use for topical treatment of AD. It suppresses the expression of MHCII and costimulatory molecules and FcεRI on LC from AD patients in vitro, and reduces the number of IDECs in lesional skin. Recently, several other new molecules have surfaced that may alter DC function in allergic inflammation and thus treat disease. In one report, administration of CpG-containing immunostimulatory DNA sequences to the lungs of allergenchallenged mice inhibited the upregulation of these costimulatory molecules, suggesting that this is one mechanism by which they suppress inflammation (Hessel et al. 2005). The S1P analog FTY720 is currently used in clinical trials for multiple sclerosis and transplant rejection. When given to the lungs of mice with established inflammation, it strongly reduced inflammation by suppressing the T-cell stimulatory capacity and migratory behavior of lung DCs (Idzko et al. 2006). Also, selective agonists of particular prostaglandin series receptors might suppress DC function. The DP1 agonist BW245C strongly suppressed airway inflammation and bronchial hyperreactivity when given to allergic mice by inhibiting the maturation of lung DCs. DCs thus exposed to DP1 agonists induced the formation of Foxp3+ Treg cells that suppressed inflammation upon adoptive transfer (Hammad et al. 2007). A very similar mechanism was described for inhaled iloprost, a prostacyclin analog acting on the IP receptor expressed by lung DCs (Idzko et al. 2007). A specific small-molecule compound (VAF347) that blocks the function of B cells and DCs was also shown to be effective in suppressing allergic airway inflammation in a mouse model of asthma (Ettmayer et al. 2006). Finally, specific inhibitors of syk kinase were shown to suppress DC function and cure established inflammation (Matsubara et al. 2006).

Origin and function of macrophages The lung contains a large variety of macrophages, of which the phenotype and function varies considerably in baseline and inflammatory conditions. The vast majority in the lung are, however, alveolar and interstitial macrophages. AMs originate from a CD34+ hematopoietic stem cell and differentiate along the myeloid pathway under the influence of MCSF and IL-6. These cells have therefore a lineage relationship with DCs. The immediate precursors of lung macrophages are blood monocytes, which have the potential to differentiate into macrophages upon arrival in the lung tissues and

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alveolar compartment. Apart from a bone marrow supply, it has been demonstrated by bone marrow irradiation experiments that alveolar macrophages derive also from a local proliferating pool of precursor macrophages that respond to M-CSF. The predominant function of AMs is to phagocytose inhaled particulate antigens, and to effectively sequester these antigens from the immune system. Therefore, AMs are endowed with many receptors, such as the CR1 and CR3 complement receptors and Fc receptors for opsonized antigens, and the macrophage mannose receptor, as well as scavenger receptors. A very efficient system of phagolysosomes fuses with endocytosed particles to neutralize the ingested material. Kradin has very elegantly demonstrated that AMs sequester inhaled particulate antigens to shield them off from the specific immune system induced by DCs. A pulmonary cellular immune response is generated to an inhaled particulate antigen when the protective phagocytic capacities of the lung are exceeded and antigen is able to interact directly with interstitial DCs. The diversion of particulate antigens by pulmonary phagocytes may help to limit undesirable pulmonary inflammation while allowing the generation of antigen-specific immune lymphocytes in vivo (MacLean et al. 1996).

Homeostasis in the alveolar compartment is maintained by alveolar macrophages Macrophages are specialized hematopoietic cells, distributed throughout different tissues of the body where they play a central role in homeostasis, tissue remodeling, host defense, and the response to foreign materials, including particulates. One of the key functional characteristics of macrophages is that, depending on their state of differentiation and the microenvironmental factors that they encounter in a particular tissue, they can be modulated to express whatever functions are required to deal most effectively with a given kind of inciting stimulus. In the lung, resident AMs are continuously encountering inhaled substances due to their exposed position in the alveolar lumen. To avoid collateral damage to type I and type II alveolar epithelia cells in response to harmless antigens, they are kept in a quiescent state, producing few inflammatory cytokines, and displaying poor phagocytic activity, as evidenced by downregulated expression of the phagocytic receptor CD11b (Holt 1978). In addition, AMs actively suppress the induction of adaptive immunity, through effects on alveolar and interstitial DCs and T cells. Elegant studies have demonstrated that in vivo elimination of AMs using clodronate-filled liposomes leads to overt inflammatory reactions to otherwise harmless particulate and soluble antigens (Thepen et al. 1989). AMs adhere closely to airway epithelial cells (AECs) at the alveolar wall and are separated by only 0.2–0.5 μm from interstitial DCs. In macrophage-depleted

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mice, the DCs have a clearly enhanced antigen-presenting function (Holt et al. 1993). When mixed with DCs in vitro, AMs suppress T-cell activation through release of NO (mainly in rodents), prostaglandins, IL-10, and TGF-β. The T cells that have been exposed to human or rodent AMs have a remarkable phenotype as they are locked in G0/G1 arrest, unable to proliferate, yet after T-cell receptor stimulation demonstrate normal calcium fluxes, produce IL-2 and IFN-γ, and express the IL-2 receptor CD25. However, this phenotype is completely reversible, and T cells resume dividing when AMs are removed. Under homeostatic conditions, AMs closely adhere to AECs and this in turn induces the expression of the integrin αvβ6 on AECs, in a TGF-β-dependent manner. It was previously shown that αvβ6 integrin-deficient mice have activated AMs. This integrin has the potential to activate latent TGF-β by binding to the latency-associated peptide (LAP), an N-terminal inactivating fragment of TGF-β. Binding of activated TGF-β to its receptors expressed on macrophages induces phosphorylation of SMAD2 and SMAD3, and to suppression of macrophage phagocytosis and cytokine production. The inhibition of macrophage function by αvβ6–TGF-β complex is unique to the lung, illustrating the microenvironmental specializations of macrophages to meet the needs of the tissue. AEC expression of the αvβ6–TGF-β complex could also suppress the function of alveolar DCs and adaptive immunity. The mechanism of immune homeostasis and tonic inhibition of macrophage function in the lung is so robust that it was long enigmatic how infection might lead to macrophage activation triggering of TLRs or, via non-TLR-mediated stimulation of innate immune receptors on macrophages, open a window of opportunity for macrophage activation. TLR stimulation of macrophages leads to a rapid loss of contact with AECs, in turn inducing a rapid loss of expression of αvβ6 integrin expression on AECs. Under these conditions, TGF-β is no longer activated, releasing the brakes over macrophage activation and innate immune function, and macrophages become primed to secrete proinflammatory cytokines (TNF-α, IL-6) and to phagocytose particulate matter. Once activated, AMs can clear the infectious threat on their own, at the same time avoiding collateral damage to the alveolus. In addition, many infectious agents lead to recruitment of CCR2+ inflammatory monocytes to the alveolar space. These freshly recruited monocytes are clearly proinflammatory and display phagocytosis and killing, and promote rather than suppress T cell and DC activation. It takes a few days before these monocytes acquire the suppressive phenotype of alveolar macrophages, allowing for another ‘window of opportunity’ for initiation of innate and adaptive responses in the lung. To avoid collateral damage and to restore gas exchange as quickly as possible, there needs to be a mechanism keeping macrophage activation in check. Activated lymphocytes secreting IFN-γ stimulate the production of matrix metalloproteinase (MMP)-9. This particular MMP has the potential to activate latent TGF-β and, in this way, tonic inhibition of macrophage

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function is restored, macrophages again adhere to AECs, and αvβ6 integrin expression is restored. It has been described previously by Holt and colleagues that freshly recruited monocytes gradually acquire the phenotype of resident suppressive AMs over a period of days (Bilyk & Holt 1993). Whether this would be a predefined process or an instruction by the lung TGF-β-rich environment remains to be shown. Certainly, the prolonged presence of activating cytokines such as GM-CSF keeps inflammatory monocytes from acquiring suppressive activities (Bilyk & Holt 1993). An additional advantage of enhanced TGF-β production would be the stimulation collagen synthesis in interstitial fibroblasts, necessary for restoring alveolar wall architecture (Lambrecht 2006b).

Function of alveolar macrophages in inflammatory conditions and asthma Under inflammatory conditions, fresh monocytes and DCs are recruited to the airways and these cells “dilute” the immunosuppressive AMs, allowing a window phase in which T-cell responses can be induced. Freshly recruited monocytes and AMs produce a variety of inflammatory mediators such as cytokines, cytokine antagonists, coagulation products, lipid mediators, and growth factors. The local production of GM-CSF in these conditions can also switch the immunosuppressive AMs into a stimulatory cell (Bilyk & Holt 1993). In humans, the recently recruited AMs can be discriminated from the resident AMs by means of staining with the combination of RFD1/RFD7, initially described by Spiteri et al. (1992). Using these antibodies, it was shown that RFD1+/RFD7 − AMs have a stimulating function, whereas the RFD1+/RFD7+ and RFD1−/RFD7+ subset has a suppressive function on T cells. Strikingly, the RFD1 marker is also expressed on lung DCs (Spiteri et al. 1992). The airways of AA patients and of sarcoidosis patients contain increased amounts of RFD1+ AMs. Numerous studies have now demonstrated that the phenotype and function of AMs in patients with asthma are fundamentally different from those of healthy controls. Most notably has been the increased expression of CD80 and/or CD86, CD1a, ICAM-1, and LFA-1 and of the low-affinity receptor for IgE on AMs from patients with AA (Aubas et al. 1984; Gosset et al. 1991; Chanez et al. 1993; Tang et al. 1998). Conversely, the expression of CD40 is lower in the AMs of asthma patients. When compared with healthy control subjects, the AMs of patients with AA produce more IL-1, IL-6, TNF-α, and IL-10, and less IL-12, explaining their preferential induction of Th2 responses. When AMs of AA patients were cocultured with syngeneic CD4+ T cells, they induced the production of IL-5 (Tang et al. 2001). In the same experiments, the AMs of healthy controls suppressed IL-5 production. In some experiments, the production of IL-5 could be inhibited by blocking antibodies against CD80 and/or CD86, IL-1, IL-6, or TNF-α (Balbo et al. 2001). Conversely, antibodies to CD40


enhanced the AM-induced IL-5 production by T cells (Tang et al. 2001). These studies suggest that AMs from AA patients can activate effector function in Th2 cells, at least in vitro.

Conclusion DCs and macrophages are crucial in determining the functional outcome of allergen encounter in the lung, nose, and skin, and antigen presentation by mDCs leads to Th2 sensitization typical of allergic disease, whereas pDCs appear likely to play a more subtle regulatory role. It is increasingly clear that DCs have an antigen-presenting function beyond sensitization. DCs therefore constitute a novel target for the development of antiallergic therapy aimed at the origin of the inflammatory cascade.

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Innate Immunity in Allergic Disease Ian Sabroe

Summary The innate immune system comprises a complex network of overlapping defenses against infection, ranging from humoral to cellular components, and from structural barriers such as the epithelium to mobile response elements such as phagocytic leukocytes. Recent rapid expansion in our knowledge of many key pathways regulating innate immune activation (Toll-like receptors, Nod-like receptors, RIG-I-like helicases) has stimulated a resurgent interest in the mechanisms of innate immunity. This immune network regulates our every interaction with the pathogenic world around us, and has important capability in the sensing of tissue damage. Lifelong engagement of the innate immune system feeds into control and modulation of the function of the adaptive immune system. The timing and nature of activation of the innate immune system is an important determinant of our risks of developing allergic disease in childhood; continual engagement of innate immunity throughout our lives directly influences the severity of allergic disease once established. The most promising new treatments for allergic disease directly engage with innate immunity to rebalance the activity of the adaptive immune system. Allergic disease cannot be understood without reference to the central role of the innate immune system. This chapter describes both the basic science of innate immunity, and its dialogue with disease, highlighting the potential for targeting of these systems to dramatically influence the future management of allergic disease.

What constitutes the innate immune system? Present long before birth, our innate immune system provides intrinsic protection against a broad range of pathogens without the need for preexposure to the pathogen or formation of immunologic memory. This lack of targeted memory leads authors to describe the innate immune system as Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

“nonspecific,” though a series of proteins, receptors, and cells provide tailored responses to microbes, leading to effective responses to many different pathogens. Thus, the innate immune system exhibits some specificity, encoded through a large germline family of genes that are not rearranged as part of a memory-invoking process. The absolute requirement of the innate system for generation of effective memory further complicates the arbitrary division of immunity into innate and adaptive systems.

Humoral factors Innate immune responses are seen in a very broad range of tissues. Indeed, the Toll-like receptors (TLRs, one of the most important series of innate immune response proteins, described in detail below) are probably represented at some level in every cell in the body. Even before such systems are engaged, however, other levels of defense have important roles in mediating successful immunity. Barrier functions served by intact epithelia are fundamental to the prevention of infection. Such barriers are augmented by secreted milieu such as airway mucus, with its ability to bind and neutralize pathogens resulting in their clearance via the mucociliary escalator. Epithelial lining fluids often contain potent microbicidal proteins such as cathepsins, collectins, lysozyme, and defensins (Bals 2000; Ganz 2004; Hickling et al. 2004), with roles in pathogen neutralization, killing, and opsonization. A key part of any inflammatory response is the induction of capillary permeability, resulting in the delivery of further humoral factors such as complement to the site of tissue damage/infection (Walport 2001a,b). In addition to its roles in bacterial killing, complement is important in the regulation of leukocyte trafficking and inflammation. Mice deficient in the complement protein C3 show reduced production of Th2 cytokines in asthma (Drouin et al. 2001), though interestingly if allergen sensitization is performed epicutaneously, C3aR knockout mice show the opposite phenotype with enhanced Th2 responses perhaps mediated by alterations in antigen-presenting cell responses (Kawamoto et al. 2004). This interaction of innate and adaptive immunity is clearly complicated, and can involve multiple components of the complement system, since C5-deficient mice also show enhanced Th2-type allergic inflammation (Drouin et al. 2006). The role

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Contact of pathogens with the innate immune system will most frequently occur at epithelia, and the biology of the airway epithelium is of considerable importance in asthma (Davies et al. 2003). Airway epithelia express a range of innate immune receptors, allowing them to function as a line of first response to pathogens: their ability to detect and respond to pathogens must clearly be substantial, given that they form the main target for most respiratory viruses. There are also potentially close relationships between epithelial cells and other cells of the innate immune system such as DCs and macrophages (Rescigno et al. 2001; Morris et al. 2005, 2006; Sabroe et al. 2006). Cooperative networks that regulate airway inflammation are discussed in more detail below. Interestingly, defective responses to respiratory viruses are evident in epithelial cells from asthmatics (Wark et al. 2005), which may be relevant in the pathology of asthma exacerbations, and phenotypic differences in epithelia between asthmatics and normal subjects have been demonstrated (Kicic et al. 2006). Effective barrier functions result from a combination of humoral and cellular mechanisms, as classically illustrated in the respiratory tract, where mucus and lining fluid serve to neutralize and clear pathogens, and epithelial cells act to impede pathogen access to the body, as well as providing a first line of response to pathogens.

2006), and alveolar macrophages can inhibit DC maturation in the lung (Holt et al. 1993). These are long-lived cells derived from monocytes, but in chronic inflammation repeated rounds of cell recruitment may result in replacement of macrophages by cells with a more monocytic phenotype (Maus et al. 2006). Data from the gut and lung has suggested that DCs may sample the local microenvironment by extruding processes through epithelial cell tight junctions (Rescigno et al. 2001; Jahnsen et al. 2006): such mechanisms might also be theoretically important in the lung. Clearly the lung is a major site of antigen exposure, and resident DCs have vital roles to play in the regulation of the subsequent inflammatory and immunologic response, acting as a bridge between innate and adaptive immunity (discussed in more detail below). Again, subsets of DCs in the lungs show selective patterns of expression of innate immune receptors such as the TLRs, and differential ability to support T-cell proliferation (Demedts et al. 2006). Other cells which act as long-term resident early-warning cells include the lung mast cell. Mast cells respond to pathogenic stimuli in species- and tissue-specific patterns (Supajatura et al. 2002; Ikeda & Funaba 2003; McCurdy et al. 2003; Okumura et al. 2003; Kulka et al. 2004; Matsushima et al. 2004; Orinska et al. 2005; Nigo et al. 2006), and through dual signaling via pathogen-response systems and IgE-mediated mechanisms have the potential to integrate innate and adaptive responses into a regulation of Th2 immunity. Resident T-cell populations that form part of the innate immune system, such as natural killer (NK) and NKT cells, may also exert important roles in allergic inflammation. These cells again respond to agonists stimulating pathogen response systems such as TLRs, though some of these responses may be mediated by indirect networks (Herzyk et al. 1992; Korsgren et al. 1999; Hart et al. 2005; Akbari et al. 2006; Gorski et al. 2006; Kay 2006).

Resident leukocyte populations

Recruited leukocyte populations

Innate immunity depends on both resident and recruited leukocytes. The macrophage without doubt plays an important role in the detection of pulmonary infections. Low inocula of pneumococci are cleared by macrophages (Dockrell et al. 2003). Alveolar macrophages also have an important role in the initiation of responses to inhaled lipopolysaccharide (LPS) (Hollingsworth et al. 2005), and their function is altered by exposure to irritants such as cigarette smoke (McCrea et al. 1994; Medvedev et al. 2006). Alveolar macrophages may also have a role to play in the regulation of airway hyperresponsiveness. These cells can exhibit strain-dependent bias toward the support of Th1 or Th2 phenotypes (Mills et al. 2000; Careau & Bissonnette 2004; Peters-Golden 2004) and be rendered tolerogenic, resulting in downregulation of allergic airways disease (Korf et al. 2006). There is also evidence that macrophages have additional roles in the maintenance of normal peripheral tolerance in vivo (Hoves et al.

Once local defenses are activated, rapid recruitment of additional leukocytes is the rule, aiming for fast and effective pathogen clearance with rapid restoration of normal tissue architecture. Neutrophils are the most numerous early phagocyte recruited to inflammatory sites. Their principal role is the neutralization of bacterial and fungal pathogens, but they also interact with many other cell types to influence the development of the inflammatory lesion (discussed below). Their role in asthma is becoming increasingly discussed (Kamath et al. 2005), potentially contributing to specific disease phenotypes evident at a pathologic (Wenzel et al. 1999) or clinically relevant (Green et al. 2002) level. Clinical phenotypes associated with airways neutrophilia include exacerbations induced by viruses (Grissell et al. 2005), severe (Jatakanon et al. 1999) or steroid-resistant disease (Wenzel et al. 1999), and potentially fatal disease (Lamblin et al. 1998). These cells are not just there in their capacity as phagocytes,

of complement fragments is thus context dependent, and recent work suggests that, for example, C5a may reduce the establishment of Th2 responses, but in models in which allergic sensitization has already occurred, inhibition of C5a might reduce disease severity (Kohl et al. 2006; Lambrecht 2006). Again, these effects may involve, but are not exclusively dependent on, alteration in dendritic cell (DC) trafficking and function (Kohl et al. 2006; Lambrecht 2006).

Barrier cells, innate immunity, and allergic inflammation

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since they can contribute to the generation of delayed-type hypersensitivity immune responses (Molesworth-Kenyon et al. 2005), modify cytokine production from macrophages (Daley et al. 2005), activate epithelia (van Wetering et al. 2005), induce the death of smooth muscle cells (Oltmanns et al. 2005), and even in death influence the subsequent healing or proinflammatory response (Haslett 1997; Zheng et al. 2004; Serhan & Savill 2005). Thus, the subsequent influx of both innate immune cells (eosinophils, monocytes, mast cells, NK cells) and adaptive immune cells (T cells, B cells) is heavily influenced by the early innate immune response. Additional recruitment of monocytes rapidly follows the neutrophil, changing the monocyte/macrophage balance of the lung (Maus et al. 2006) and providing cells that engage in phagocytosis of pathogen and apoptotic neutrophil alike, regulating the balance between ongoing inflammation and injury resolution. Eosinophil recruitment, a hallmark of allergic inflammation, provides a further phagocytic cell whose biological role in immunity and disease still remains an uncertain area (Flood-Page et al. 2003a,b). The presence of a diverse and responsive resident leukocyte population thus enables a sophisticated early response to pathogens. Many day-to-day routine microbial encounters may never “make it through” to interaction with the adaptive immune system, effectively being targeted by the highly efficient innate response. Activation of the innate response is an inescapable component of inflammatory reactions pertaining to epithelial and mucosal surfaces, and a potentially important contributor to the pathology of allergic disease.

How does the innate immune system recognize pathogens? Many molecules contribute to pathogen recognition, from well-described families of receptors such as the TLRs (Basu & Fenton 2004; Chaudhuri et al. 2005), Nod family/Nod-like receptors (NLRs) (Meylan et al. 2006), and scavenger receptors, through to integrins and sometimes apparently surprising moieties such as the platelet-activating factor receptor (Fillon et al. 2006). These signaling systems are often studied in the context of investigation of responses to their purified putative ligands or agonists. While this gives a picture of the contribution of individual signaling systems to a given response, it is important to remember that pathogens or pathogen remnants will typically interact with multiple receptor systems, sometimes across a membrane surface and sometimes within a phagolysosome. The processes of phagocytosis, and the interaction of pathogen surfaces and soluble components presented simultaneously potentially across a measurable portion of an innate immune cell’s surface, are likely to generate summated signaling and responses driven by a range of mechanisms.

Innate Immunity in Allergic Disease

Bacterium Virus

Lipoproteins

Flagellin ssRNA

LPS Proteins

1/2

3/3

4/4

5/5

2/6

dsRNA

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Fig. 9.1 Toll-like receptor (TLR) agonists. TLRs divide into two broad groups: those whose predominant role appears to be in the mediation of antibacterial responses, and those whose role appears more tied to meeting responses to viruses. TLRs colored dark blue appear more linked to antibacterial immunity. TLR1/2 heterodimers and TLR2/6 heterodimers mediate responses to a range of bacterial lipoproteins. Other TLRs function as homodimers. TLR4 responds to lipopolysaccharide (LPS), TLR5 to flagellin, and TLR9 to bacterial and viral DNA. TLRs colored pale blue are more central to responses to viruses. TLR3 mediates responses to double-stranded RNA, whereas TLR7 and TLR8 have been linked to responses to single-stranded RNA. As noted, TLR9 responds to some viral DNAs. The roles of TLR10 (white) have yet to be clarified. (See CD-ROM for color version.)

Toll-like receptors (Figs 9.1 & 9.2) The discovery of a germline-encoded family of pattern recognition receptors mediating responses to pathogens across species as evolutionarily distinct as fly and human has transformed our understanding of innate immunity. The story began when knockout of a gene in Drosophila led to a failure of fly embryos to undergo normal polarization (Anderson et al. 1985); in German, the language in which the protein underlying these abnormalities was named, toll has many meanings, including “great,” “weird” and “crazy.” A role for this molecule in fly development was later complemented by identification of a role for this protein in fly immunity (Lemaitre et al. 1996). The link to mammals came with the discovery of constitutive activation of human homologs of Toll-activated immune responses (Medzhitov et al. 1997) and that a human family of these proteins existed (Rock et al. 1998). Natural mouse strains unable to respond to LPS were subsequently shown to be lacking in a functional TLR4 (Poltorak et al. 1998; Qureshi et al. 1999), providing the key proof of the role of this family of proteins in innate immune signaling. In a remarkably short time we have come a long way in our understanding of TLRs. We now know that there are at least 10 TLRs in the human, with an eleventh that is

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Environmental microbial products

Allergens

Infections

Spectrum of contiguous immunity

Th1/innate immunity-type

Stimuli

Smoking, pollution

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Initiation signals Antigen presentation

Neutrophil recruitment

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Monocyte recruitment

Th2-type T-cell activation Processes

Mast cell recruitment

NK/NK T-cell stimulation Tissue cell activation

Bronchial hyperreactivity

functional in the mouse. Putative agonists or ligands have been assigned for each of these, except TLR10, whose function is still somewhat enigmatic, though its genetics appear to contribute to asthma risk (Lazarus et al. 2004). While not the only receptors employed for the detection of pathogens, they are clearly extremely important. In common with most pathogen-response elements, TLRs recognize not single specific ligands but molecules that have in common some molecular pattern. For example, TLR4 recognizes a range of LPS species, whereas TLR2 responds to lipoproteins, TLR5 to flagellins, and TLR9 to bacterial DNA. There are only limited data describing the molecular interaction of putative TLR ligands with their receptors, and further complications arise from the use of accessory proteins

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Fig. 9.2 Contiguous immunity in allergic disease. The pathology of chronic inflammatory diseases such as asthma are to some extent poorly described by traditional descriptors such as innate or adaptive disease, or Th1 vs. Th2 immunological phenotypes. These chronic diseases are perpetuated by repeated exposure to a variety of stimuli, activating a large number of inflammatory subsystems involving innate and adaptive, Th1 and Th2, type processes. Continual evolution of the inflammatory state occurs through dialogue between these inflammatory nodes, generating cooperative and regulatory networks that determine the disease phenotype and its progression or resolution. With time, as pathology evolves and the nature of the tissue changes in response, the components of the network alter. Such alterations occur both with respect to numbers and types of cells, and activation or differentiation status. Inflammation is regulated by networks that are physically adjacent with the potential for constant communication and dialogue (spatially contiguous), which may evolve in complex sequences over time (temporally contiguous). The relative contribution of each component is flexible, and understanding their different contribution to separate phases of the disease is essential to facilitate effective therapeutic targeting. (From Sabroe et al. 2007, with permission.) (See CD-ROM for color version.)

to enable signaling. For example, LPS signaling appears to require monomerization of LPS by LPS-binding protein, presentation to CD14 (a transmembrane or soluble nonsignaling protein), and transfer to a complex of MD-2 (a secreted protein) and TLR4 (Shimazu et al. 1999; Visintin et al. 2001a; Nagai et al. 2002; Gioannini et al. 2004; Kennedy et al. 2004). Under some circumstances, integrins such as CD11b/CD18 may play roles in LPS responses (Perera et al. 2001), and TLRs may aggregate in lipid rafts with other molecules that could facilitate signaling (Triantafilou & Triantafilou 2002; Triantafilou et al. 2004, 2006). Other TLRs broaden their range of agonists by heterodimerization: this is particularly seen for TLR2, which dimerizes with TLR1 or TLR6 (Ozinsky et al. 2000). The mechanism of interaction that enables broad

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detection of pathogens, while maintaining discrete nonrecognition of self, is still poorly understood with relatively little available pharmacologic data on the behavior of the TLRs. These receptors also show some ability to detect host damage, since self molecules including hyaluronan, fibrinogen, fibronectin, heat-shock proteins, and transcription factors can all potentially activate TLR4 (Ohashi et al. 2000; Okamura et al. 2001; Smiley et al. 2001; Guillot et al. 2002; Park et al. 2004; Tsan & Gao 2004; Jiang et al. 2005; Ohya et al. 2006). This literature has been bedevilled by the potential contamination of putative TLR4 agonists by LPS (Tsan & Gao 2004), an area fraught with difficulty (Wakelin et al. 2006). It appears true that TLR4 in particular can act as a sensor of tissue damage in the absence of pathogen exposure, but this is an area in which the evolution of our understanding remains hard to predict. TLRs are expressed widely, though some curiosities to these expression patterns are seen. In general, innate immune cells likely to come into contact with bacteria respond well to agonists of TLR2 (principally bacterial lipoproteins) and TLR4 (LPS), as well as to flagellin (TLR5), and often to bacterial DNA (TLR9). Many barrier cells such as airway epithelial cells also show similar responses and express similar patterns of TLRs (Hertz et al. 2003; Adamo et al. 2004; Armstrong et al. 2004; Guillot et al. 2004; Sha et al. 2004; Guillot et al. 2005). DCs show select patterns of TLR expression, with monocytederived DCs showing a monocyte-like expression pattern including TLR2 and TLR4, whereas plasmacytoid DCs tend to favor expression of TLR7 and TLR9 (Demedts et al. 2006). A wide variety of tissue cells, including epithelia, smooth muscle, and endothelial cells, respond to synthetic analogs of double-stranded RNA. Often generated as an intermediate in viral replication, double-stranded RNA is sensed by a variety of mechanisms, including protein kinase R (PKR), TLR3, and also the cytosolic receptors of the RIG-I/mda5 pathway (see below). TLR3 expression seems relatively widespread, although it is only found in rare leukocyte populations in the blood, including immature DCs (Muzio et al. 2000; Visintin et al. 2001b; Matsumoto et al. 2003). In the lung, monocytederived DCs express TLR3 (Demedts et al. 2006), as do alveolar macrophages, though unusually human alveolar macrophages do not show the classical induction of type I interferons (Punturieri et al. 2004) that is a hallmark of activation of the antiviral TLRs (TLR3, TLR7, TLR8). TLR3 also has a putative endogenous agonist (mRNA) (Kariko et al. 2004), and thus its common tissue expression may also indicate other roles in host defense or responses to tissue damage (Brentano et al. 2005) as yet relatively unexplored. In contrast to the “antiviral” receptor TLR3, TLR7 and TLR8 mediate responses to single-stranded viral RNA (Heil et al. 2004) and seem to show expression in selected leukocyte populations, including mast cells, neutrophils (Hayashi et al. 2003) and eosinophils (Nagase et al. 2003) but most notably plasmacytoid DCs, rather than in tissue cells (Hemmi et al.


2002; Mohty et al. 2003; Nagase et al. 2003; Diebold et al. 2004; Matsushima et al. 2004; Berkeredjian-Ding et al. 2005; Hart et al. 2005; Demedts et al. 2006; Morris et al. 2006). These receptors seem to be dependent on acidification of their intracellular phagosomes for effective signaling (de Bouteiller et al. 2005; Gibbard et al. 2006). TLRs are also important in T-cell function, providing another point of interaction between the innate and adaptive immune systems. The role of TLRs in T-cell function is discussed in more detail below. TLRs signal through a family of adapter proteins. Currently five adapters have been identified. MyD88 is the prototypic adapter, shared with the interleukin (IL)-1 receptor. Utilized by all TLRs except TLR3, it forms a signaling complex with a series of other proteins to activate mitogen-activated protein kinase (MAPK) and NF-κB cascades. A further adapter, MAL/TIRAP, shares in this signaling cascade for the receptors TLR2 and TLR4, but MAL/TIRAP is not involved in the signaling of TLR5, TLR7, and TLR9. TLR3 uses the adapter TRIF to activate NF-κB and the generation of type I interferons, coordinating antiviral responses. TLR4 also uses TRIF, but couples to it via the adapter TRAM. The exact pattern of signaling, and potentially of adapters used, may vary from cell to cell. A fifth adapter, SARM, appears to be a negative regulator of TRIF signaling (Carty et al. 2006). Other pathways initiate signaling via tyrosine kinases (Jefferies et al. 2003; Doyle et al. 2005; Horwood et al. 2006), small GTPases, and phosphatidylinositol 3-kinases: such pathways are still relatively poorly understood. Signaling is very carefully regulated by some extraordinarily complex pathways. Adapter function and localization is regulated (Gray et al. 2006; Mansell et al. 2006; Rowe et al. 2006). TLR8 (Wang et al. 2006) and the IL-1R superfamily member T1/ST2 (Brint et al. 2004) can negatively regulate TLR signaling. A variety of further negative regulators of TLR signaling, including members of the IRAK family and SOCS families, control responses, and TLR signaling can be rapidly tolerized (Medvedev et al. 2006). Presumably these very complex patterns of signaling and regulation thereof all enable tailored and controlled responses to specific pathogens. The evolving map of TLR signaling is complex indeed (Oda & Kitano 2006), and remains to be fully elucidated. Many of these pathways are actively targeted by viruses, in order, presumably, to promote their survival in host tissues (Bowie et al. 2000; DiPerna et al. 2004).

Nod-like receptors The NLRs form another family of less well understood intracellular pattern-recognition receptors (Inohara et al. 2005), with the potential to cooperate with TLRs in the induction of effective immune responses (van Heel et al. 2005). NOD1 and NOD2 recognize components of Gram-positive bacterial peptidoglycans. The best characterized of these interactions is the ability of NOD2 to respond to muramyl dipeptide, a

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peptidoglycan fragment generated during bacterial replication and host degradation of phagocytosed bacteria (Meylan et al. 2006).

Other sensors of viral infection Recently, a further family of cytoplasmic receptors have been identified that respond to viral infection. RIG-I and mda5 sense viral infection, presumably through the detection of double-stranded RNA, and activate IRF and NF-κB signaling pathways through another, mitochondrial-bound, adapter, Cardif (also known as VISA, MAVS, and IPS-1, having been discovered almost simultaneously by several groups). Early data indicate potentially specific roles for mda5 and RIG-I in responses to individual viruses, but this remains a very new field with much work to be done in defining their contribution to antiviral responses in a range of settings (Meylan et al. 2006).

Other pattern-recognition receptors While TLRs, NLRs, and the helicase-like proteins RIG-I and mda5 are currently major targets of research, it is important to recall that the family of pattern-recognition molecules is broad. Humoral factors such as complement are important, scavenger receptors contribute to recognition of bacteria such as Neisseria meningitidis and the pneumococcus (Hampton et al. 1991; Haworth et al. 1997; van der Laan et al. 1999; Fitzgerald et al. 2000; Peiser et al. 2000; Thomas et al. 2000; Gordon 2002; Peiser et al. 2002; Arredouani et al. 2004; Mukhopadhyay et al. 2004), and integrins may contribute to TLR signaling (Perera et al. 2001). Responses to viruses also involve PKR (Saunders & Barber 2003), and foreign DNA may also involve complex sensing pathways beyond TLR9 (Alvarez et al. 2006; Wagner & Bauer 2006), which may be cell type dependent.

How does the innate immune system become activated? The first thing to note is that very often the innate immune system will not become activated on pathogen exposure, because barrier components of the innate immune system will control the stimulus effectively without recourse to activation of cell signaling and responses. Mucociliary clearance, interaction with neutralizing proteins in skin or lung lining fluid, and impermeable barriers all conspire to prevent stimuli ever reaching a cellular surface in a form that requires an active response. However, it should be apparent from the description of the innate immune system above that innate immunity is far from a simple mechanism that provides basic barrier functions, or one whose role has been apparently surpassed by the more complex adaptive immune response. Indeed, the innate immune system functions to maintain our health on an hour-by-hour basis, continually regulating interactions with both pathogens and commensals in our

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environment, with no need of recourse to immunologic memory. It is also becoming apparent that effective immunity is not the function of a single cell, protein, or pathway, but rather depends on the coordinated action of a complex interdependent system whose evolutionary roots probably go back to the first unicellular organisms. In the context of single exposures to specific insults, it is possible to begin to define the networks involved in the generation of effective innate immunity. Signaling from sentinel cells such as the macrophage appears to be important in the initiation of responses to bacteria and LPS. These signals cooperate with responses from tissue cells, generating rapid amplification of signals favoring an efficient innate response. Interestingly, signaling from tissue cells and leukocytes in response to molecules such as LPS may play separate roles, since there is evidence that signaling initiated by macrophages is important for leukocyte recruitment, whereas signaling from lung tissue cells is needed for LPS to induce bronchoconstriction (Noulin et al. 2005). Activation of macrophages leads, via secretion of molecules such as IL-1, to rapid activation of tissue cells, switching epithelia and smooth muscle into prosynthetic phenotypes, and driving rapid amplification of cytokine production. Rapid recruitment of granulocytes, monocytes, and lymphocytes ensues. Each cell type in turn may modify the immune response, through the release of cytokines, tissue-damaging proteases, and reactive oxygen species, and ultimately through their death. This appears to be particularly true for neutrophils, whose death by apoptosis is a crucial injury-resolving stimulus (Haslett et al. 1994; Haslett 1997; Bianchi et al. 2006). The epithelial cell provides another immunocompetent sensing cell with considerable potential to initiate responses to bacterial infection, through its expression of molecules such as TLR4. While it can undoubtedly respond to LPS, there is now evidence that the magnitude of this response is dwarfed by that which may be achieved when sentinel leukocytes such as monocytes use the epithelial cell to amplify their response to LPS (Tsutsumi-Ishii & Nagaoka 2003; Morris et al. 2005, 2006). In chronic inflammation, the repopulation of the lung with relatively young monocytes/macrophages (Maus et al. 2006) amplifies the potential for potent responses to microbial pathogen exposure. The epithelium, and other TLR-responsive tissue cells, perhaps play a much greater role in the initiation of responses to viruses. Expression of a variety of viral sensors by this cell type allows it respond rapidly to viral infection, though again there is potential for cooperation with other innate immune cells such as monocytes/ macrophages in the initiation of effective antiviral immunity, through expression of TLR7/8 on some leukocyte populations. In reality, exposure to single TLR agonists probably never occurs, since bacteria and viruses will interact with components of the innate immune system through multiple molecular recognition systems. In these settings, it is becoming increasingly apparent that cooperativity between cells

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and receptors is essential for rapid and efficacious signaling and activation of innate immunity (Morris et al. 2006).

How do the innate and adaptive immune systems interact? Interactions with T and B cells Activation of innate immunity is key to the generation of effective adaptive immunity. TLR signals are among the most potent inducers of DC maturation and trafficking, making their signaling central to new treatments for allergic disease (Creticos et al. 2006; Hayashi & Raz 2006; Krieg 2006). Specific patterns of TLR expression between monocyte-derived DCs and plasmacytoid DCs suggest that these cells may serve different functions in innate immunity, and there is some evidence to support this contention in vivo (De Heer et al. 2004; Demedts et al. 2006). Some TLRs may be more naturally biased to a specific cytokine phenotype: there is evidence, for example, that TLR2 signaling may favor a Th2 response (Redecke et al. 2004), though this is not a unanimous finding (Sieling et al. 2003; Komai-Koma et al. 2004). TLR4 signaling has been associated with prevention of airway inflammation (Rodríguez et al. 2003), but TLR4 knockouts may show reduced Th2 responses, which appears contrary to these data (Dabbagh et al. 2002). Both TLR2 and TLR4 agonists may reduce risks of allergic sensitization (Velasco et al. 2005). The key to understanding this complicated area lies in appreciating the timing, duration, and intensity of the DC-stimulating signal. Low amounts of LPS, acting via TLR4, may actually favor Th2 responses during sensitization, whereas higher doses drive Th1-type inflammation (Eisenbarth et al. 2002, 2003). Once disease is established, the picture may be different again, since low amounts of endotoxin administered with allergen may downregulate Th2 inflammation whereas high doses enhance it (Hollingsworth et al. 2006). Variations between mouse strains, amounts of endotoxin administered, sensitization protocols (which might have a profound impact depending on route of administration), and levels of endotoxin contamination of classical model allergens such as ovalbumin all conspire to generate a field that is still confusing and unclear. Innate immune signals also directly regulate the function of cells of the adaptive immune system. LPS is a stimulator of T-cell proliferation (Vogel et al. 1983) and TLR2 can function as a costimulatory molecule on memory T cells (Komai-Koma et al. 2004). TLR2 signaling can also temporarily reduce the inhibitory potential of CD25+ T regulatory (Treg) cells, as well as rendering effector T cells less susceptible to Treg suppression of function (Liu et al. 2006). Again, contradictory data in this rapidly emerging area also suggest a potential for TLR2 signaling to enhance the suppressive ability of CD4+CD25+ Treg cells (Zanin-Zhorov et al. 2006). LPS is, additionally, an effective stimulus of B-cell proliferation, via TLR4 and the


related receptor RP105 (Ogata et al. 2000). It is again important to understand that TLR-mediated signaling into B- and T-cell function may depend on direct stimulation, but also on indirect signaling. Inflammatory sites to which T cells are recruited will contain many cytokine signals from activated leukocytes and tissue cells that directly modulate T-cell function and proliferation, and in the context of antigen presentation the TLR-driven activation status of the antigenpresenting cell may have profound consequences for T-cell function.

Interactions at the level of the mast cell The mast cell probably represents an important interface between innate and adaptive immunity. Mast cells are clearly capable of expressing a range of TLRs, though there are likely to be differences in TLR expression between tissues and species (Applequist et al. 2002; Supajatura et al. 2002; Ikeda & Funaba 2003; McCurdy et al. 2003; Okumura et al. 2003; Rodríguez et al. 2003; Kulka et al. 2004; Matsushima et al. 2004; Orinska et al. 2005; Nigo et al. 2006). TLR4 signaling may be able to significantly enhance Th2 cytokine production in response to IgE stimulation (Nigo et al. 2006), which may also explain the ability of LPS to increase allergic inflammation in some settings. Mast cell recruitment, a hallmark of allergic asthma (Brightling et al. 2002), may also be driven by TLR-mediated responses to viral infection (Morris et al. 2006), through cytokine generation from airway smooth muscle cells, potentially in cooperation with signals from activated leukocytes.

How do we translate this information into an understanding of human allergic disease? It should now be apparent that the innate immune system is crucial to health, and has immense potential to play important roles in human allergic disease by the regulation of inflammation, the control of sensitization, and close liaison with the function of the adaptive immune system. Despite the huge contribution of the innate immune response to allergic disease, the subject has to some degree, until the excitement of the TLR field, been something of a Cinderella speciality. There are currently substantial problems in translating our understanding of innate immunity in vitro and in vivo into a clear view of the pathology of allergic diseases. Most models of allergen challenge employed in vivo are relatively biased to an understanding of the mechanisms of Th2-driven inflammation. It has only recently become apparent that levels of endotoxin in the commonest allergen used in these studies, ovalbumin, is of considerable significance in understanding these processes (Eisenbarth et al. 2002; Hollingsworth et al. 2006). While these models have made a very great contribution to our understanding of allergic inflammation, they have

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not typically been geared to investigating the contribution of the innate immune response to these processes; moreover, the stimulation of exacerbations of allergic diseases such as asthma by viral infections, driving neutrophilic inflammation, is harder to model. Likewise, in vitro studies of human or mouse cells are often limited in their scope, and hard to use to make general inferences. The complexities involved are complicated by important differences between the innate immune response in mouse and human, as illustrated by studies of IRAK-4, a key signaling protein in the TLR cascade. While knockout of this protein in humans is associated with profound susceptibility to bacterial infections (Medvedev et al. 2003; Picard et al. 2003), in IRAK-4–/– mice there is marked susceptibility to an even greater range of pathogens (Suzuki et al. 2002). It is also likely that species-specific evolution of innate immune systems has occurred to tailor the responses of individual species, such as mouse and human, to their principal pathogens and commensals. That this is true is borne out by the chaos that ensues when a pathogen jumps species, as illustrated by the current fears over avian influenza and the havoc wreaked by bovine spongiform encephalopathy. Nonetheless, we can begin to discern how innate immunity will modify allergic disease.

Disease phenotypes It is important to note that no one process explains all disease. Within the field of asthma, it is clear that there are several pathologic phenotypes, as yet poorly characterized. These may include phenotypes in which certain innate immune systems, such as neutrophilic inflammation, are more represented (Lamblin et al. 1998; Wenzel et al. 1999; Green et al. 2002; Kamath et al. 2005), potentially requiring individualized therapy (Green et al. 2002). We are yet to distinguish these phenotypes reliably, which hampers their modeling in vitro and in vivo. In some situations, such as asthma induced by LPS exposure in certain occupational groups, the contribution of the innate immune system is easy to discern. Similarly, the dominant role of infections in the initiation of asthma exacerbations, and their role in acute exacerbations of eczema, should point to an important role for innate immunity in the expression of established disease. The evidence of the hygiene hypothesis suggests that innate immunity plays an important role in disease initiation as well. Equally, it is feasible that some sufferers of allergic disease, e.g., those with asthma induced by exposure to small domestic or research animals, have a disease that is almost entirely dominated by purely allergic triggers with less of a role for innate immune responses. The role of the innate immune system in disease may therefore be broken down into specific areas.

The innate immune system in disease initiation The data above demonstrate that the actions of the innate immune system are crucial in determining patterns of sensitization. Although the relative contribution of differing

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amounts of TLR agonists to the severity of allergic disease is still a complicated area of much research (Eisenbarth et al. 2002, 2003; Hollingsworth et al. 2006), it is absolutely clear that exposure to microbial stimuli modulate sensitization. This has been well illustrated in humans, as evidenced by epidemiologic data and expressed in the hygiene hypothesis (von Mutius 2001; Braun-Fahrlander et al. 2002). Exposure to endotoxin and other microbial compounds, at appropriate doses and in early life, appears to be highly protective against development of atopy (Braun-Fahrlander et al. 2002; Gehring et al. 2002; Matricardi et al. 2002; Weiss 2002). To what degree this is by induction of Th1 cell function, or upregulation of Treg cell function, remains an active area of investigation. Importantly, polymorphisms in genes associated with detection of pathogens modulate the risks of developing atopic disease. In farming children, polymorphisms in TLR2 predict risk of developing allergic disease (Eder et al. 2004). An interesting association has been seen between polymorphisms in the TLR4 coreceptor, CD14: those with a specific CD14 genotype show a negative association between environmental endotoxin and risk of allergic disease but a positive association with nonatopic wheeze (Simpson et al. 2006), and provide support for the notion that low levels of endotoxin may favor a Th2 pattern of immunity (Simpson et al. 2006). Interestingly, activation of the epithelium can modify local DC responses, potentially affecting risks of sensitization or amplification of established disease (Pichavant et al. 2006). Finally, although a controversial area, childhood viral infections are associated with, or modify later risks of, allergic disease (Singh et al. 2006).

The innate immune system in disease perpetuation Animal studies have demonstrated that high doses of LPS may exacerbate airway inflammation (Hollingsworth et al. 2006). These studies are consistent with observations that endotoxin levels in house dust are correlated with disease severity (Michel et al. 1996), and the link between viral infections and exacerbations of allergic airways disease is well established. In most patients with asthma, exacerbation triggers are predominantly infective or nonspecific, and allergens are rarely inhaled in the absence of TLR agonists such as endotoxin (Michel et al. 1996). Repeated rounds of inflammation, most often triggered by pathogens and perhaps endotoxin, will drive activation of the innate immune response, triggering recruitment of innate and adaptive immune cells. Chronic inflammation will cause the lung to be repopulated with relatively young and biologically active monocytes (Maus et al. 2006). Acute and chronic exposure to even relatively low-level innate immune stimuli can initiate cooperative signaling between leukocytes and tissue cells that can result in the potent induction and perpetuation of inflammation (Morris et al. 2005, 2006; Sabroe et al. 2006). These inflammatory networks are likely to evolve over time, as the local tissue environment changes. For example,

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airway remodeling and smooth muscle hypertrophy in the airway results in a greater potential for airway smooth muscle to contribute to the inflammatory response and, given its huge cytokine-secreting potential in cooperation with monocyte activation (Morris et al. 2005, 2006; Sabroe et al. 2006), this is potentially very significant. Alterations or differences in smooth muscle and epithelial phenotypes between asthmatics and normal subjects may also underpin disease and affect disease phenotype as processes evolve over time. Chronic recruitment of neutrophils may exert multiple effects on disease, through their ability to secrete chemokines and cytokines, contribute to Th1-type cell recruitment (Molesworth-Kenyon et al. 2005), modulate the cytokine response of macrophages (Daley et al. 2005), and directly induce tissue cell activation and damage (Oltmanns et al. 2005; van Wetering et al. 2005). Thus it can be seen that during chronic disease, multiple interrelated networks will perpetuate inflammation. These networks involve continual dialogue between innate and adaptive immune systems, and are modulated with time as the nature of the tissue changes with remodeling and chronic inflammation. Repeated rounds of acute pathogen stimulation will interact with chronic inflammation that has often become self-perpetuating. Importantly, components


of this inflammatory network may play different roles at different points in the disease, influencing acute inflammation, airway remodeling, leukocyte survival, and chronic inflammatory responses. In the setting of chronic allergic diseases such as asthma, simple terms of innate and adaptive immunity describe the pathologic processes poorly. We have therefore recently proposed that such pathologies represent a process we have named “contiguous immunity,” where multiple networks are both physically adjacent and cooperating (physically contiguous) and evolving over time (temporally contiguous) (Sabroe et al. 2006) (Fig. 9.2). These networks also allow modeling of other environmental stimuli with the chronic inflammation of allergic disease. For example, ozone-induced hyperresponsiveness is partly dependent on TLR4 signaling (Hollingsworth et al. 2004). Significant single environmental exposures, such as seen in emergency workers at the World Trade Center collapse, can also result in dramatic effects on lung function (Banauch et al. 2006). Whether any of these effects are mediated by activation of the innate immune system is, of course, not clear, but it is tempting to speculate that cell networks incorporating elements of innate immune responses to inhaled stimuli and local tissue damage are likely to have participated in these significant episodes of lung damage.

INNATE IMMUNE RESPONSE Inflammation

Bacterial killing

PMN

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Antimicrobial peptides (epithelial cells)

EO

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Activation MNC

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Fig. 9.3 Immune pathways activated by TLR signaling. TLR signaling exerts its effects on multiple cell types to regulate innate immune inflammation. Direct effects on individual cell types are indicated by solid lines, indirect effects by actions exerted by intermediary cells. EO, eosinophil; MNC, mononuclear cell; PMN, neutrophil. See text for definition of other abbreviations. (From Parker et al. 2006, with permission.) (See CD-ROM for color version.)

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Choosing targets

Impact of disease on innate immunity While our primary concern is the impact that the innate immune system has on allergic disease, it is essential to recognize that allergic disease also impacts on the functioning of the innate immune system. In part this is through treatment, since immunosuppressive therapies inevitably contribute to impairment of immunity at the levels of the innate and adaptive systems. Induction of inflammation might be thought to generate a local environment that is hostile to pathogens, and allergen provocation of the nose can, for example, enhance subsequent responses to endotoxin (Eldridge & Peden 2000). Beyond this, asthma itself is a risk factor for invasive pneumococcal disease (Talbot et al. 2005). Macrophages are crucial in defense against pneumococccus (Dockrell et al. 2003). It is interesting to note, therefore, that the phenotype of allergic disease and the response to infection may rest in part in macrophage phenotypes that may be better or worse at supporting Th2 and Th1 responses (Mills et al. 2000). Susceptibility to pneumococcal disease in different mouse strains is potentially associated with their ability to recruit and activate neutrophils (Gingles et al. 2001). Allergic inflammation further degrades the function of innate immunity, through suppression of the generation of antimicrobial peptides such as defensins from the epithelium, resulting in increased susceptibility to Pseudomonas aeruginosa infections in mouse models (Beisswenger et al. 2006). Abnormalities in the bronchial epithelium also appear to underlie risk factors for viral infection, since asthmatic airway epithelium shows an impaired interferon response to viral infection (Wark et al. 2005; Contoli et al. 2006). It is interesting to note that local immunity may be impaired even in the presence of a potentially vigorous chronic inflammatory response. Indeed, chronic inflammation may for other reasons provide an environment with potentially advantageous components from the point of view of the respiratory pathogen. Barrier disruption by epithelial damage may favor pathogen access, and upregulation of tissue cell adhesion molecule expression may make viral attachment and infection easier.

The complex immunopathology of asthma and other allergic diseases remains poorly understood. Making the link between the innate immune system and allergic disease has now allowed us to consider targets that had previously been overlooked. Each component of the innate immune system may be a feasible drug target, but we need to consider carefully how such targets should be handled. For example, if the innate immune system is activated repeatedly over time, generating repeated acute waves of high levels of activation (potentially on a background of chronic activation), it may be appropriate to target these separately from the underlying chronic disease. This might be achieved, for example, by neutralizing IL-1 or other important upstream cytokines during acute exacerbations. The long-lived nature of the immune response also requires serious thought when designing interventional therapies and drug trials. This is well illustrated by a cursory consideration of the biology of the macrophage. It is clear that monocytes and macrophages perform important functions as immune surveillance cells, with great potential to amplify inflammatory responses through cooperative signaling via tissue cells such as epithelium and smooth muscle (Morris et al. 2005, 2006). Macrophages are very long-lived cells, and a single inflammatory stimulus can result in the gradual repopulation of the lung by young monocytes that may have a different activation phenotype, and will persist for months or years in the lung tissue (Maus et al. 2006). Thus, preventing monocyte trafficking to the lung with drugs such as chemokine receptor antagonists is a potentially very useful therapy, but (i) the effects of these drugs may take months of administration to become apparent and (ii) their efficacy may be different on background chronic disease compared with the disease of acute exacerbations. However, targeting the processes of innate immunity is clearly effective and feasible. Considerable interest and excitement has been generated by the use of anti-tumor necrosis factor (TNF)-α strategies in the treatment of asthma (Howarth et al. 2005; Berry et al. 2006). Neutralization of IL-1 may also represent a useful treatment (Sabroe et al. 2006). Importantly, not all therapeutic options will involve the neutralization of cytokines: targeting innate immune deficiencies that lead to impairment of viral responses in asthmatics by replacement of absent cytokines is also theoretically feasible (Wark et al. 2005).

Therapeutic exploitation of the biology of the innate immune system

Taming the adaptive response

Activation of the innate immune system is an integral part of the pathology of allergic diseases such as asthma, with a dual role that has different emphases in disease initiation and disease perpetuation. Sadly underappreciated in the past, the resurgence in interest in innate immunobiology has been spearheaded by the identification of the TLR system and its huge contribution to health and disease.

One of the most exciting areas in the treatment of allergic disease is the prospect of using the power of the innate immune response to ameliorate allergic responses. Conventional immunotherapy aims to downregulate Th2 responses, while upregulating Th1 or Treg responses, by judicious administration of allergen. Although effective, it is timeconsuming and requires considerable clinical skill, being most effective in cases where single allergens are clearly dominant

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(Durham et al. 1999; Robinson et al. 2004). An understanding of TLR biology now allows the development of potentially more efficacious immunotherapy strategies. While much TLR signaling induces a Th1-type response, interest has focused on the biology of TLR9. This receptor responds to sequences in DNA that are more common in bacterial and viral DNA than human DNA, named “CpG motifs” (Hemmi et al. 2000; Bauer, S. et al. 2001; Lund et al. 2003). Short oligonucleotides containing CpG motifs are remarkably potent activators of TLR9-positive cells, including B cells (Bourke et al. 2003) and DCs, particularly the plasmacytoid DC subset (Bauer, M. et al. 2001; Kadowaki et al. 2001; Boonstra et al. 2003; Demedts et al. 2006). Other cells, such as the epithelium, also express TLR9 (Li et al. 2004; Sha et al. 2004). There is some variation between species and cell type in the nature of the optimal form of the CpG motif (Ballas et al. 2001; Roberts et al. 2005), but some form of secondary structure to the short oligonucleotide, such as the formation of hairpin loops, appears to facilitate immune responses. This activation induces a very strong Th1 response and can be used to induce Treg activity (Moseman et al. 2004). Add to these facts the potential to couple such short oligonucleotides directly to allergen (Marshall et al. 2001), minimizing the CpG dose and reducing the risk of nonspecific immune priming, and the potential to generate a very effective immunomodulatory therapy arises. Such strategies have been shown to be promising in animals (Tighe et al. 2000): there is now early human data showing that immunotherapy with CpG-conjugated allergen may be a very simple, safe, and effective treatment to reduce human allergic disease (Creticos et al. 2006). It is also feasible that administration of CpG motifs alone (also known as immunostimulatory DNA motifs) to the whole organism or to DCs ex vivo may be a useful strategy (Chiang et al. 2003; Ikeda et al. 2003; Fanucchi et al. 2004), but the potential for TLR9 signaling to contribute to disease pathologies such as systemic lupus erythematosus suggest that caution is needed with TLR-based therapies (Boulé et al. 2004; Barrat et al. 2005; Means et al. 2005), and dose limitation by allergen conjugation may be wise, though experience in humans has been generally reassuring to date (Krieg 2006). These data are obviously extremely exciting, but a note of caution needs to be sounded. Immunotherapy is traditionally most effective in individuals with a single dominant allergic disorder, such as grass pollen-induced hay fever. In chronic disease, allergen avoidance has been a disappointing treatment, and while the reasons for this may be legion, chronic allergen-independent disease mechanisms, which may in significant part be dependent on the innate immune system, may be dominant. Nonetheless, for hay fever, asthma related to single dominant allergens (e.g., animal danders), or venom allergies and anaphylaxis, TLR9-directed therapies are an extremely exciting area and several drugs are in clinical trials (Krieg 2006).


Conclusion The innate immune system is a multilayered, complex, germline-encoded system that interacts with the adaptive immune system at multiple levels. Its activation may underpin allergic disease, while serving different roles in disease initiation and disease persistence. Targeting innate immunity is feasible and already underway, with the potential to generate new and effective therapies for allergic disease.

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Signal Transduction in Allergic and Inflammatory Cells Rafeul Alam

Summary Signal transduction is a fundamental cellular process that is essential for interpreting environmental cues and mounting an appropriate cellular response. Signal transduction is initiated by cell-surface receptors and involves a variety of enzymes, adapter molecules, chaperones, nucleotides, nucleotidebinding molecules, cytoskeleton, motor proteins, and ions. The generation of initial signal requires the activation of a kinase or a GTP-binding protein. The initial signal is then amplified through a variety of mechanisms including the recruitment of adapters and downstream targets. Many signaling pathways converge on a few final common pathways, including the NF-κB, Ca2+-calcineurin, mitogen-activated protein kinase (MAPK), Jak–STAT, and phosphatidylinositol 3-kinase (PI3K)–AKT pathways. Allergic inflammation is characterized by the presence of Th2 cells, eosinophils, and mast cells. Studies have identified important signaling molecules and master regulators of these inflammatory cells. Many cell-surface receptors, instead of stimulating, actually inhibit the cell through the generation of an inhibitory signal. Specific molecules have been identified that mediate this inhibitory signal. Therapeutic modalities are now being designed that either inhibit the activating signal or augment the inhibitory signal in order to control inflammatory diseases.

Table 10.1 Types of protein modification and the structural domains that recognize the modification. Amino acid

Modification

Signaling domain

Serine

Phosphorylation

14-3-3, KIX, MH2, WW, WD40, LRR, arrestin domain, PBD, BRCT, CID

Threonine

Phosphorylation

14-3-3, WW, FHA, PBD, BRCT

Tyrosine

Phosphorylation Nitration Sulfation

SH2, PTB, C2 ? ?

Lysine

Acetylation

Bromodomain

Arginine

Methylation Ubiquitination Sumoylation Neddylation

Chromodomain UIM, UBA, CUE, UEV, NZF, PAZ SIM, SHD ?

Proline

Isomerization

?

Aspartate

ADP-ribosylation

?

Glutamate

Methylation

?

Signal generation Introduction Many immune cells as well as resident tissue cells participate in allergic diseases. There are significant differences in signaling pathways leading to their activation. It is not the objective of this chapter to describe signaling mechanism of all allergic cells. Instead it provides a broad outline of general signaling mechanisms and then focuses on the signal transduction mechanisms of select cells of the immune system.


Most proteins have an innate ability to function as environmental sensors. They sense subtle changes in the surrounding microenvironment (at the cell membrane as receptors or inside the cell) and in response undergo a conformational change. This conformational change lies at the heart of signal generation. As a result of this conformational change the protein may become catalytically active (if the protein is an enzyme) and modify another protein or acquire affinity to interact with a new partner. This change(s) is sensed by other proteins, which creates a ripple effect and leads to signal transduction. The ultimate goal of this process is to read, generate/amplify, and convey information. Proteins are amenable to secondary modification on many residues (Yang 2005). A list of amino acid residues that undergo secondary modification is shown in Table 10.1. The secondary

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modification of proteins is sensed by an appropriate interactive domain (e.g., SH2 domain recognizing phosphorylation of tyrosine residue) leading to protein–protein interaction and exchange of information. A major focus of signaling research is to understand how a cell receives signals from the environment and conveys this signal to intracellular destinations for an appropriate biological response. Cell-surface receptors are primarily responsible for generating intracellular signals in response to extracellular cues. Intracellular molecules that generate and transduce signals can be classified into the categories shown in Table 10.2. These molecules utilize a variety of mechanisms to generate signals. Examples include association with GTP or GDP, phosphorylation by kinases and dephosphorylation by phosphatases, ubiquitylation (mono or poly), sumolation, and

Table 10.2 Intracellular molecules that generate and transduce signals. Enzymes Kinases Tyrosine Serine/threonine Leucine Threonine/tyrosine kinases Phosphatases Proteases Ligases (e.g., ubiquitin ligase) GTPases Heterotrimeric G proteins (a, b, g) p21 G proteins Ras (oncoprotein Ras) Rho (Ras homolog) Rab (Ras-related genes expressed in rat brain) ARF (ADP ribosylation factor) Adapter/scaffolding proteins Grb2 (growth factor receptor-bound protein 2) MP1 (MEK1 partner 1) MyD88 (myeloid differentiation factor 88) TIRAP (TIR domain-containing adapter protein) Regulatory/inhibitory proteins SOCS (soluble inhibitor of cytokine signaling) RGS (regulator of G-protein signaling) Lipids Inositol trisphosphate Nucleotides Cyclic AMP Cyclic GMP Ions Ca2+ Transcription factors

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acetylation/deacetylation. These modifications significantly alter the physiochemical behavior of the protein. A frequent outcome of this process is dimerization/oligomerization of signaling molecules, association with other signaling proteins and chaperones, and translocation to specific destinations. The secondary modification of amino acid residues of a protein or its conformational changes are recognized by specific structural domains that play a critical role in signal transduction. The PFAM (protein family database alignment and hidden Markov models) database (www.sanger.ac.uk/Software/Pfam/) lists 84 different modular domains. A few examples of these modular domains and their target amino acid residues are shown in Table 10.1. A typical example of this domain-based interaction is phosphotyrosine. When a protein is tyrosyl phosphorylated, it is recognized by three or more distinct modular domains: SH2, C2, and phosphotyrosine-binding domain. Which of these domains interacts with phosphotyrosine depends on conserved amino acid residues adjacent to the phosphotyrosine residue and availability of domaincontaining proteins in the surrounding milieu.

G protein-coupled signaling Heterotrimeric G proteins Activation of specific proteins through GTP binding represents a major form of signal transduction (reviewed by Hubbard & Hepler 2006; Pierce et al. 2002). There are two families of G proteins: heterotrimeric (big) G proteins and small G proteins. Heterotrimeric G proteins transduce signals from G protein-coupled receptors (GPCR). GPCR represent the biggest family of cell membrane receptors. The Swiss-Prot database (www.expasy.org/sprot) has 2841 entries, encompassing all species. GPCR relevant to allergic diseases include receptors for histamine, leukotrienes, prostacyclins, kinins, adenosine, neurokinins, chemokines, and most hormones and neurotransmitters (including epinephrine and acetylcholine). The heterotrimeric G proteins comprise three subunits: α, β, and γ. The Gα subunit has 25 family members (examples include Gi, Gs, Gq, and G12), Gβ has five members, and Gγ has 10 members. In unstimulated cells GPCR are associated with the GDP-bound form of Gα (Fig. 10.1). On ligand binding GDP is exchanged for GTP, which triggers dissociation of Gα from the β and γ subunits. Activated Gα then associates with and stimulates adenyl cyclase, phospholipase C (PLC)-β, Src family kinases, and Bruton’s tyrosine kinase (Btk), among others. The β and γ subunits activate PI3K-γ. The activation of these effectors transduce signals downstream. The GTP bound to Gα is rapidly hydrolyzed to GDP. As a result the GDP-bound Gα regains affinity for the β and γ subunits and homeostasis is restored. The G proteins represent a major mechanism of cAMP- and Ca2+-dependent signaling in the cell. PLC-β hydrolyzes phosphatidylinositol bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG), leading to the elevation of calcium and activation of protein kinase C (PKC). In addition, G protein activation is linked to

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H

Receptor

IP3

PLCb,g Ga

Ga family (23 members) • Gs • Gi • Gq • G12 Gb (5 members) Gg (10 members)

Ga

b g

c

b g

GTP GDP PI3Kg Src

GDP-a

b g

GTP-a

Effector Phospholipase Cb Adenyl Cyclase Bruton’s tyrosine kinase (Btk) Src-family

Fig. 10.1 Signal transduction mechanism of G protein-coupled receptors. Ligand binding to the receptor induces a conformational change in Ga and an increase in its affinity for GTP. The GTP-bound Ga dissociates from the b and g subunits. The dissociated subunits activate downstream signaling molecules. The GTPase activity of Ga hydrolyzes GTP into GDP. The GDPassociated Ga regains affinity for b and g subunits and homeostasis is restored. The ribbon diagram in the upper right corner shows the crystallographic structure of rhodopsin, a prototypic G protein-coupled receptor. (See CD-ROM for color version.)

the MAPK pathway (Kampen et al. 2000). Inhibition of Gi with pertussis toxin blocks the eotaxin-induced activation of ERK and p38 in eosinophils. There are also reports suggesting that the βγ dimer of G proteins can bind and activate PI3K-γ. Elevated intracellular Ca2+ activates a variety of downstream effectors including calcineurin, calmodulin, and myosine light chain kinase. Calcineurin is a phosphatase that dephosphorylates NFAT, a transcription factor. Phosphorylated NFAT is sequestered in the cytosol. Dephosphorylation allows its translocation to the nucleus, association with other transcription factors (e.g., AP1), and induction of new gene transcription (Fig. 10.2). Myosin light chain kinase activates myosin, an important motor protein involved in cell motility, contraction, degranulation, and cytoskeletal changes.

Small G proteins Small G proteins represent a large family of signaling molecules. Unlike the big G proteins, small G proteins do not associate with β and γ subunits. However, like big G proteins their activation status is determined by the association with GTP. There are multiple subfamilies including Ras (rat sarcoma), Rho (ras homology), Rab, Ran, ARF, and non-Ras GTPases (reviewed by Colicelli 2004). The Ras family has 35 members, Rho 23 members, Rab and Ran 71 members, and ARF and SARA 30 members. Ras and Rac play a major role in activation of the MAPK pathway. Rac, Rho, and cdc42 are critical regulators of the cytoskeleton (Etienne-Manneville & Hall 2002). Rac controls actin polymerization, membrane ruffles forma-

Ca2+ Calcineurin NFAT-p

Nucleus NFAT

NFAT

Fig. 10.2 Calcium and calcineurin signaling pathway. Receptor stimulation leads to activation of phospholipase C (PLC) b or g isoform, which then acts on membrane lipids and generates inositol trisphosphate (IP3). IP3 releases Ca2+ from intracellular stores. Increased Ca2+ activates calcineurin, which is a phosphatase. Calcineurin dephosphorylates NFAT (nuclear factor of activated T cells), which then migrates to the nucleus. (See CD-ROM for color version.)

tion, and cell movement, whereas Rho is essential for focal adhesion formation. Cdc42 is a master regulator of cell polarity and directional cell movement. Broadly, this family of G proteins determines cell shape, polarity, movement, phagocytosis, and secretion. Proteins of the Rab (reviewed by Jordens et al. 2005) and ARF (Randazzo et al. 2000) families are involved in endomembrane movement and vesicular transport and as such play a vital role in endocytosis and exocytosis.

Tyrosine kinases Tyrosine kinases catalyze the transfer of the γ phosphate of ATP to the hydroxyl group of tyrosine on proteins. This family of kinases consists of nearly 500 members. Most kinases, including tyrosine kinases, are maintained in an inactive conformation through intramolecular interactions. Activation of kinases requires disruption of these intramolecular bonds by interaction with other molecules/ligands. In the case of receptor tyrosine kinases (RTKs) extracellular ligands induce this conformational change and induce their catalytic activity. Examples of RTKs include receptors for growth factors such as epidermal growth factor (EGF), platelet-derived growth factor, and nerve growth factor. The structural mechanism of activation of some of these kinases has been elucidated. The EGF receptor undergoes a dramatic conformational change in its extracellular domain (Zhang et al. 2006), which is transmitted to the intracellular domain. This conformational change in the intracellular domain leads to head-to-tail positioning of the kinase domain of two adjacent receptors (Fig. 10.3). This head-to-tail positioning disrupts their intramolecular inhibitory binding and activates the kinase. Nonreceptor tyrosine kinases (NRTKs) bind to receptors in a noncovalent fashion. Some NRTKs do not bind cell

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Immunology of the Allergic Response EGF (or other ligands)

EGFR ErbB2 ErbB4

EGFR ErbB2 ErbB3 ErbB4

membrane receptors and their activation occurs in the later phase of the signaling cascade. NRTKs include the following families: ACK, Csk, FAK, Fes, FRK, JAK, Src, Syk, and Tec. The Src family is one of the larger tyrosine kinase families. There are 10 family members: Blk, Brk, Fgr, Fyn, Hck, Lck, Lyn, Src, Srm, and Yes. Lck and Fyn play an essential role in signal generation by the T-cell receptor (TCR) (Palacios & Weiss 2004). Lck and Fyn associate, respectively, with CD4 and CD3 of the TCR complex and are critical for activation of T cells (Fig. 10.4). Activation of these kinases leads to phosphorylation of ZAP70 and propagation of downstream signals via the MAPK, PLC-γ and Ca2+ signaling pathways. Lyn, Fyn, and Syk kinases play a similar signal-generating role in B-cell receptor (BCR) and FcεRI signaling. Lyn and Syk are important for growth factor-mediated signal generation in eosinophils. Src family kinases also play an essential role in signaling processes mediated by cell–cell adhesions. The Janus family represents an important family of NRTKs. Most cytokine and many growth factor receptors utilize this family of tyrosine kinases to transduce signals. There are four members: Jak1, Jak2, Jak3, and Tyk2 (Aaronson & Horvath 2002). These kinases are especially important for hematopoiesis and development of the immune system. Jak kinases phosphorylate the STAT family of transcription factors and regulate cellular response to cytokine and growth factor stimulation (Kisseleva et al. 2002).

Clinical relevance The deficiency of many tyrosine kinases leads to severe immunodeficiency. Jak3 deficiency causes severe combined immunodeficiency (SCID) (Macchi et al. 1995). Tyk2 mutation has been linked to a patient with hyper-IgE syndrome (Minegishi et al. 2006). Lck deficiency causes T-cell deficiency (Goldman et al. 1998). ZAP70 deficiency/mutation has been linked to SCID and CD8 T-cell deficiency (Chan et al. 1994). Btk deficiency or mutation causes X-linked agammaglobulinemia (Rawlings et al. 1993). Mutation of EGF receptor family members is the cause of a variety of cancers.

Mechanism of activation of Src family kinases Most kinases are inactive in their native conformation. This inactive state is maintained through multiple intramolecular

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Fig. 10.3 Schematic presentation of activation of the epidermal growth factor (EGF) receptors: EGFR, ErbB2, ErbB3, and ErbB4 (ErbB is homologous to the viral erythroblastoma oncogene v-erbB). Following the binding of the ligand EGF, the intracellular domains of the dimerized receptor position themselves in a head-to-tail position. This conformational change opens up the activation loop, which results in activation of the kinase. (See CD-ROM for color version.)

CD4

Unc Lck

TCR ab d d zz

Fyn

T-cell signaling LAT

I ZAP70 PKC8

Grb2

P

PI4,5P2

P PLCg Ras

P P GADS SOS P SLP76

NFkB

IP3 MAPKKK

MAPK

Ca++ NFAT

IL-2 transcription Proliferation

Fig. 10.4 Simplified schematic presentation of signal transduction mechanism of the T-cell receptor (TCR). Antigen presentation leads to the formation of a multimolecular complex involving CD3g (not shown), d, e, two z subunits and CD4. The CD4-associated adapter protein Unc119 activates the tyrosine kinases Lck and Fyn, which then phosphorylate ITAM (I) residues on z subunits. Phosphorylated ITAMs recruit ZAP70. Activated ZAP70 then phosphorylates the transmembrane adapter protein LAT. Phosphorylated LAT recruits a number of signaling molecules including PLC-g, Grb2, GADS, SLP76, and SOS. The latter activates Ras and transduces signals via the MAPK pathway. The action of PLC-g leads to the generation of Ca2+ signals and activation of the transcription factor NFAT. TCR also activates PKC-q, which transduces signals via the NF-kB pathway. GADS, Grb2-like adaptor downstream of Shc; Grb2, growth factor receptor-bound 2; ITAM, immunoreceptor tyrosine-based activating motif; LAT, linker of activated T cells; NFAT, nuclear factor of activated T cell; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; SLP76, SH2-containing leukocyte-specific protein of 76 kDa; SOS, son of sevenless; ZAP70, z chain-associated protein of 70 kDa. (See CD-ROM for color version.)

bonds that impose a constrained conformation. An essential step in kinase activation is the disruption of these inhibitory intramolecular bonds, allowing a relaxed conformation and catalytic activation. The crystal structure of Src kinases has provided a basic understanding of kinase activation (Boggon & Eck 2004). Src kinases are composed of five structural components: a unique region, SH2 (SH, Src homology) and SH3 domains, a linker region, and the kinase domain. There are two inhibitory intramolecular bonds that keep the kinase in a closed “off” conformation. The first is a tyrosine residue

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at the C-terminus, which when phosphorylated binds to the SH2 domain. The linker region contains a proline-rich SH3-binding motif, which binds to the SH3 domain and creates a second intramolecular bond. Activation of the kinase requires a stepwise approach. The engagement of a tyrosine phosphatase (e.g., CD45) dephosphorylates the C-terminal phosphotyrosine and disrupts the first bond. The second step is disruption of the SH3–linker interaction. This involves interaction with a high-affinity SH3 motif-containing ligand, which disrupts the internal low-affinity binding and moves the SH3 domain away from the activation loop. This allows a relaxed conformation leading to kinase activation. The identity of the endogenous ligand was until now unknown. We recently reported the cloning and characterization of the new SH3 ligand called Unc119 (Cen et al. 2003; Gorska et al. 2004). Unc119 is associated with many membrane receptors including CD3, CD4, and interleukin (IL)-5 receptor α chain (see Fig. 10.4). On receptor stimulation Unc119 binds to the SH3 domain of Src kinases (e.g., Lck, Fyn, Lyn) and activates the kinase, which results in its autophosphorylation.

Signal amplification Adapter proteins The initial activation of receptor-associated signaling molecules is usually transient and weak in nature. In order to generate a conducive signal this initial receptor-associated signal is amplified through a variety of mechanisms. Adapter proteins are one class of molecules that function as amplifier of signals (Wonerow & Watson 2001; Lindquist et al. 2003). Adapter proteins are signaling molecules that do not have catalytic domains, but instead contain modular protein–protein and protein–lipid interaction domains or motifs. Adapter proteins are involved in the proper organization and activation of protein complexes. Following activation many receptors are phosphorylated. This phosphorylation causes recruitment of adapter proteins. The recruited adapters amplify and relay signals to a next set of signaling molecules. Examples of adapters include LAT, Shc, Grb2, GAD, Nck, SLP76, and MyD88. In the case of T cells or mast cells, initial tyrosine kinase activation leads to phosphorylation of LAT on up to nine different tyrosine residues (see Fig. 10.4). These phosphotyrosine residues then serve as docking sites for additional signaling molecules such as GAD, SLP76, and others. The recruited GAD and SLP76 also undergo phosphorylation and relay the signal to another set of signaling molecules.

Clinical relevance BLNK (B-cell linker protein, also known as BASH and SLP65) is an adapter protein that is expressed in B cells and is important for B-cell signaling. Deficiency of BLNK is a cause of hypogammaglobulinemia (Minegishi et al. 1999). Deficiency of another adapter protein IL-1 receptor-associated kinase


(IRAK)-4 causes pyogenic bacterial infections (Picard et al. 2003). The mutation of SLAM-associated protein (SAP), also known as SH2 domain-containing adapter protein (SH2D1A), which is expressed in T cells and natural killer cells, causes X-linked lymphoproliferative disorder (Morra et al. 2001).

Lipid rafts Another mechanism of signal amplification is signaling through the lipid raft. Lipid rafts are freely diffusing, stable, lateral assemblies of sphingolipids and cholesterol. They constitute an important organizing principle for the plasma membrane. The basic concept is that lipid rafts facilitate selective protein–protein interactions by selectively excluding or including proteins. An example of this lipid raftfacilitated signaling is the concentration of Lck, CD2, CD28, and Cbp in T cells (Rodgers et al. 2005).

ITAM, ITIM, and ITSM Some transmembrane receptors have two tyrosine residues that are spaced at an appropriate distance and placed in the context of conserved residues: D/E-X7-D/E-X2-Y-X2-L-X7Y-X2-L (where X indicates any amino acid residue). These tyrosine residues, when phosphorylated, are able to recruit and activate downstream signaling molecules that contain phosphotyrosine-binding domains (e.g., SH2, C2, and the phosphotyrosine binding domain). This conserved sequence is known as ITAM (imunoreceptor tyrosine-based activation motif) (Flaswinkel et al. 1995). The following receptors contain ITAM in their cytosolic fragment: TCR ζ, γ, δ and ε; BCR α and β; FcεRI β and γ; and FcγRIIa. Another conserved tyrosine residue-based motif recruits signaling inhibitors (e.g., tyrosine and lipid phosphatases: SHP-1, SHP-2, SHIP) instead of signaling activators and thereby terminates signal transduction (Isakov 1997). This motif has the following sequence: I/L/V-X-Y-XX-L/V. This motif is preceded and followed by hydrophobic residues unlike the tyrosine residues in ITAM, which is preceded by acidic residues. ITIM (imunoreceptor tyrosine-based inhibitory motif) is present in the following receptors: FcγRII, KIR, NKG2A/B, mLy-49A, gp49B1, SIRPα, Siglec-3, -5, and -8, PIR-B, and MAFA. An example of ITAMbased signal amplification is signaling by the TCR. The CD3 complex has two CD3ζ subunits and the cytosolic fragment of each CD3ζ has three ITAMs. Since each ITAM binds two SH2 domain-containing molecules or a single molecule with two SH2 domains, one CD3 complex can bind a total of 12 SH2 domain-containing molecules. This results in substantial amplification of the initial signal at the receptor complex. In the case of T cells and CD3ζ (see Fig. 10.4), phosphorylation of ITAM results in the recruitment of multiple ZAP70 molecules and subsequent downstream signal transduction. It should be pointed out that although in the vast majority of cases ITAM generates an activation signal (as the name implies), there are examples where ITAM actually delivers an inhibitory signal and ITIM generates activation signals

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(Barrow & Trowsdale 2006). A variation of ITIM has recently been coined, the immunoreceptor tyrosine-based switch motif (ITSM), which has the following consensus sequence: T-X-Y-X-X-V/I (Shlapatska et al. 2001). This motif is present in CD150, 2B4, CD33, Siglec-9, and PD1. The presence of this motif allows binding of the adepter protein SH2D1A to the receptor and switches its interaction from a tyrosine phosphatase (SHP-1, SHP-2) to a lipid phosphatase (e.g., SHIP).

Signaling integration MAPK pathway An example of signaling integration is the activation of MAPKs (Roux & Blenis 2004). Various extracellular and intracellular signaling pathways converge on the MAPK signaling pathway. MAPKs are activated in a cascade involving two upstream protein kinases known as MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK). There are at least 10 different members of the MAPK family. Four pathways have been well characterized and extensively studied, including ERK1/2, JNK, p38, and ERK5 (Fig. 10.5). MAPK signaling regulates major cellular functions including proliferation, differentiation, survival, apoptosis, adhesion, migration, and degranulation. In a typical growth factor-initiated signaling pathway the phosphorylated receptor serves as the docking site for the SH2 domain of the adapter protein Shc. Subsequently, Shc undergoes tyrosine phosphorylation and recruits the adapter protein Grb2. Grb2, in turn, binds to Sos (son of sevenless), a Ras GTP/GDP exchange factor. Therefore, interaction of Shc

Raf-1, A & B-Raf, Cot/Tpl2

MEKK1-4, ASK1, MTK1, TAK1, MLK4

MEKK1-4, ASK1, MTK1, TAK1, MLK4

MEKK2-3, Cot/Tpl2

MAPKK

MEK1/2

MKK4,7

MKK3,6

MEK5

MAPK

ERK1/2

JNK1–3

p38

ERK5

ELK1, Sap1 c-fos, RSK

C-Jun, ATF2

MK2, ATF2, MEF2C

MEF2C, Sap1a, SGK

MAPKKK

Fig. 10.5 Mitogen-activated protein kinase (MAPK) signaling pathway. The generic MAPK cascade starts with a MAPK kinase kinase (MAPKKK), which phosphorylates the MAPK kinase (MAPKK), which in turn phosphorylates MAPK (left panel). Four major MAPK signaling pathways, ERK1/2, JNK1–3, p38 and ERK5 and their upstream activators, are shown. ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase. (See CD-ROM for color version.)

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with the growth factor receptor results in recruitment of the Grb2–Sos complex to the membrane, where the Sos effector Ras is localized. Sos induces GDP to GTP exchange in Ras, which leads to its activation. Ras stimulates the activity of the serine/threonine kinase Raf-1, which is a MAPKKK. Raf-1, in turn, induces the activation of MEK1, a MAPKK. MEK1 phosphorylates ERK on threonine and tyrosine residues on the consensus T-E-Y motif of ERK1 and ERK2. Activated ERK1/2 kinases then phosphorylate a multitude of downstream cytosolic and nuclear effectors in the consensus motif S/T-P. The ERK1/2 module (Raf-1–MEK1/2–ERK1/2) can be activated by a variety of upstream pathways including various small G proteins (Ras, Rap), Gα and Gβγ, Ca2+ signaling and adapter proteins (e.g., TAB). Like ERK1/2, the other members of this family (JNK and p38) are activated by multiple upstream mechanisms.

Clinical relevance Ras is one of the most frequently mutated genes in human cancer (Rodriguez-Viciana et al. 2005). For this reason various strategies are being developed to interfere with this signaling pathway. Genetic, molecular, and animal studies implicate ERK1/2, p38, and ERK5 in various inflammatory illnesses including rheumatoid arthritis, chronic obstructive pulmonary disease, and asthma (Duan & Wong 2006). Low-molecularweight inhibitors of these pathways are now undergoing human trials.

NF-kB pathway This pathway represents one of the most important signaling pathways for cell activation, survival, differentiation, and oncogenesis. It demonstrates signaling integration at key signaling hubs. Various signaling pathways converge on the IKK (inhibitor of κB kinase) complex (Fig. 10.6) (Li & Verma 2002). For example, the TCR complex activates PKC-θ, which transduces signals through the complex of BCL10, MAGUK, and MALT1 to the IKK complex. The tumor necrosis factor receptor superfamily engages the adapter proteins TRADD and TRAF2 to activate RIP and MKK3, which then converge on the IKK complex. IRAK binds to the cytosolic fragment of Toll-like receptors (e.g., TLR4). Activation of TLR leads to the recruitment of TRAF6 through IRAK. TRAF6 propagates signals via TAK1 and TAB, which leads to IKK activation. The IKK complex is composed of IKKα, IKKβ, and IKKγ (NEMO). IKKγ (NEMO) is a scaffold for IKKα and IKKβ and is important for proper signal transduction. Activation of IKKα, especially IKKβ, leads to phosphorylation of IκB. Phosphorylation of IκB is a signal for its ubiquitylation and proteosomal (β-TRCP ubiquitin proteasome) degradation. IκB binds NF-κB (p65, p50) and retains it in the cytosol. Its degradation allows NF-κB to migrate to nucleus. NF-κB is frequently phosphorylated and acetylated, which facilitates its DNA-binding activity. In the nucleus NF-κB binds to its

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TNFR

TCR


TLR

Cytokine Cytokine receptor

TRAF2 RIP MKK3

IKK (a, b, g)

BCL-10 MALT1 MAGUK

PKC-8

TAK1 TAB

IRAK TRAF6

SOCS

JAK

JAK

PTP Ub

IkB P IkB U

JAK

NFkB Nucleus NFkB

STAT

Y P

Proteasome

NFkB

STAT

PY STAT

PTP

Y P

Proteosomal degradation Fig. 10.6 NF-kB signaling pathway. Activation of various receptors such as those for tumor necrosis factor (TNFR), T-cell receptor (TCR), or Toll-like receptor (TLR) leads to the generation of signals that converge on the inhibitor of kB (IKK) complex. IKK phosphorylates (P) IkB, which is then ubiquitylated (ub) and degraded. The IkB-bound NF-kB is then released and translocated to the nucleus for transcriptional activity. See text for definition of other abbreviations. (See CD-ROM for color version.)

Nucleus P Y PIAS

STAT PTP

Y P

STAT

Transcription

consensus sequence on the promoter of more than 200 genes and induces gene transcription.

Clinical relevance Mutation of IKKγ (NEMO) causes X-linked hypohidrotic ectodermal dysplasia with variable immunodeficiency (Zonana et al. 2000). NEMO deficiency may manifest as severe profound immunodeficiency or as hyper-IgM syndrome.

Jak–STAT pathway The Janus kinases (Jak1– 3 and Tyk2) phosphorylate a family of transcription factors called STAT (signal transducer and activator of transcription). There are six members of the STAT family: STAT1–STAT6 (Kisseleva et al. 2002). Following tyrosine phosphorylation, the STATs undergo homodimerization and heterodimerization, which allows them to translocate to nucleus (Fig. 10.7). Frequently the STATs are additionally phosphorylated by MAPK, which enhances their transcriptional activity. Most cytokines signal through the Jak–STAT pathway. The receptor for IL-12 and interferon (IFN)-γ activates STAT1 and STAT4, respectively (O’Garra & Robinson 2004). Activation of STAT1 and STAT4 is important for the induction of the transcription factor T-bet and for a Th1 response (Fig. 10.8). The IL-4 receptor transduces signals through STAT6. In addition to STAT6, STAT5 is also important for a Th2 response. Both these molecules induce GATA-3 transcription, which is a master regulator for Th2 differentiation (Murphy & Reiner 2002).

Clinical relevance STAT1 mutation predisposes patients to extrapulmonary mycobacterial infections (Dupuis et al. 2001).

Fig. 10.7 Jak–STAT signaling pathway. The binding of cytokines to their respective receptors causes activation of receptor-associated Jak (Janus kinase) kinases: Jak1, Jak2, Jak3, and Tyk2. Activated Jak kinases phosphorylate the receptors and the recruited STATs. Phosphorylated STATs dimerize and translocate to nucleus. Phosphorylated Jaks are inhibited by protein tyrosine phosphatase (PTP) and SOCS (suppressor of cytokine signaling). The transcriptional activity of STAT is antagonized by PIAS (protein inhibitor of activated STATs) and PTP. (See CD-ROM for color version.)

STAT1 T-Bet

STAT6 STAT5

STAT4

GATA-3 C-Maf

RORgT

FoxP3

Th1

Th2

Th17

Treg

Fig. 10.8 Transcription factors and T-cell differentiation. Major transcription factors involved in Th1, Th2, Th17, and Treg are shown. The initial transcription factors that lead to the induction of RORgT (retinoic acid-related orphan receptor g expressed in T cells) and FoxP3 (forkhead box protein 3) are currently unknown.

PI3K–AKT pathway PI3K phosphorylates phosphatidylinositol lipids at the D3 position of the inositol ring and produces phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Cantley 2002). The reaction is reversed by the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10). Cellular membranes are the sources of PI3K substrates. PIP3 creates a membrane

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docking site for pleckstrin homology (PH) domain-containing proteins, e.g., the serine/threonine kinase AKT, tyrosine kinases belonging to Tec family, PLC-γ, and the GTP/GDP exchange factor Vav. The binding of PIP3 at the cell membrane is important for activation of these molecules. The activation of AKT (also known as protein kinase B) depends on additional kinases: phosphoinositide-dependent kinase 1 and 2 (PDK1 and PDK2). AKT, in conjunction with the ERK1/2 pathway, activates S6 kinase and facilitates activation of mTOR (mammalian target of rapamycin) (Shaw & Cantley 2006). Activation of S6 kinase and mTOR promotes cell survival and proliferation. AKT regulates a number of important signaling pathways including GSK3β, Bcl2 family members, and the transcription factors p53, FOXO and β-catenin. AKT regulates cell survival through its phosphorylation of Bcl family members and inhibition of GSK3β.

Signal termination The duration and amplitude of signals are under stringent control. Short-lasting or weak signals may not elicit any cellular response. On the other hand long-lasting or very strong signals may lead to pathologic changes, e.g., cell death or excessive proliferation. Signal termination is essential for cellular homeostasis. Cells employ a number of mechanisms to terminate signaling processes. Receptor endocytosis is one such mechanism. Receptors may also undergo degradation. Activation of many receptors leads to the recruitment of a ubiquitin ligase, e.g., Cbl. IL-5 induces ubiquitination and proteosomal cleavage of the cytoplasmic portion of β chain (Martinez-Moczygemba & Huston 2001). Enzymatic reversal of modified signaling molecules is another mechanism. Targeting of phosphorylated tyrosine, serine, threonine, and lipid residues by phosphatases is an efficient mechanism of restoration of homeostasis. The tyrosine phosphatase SHP-1 associates with the phosphorylated βc chain of the IL-3 receptor on stimulation of myeloid cells and negatively regulates IL-3-induced proliferation (Paling & Welham 2002). SHIP is an SH domain phosphatidylinositol phosphatase, which inactivates PIP3 and inhibits signal transduction. SHP and SHIP are preferentially recruited to the ITIM of inhibitory receptors such as FcRγII and PIR-B, and blocks signaling through activating receptors such as FcεRI on mast cells (Uehara et al. 2001). Signal termination also involves physical blocking of active sites. SOCS (suppressor of cytokine signaling) family members bind to phosphorylated tyrosine residues of other molecules, such as receptors or Jaks and block access of downstream molecules (e.g., STATs) (Alexander & Hilton 2004). CIS1 binds to the tyrosine-phosphorylated βc chain and prevents STAT5 recruitment (Matsumoto et al. 1997). IL-5 and granulocyte–macrophage colony-stimulating factor (GM-CSF) both upregulate mRNA for CIS in eosinophils, which is likely to function as a negative feedback mechanism.

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SOCS1 has been proposed to bind to the tyrosine residue in the activation loop of Jaks (including Jak2) and block access of Jak substrates (Ilangumaran & Rottapel 2003). Recently, a negative regulator of the ERK pathway has been identified in eosinophils (Inoue et al. 2005). Spred1 is a member of the Sprouty-related family of signaling molecules. It binds to Raf-1 and inhibits Raf-1 and ERK activation through a not-so-well characterized mechanism. IL-5-stimulated eosinophil differentiation is enhanced in Spred1-deficient mice.

Signal transduction in eosinophils The eosinophil is a major effector cell in allergic inflammation. Its growth and differentiation are regulated by hematopoietins such as IL-3, IL-5, and GM-CSF. The function of mature eosinophils is regulated by chemokines. The hematopoietins have their own α receptor and share a common β receptor. The common β receptor generates and transduces the majority of the intracellular signals. Tyrosine kinases of the Src and Janus family associate with the hematopoietin receptor (Adachi & Alam 1998). For example, IL-5 receptor associates with Jak1, Jak2, and Lyn kinase (Torigoe et al. 1992; Pazdrak et al. 1995; Van der Bruggen et al. 1995). Receptor dimerization leads to the interaction of the adapter protein Unc119 with Lyn (Fig. 10.9). As a result Lyn is activated. Lyn and

IL-5 receptor a bc Unc Jak1/2 STAT5

Lyn P P

P Syk P Shc

Ras c-raf

AKT

P ERK PLA2

p38 BclxL

GSK3b

Differentiation Survival Degranulation Cytokine Fig. 10.9 Eosinophil signaling through the interleukin (IL)-5 receptor. Engagement of IL-5 to the receptor induces interaction of Unc119 with Lyn kinase, which results in activation of the kinase. Lyn and Jak kinases phosphorylate the common b receptor (bc). Phosphorylated bc recruits Syk and Shc, which indirectly activate Ras, the MAPK signaling pathways and AKT. Both ERK and p38 pathways activate phospholipase (PL)A2 to generate eicosanoids. AKT regulates eosinophil survival by activation of the Bcl family of molecules and inhibition of GSK3b. Jak kinases phosphorylate STATs, including STAT1, STAT3 and STAT5. STATs regulate eosinophil differentiation. AKT, viral oncogene v-AKT; Bcl, B-cell lymphoma-associated gene product; ERK, extracellular signal-regulated kinase; GSK, glycogen synthase kinase; STAT, signal transducer and activator of transcription; Unc119, uncoordinated gene 119. (See CD-ROM for color version.)

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other tyrosine kinases contribute to the activation of Ras, PI3K, and MAPK signaling pathways, especially that of ERK1/2 and p38 (51). Jak kinases phosphorylate and activate STAT1, STAT3, and STAT5, which then translocate to the nucleus and induce new gene transcription. STAT5, ERK1/2, and p38 MAPK play an important role in eosinophil differentiation. ERK1/2 and p38 MAPK are involved in eosinophil chemotaxis, degranulation, and cytokine production (Pazdrak et al. 1995; Kagami et al. 2000; Kampen et al. 2000). The PI3K– AKT pathway is involved in eosinophil survival. Chemokines such as eotaxin activates CCR3 and transduces signals via the Gi protein. This pathway leads to activation of PI3K and MAPK signaling pathways (Kampen et al. 2000). There are important differences in signaling between the IL-5 receptor and CCR3 in regard to the amplitude, duration, and intracellular localization of PI3K and MAPK activation. The activation of PI3K, AKT, ERK1/2, and p38 by IL-5 is of higher amplitude and longer duration than that by eotaxin. This difference translates into a qualitatively different biological response.

Signal transduction in mast cells The high-affinity IgE receptor (FcεRI) is composed of four subunits: α, β, two γ subunits. The β subunit is associated with Lyn kinase, which is an important activator and regulator of signal generation by FcεRI (Nadler et al. 2000). On IgE binding to the receptor, Lyn is activated by an unknown mechanism, which leads to the phosphorylation of ITAM on γ subunits (Fig. 10.10). Syk is then recruited to these phosphorylated ITAMs, which then phosphorylates the membrane-associated adapter protein LAT (linker of activated T cells). Phosphorylated LAT then functions as a docking site for many downstream signaling molecules including PLC-γ, GADS, SLP76, and the Grb2–Shc complex. The action of PLC-γ leads to the generation of IP3 and mobilization of intracellular calcium. Further, it releases DAG, which activates PKC. The multimolecular complexes of Grb2–Shc, GADS, and SLP76 transduce signals via ERK1/2 and p38 MAPK signaling pathways (Gilfillan & Tkaczyk 2006). Both ERK1/2 and p38 MAPK phosphorylate phospholipase A2, leading to eicosanoid synthesis via the cyclooxygenase and lipoxygenase pathways. Calcium, PKC, and MAPK signaling are all important for mast cell degranulation. MAPK signaling seems to contribute to cytokine synthesis. In addition to Lyn, recent studies point to an important contribution by the Fyn kinase. Unlike Lyn, Fyn transduces signals by engaging NTAL, an adapter protein that is similar to LAT in its signaling role (Rivera 2005). Mast cell signaling is regulated by the input from the activating receptor such as FcεRI and c-kit as well as by inhibitory receptors such as FcγRIIb (receptor for IgG) (Ono et al. 1996), gp49B1 (Daheshia et al. 2001), MAFA (Ortega


FceRI a b gg

FcgRIIb LAT

I Syk

P P

Lyn

Grb2 SLP76 MAPKKK

P

PI4,5P2 PLCg

P

SHP1

I SHIP

PI3K P GADS DAG IP3 PKC

MAPK

PLA2

Ca2+ Degranulation Cytokine secretion Eicosanoids

Fig. 10.10 Mast cell signaling through the high-affinity IgE receptor. FceRI is composed of one a, one b and two g subunits. The a and b subunits are associated with Lyn kinase. Binding of IgE to the receptor leads to activation of Lyn, which then phosphorylates the immunoreceptor tyrosine-based activating motif (ITAM) (I) residue on the g subunit. Syk is recruited to the phosphorylated ITAM and is then phosphorylated by Lyn. Activated Syk phosphorylates tyrosine residues on LAT (linker of activated T cells). Phosphorylated LAT serves as the docking site for many signaling molecules such as Grb2, phospholipase (PL)C-g, GADS, SLP76, and phosphatidylinositol-3 kinase (PI3K). PLC-g generates inositol trisphosphate (IP3) and diacylglycerol (DAG) from membrane lipids. IP3 releases Ca2+ whereas DAG activates protein kinase (PKC). GADS, Grb2, and SLP76 lead to the activation of various mitogen-activated protein kinase (MAPK) signaling pathways. ERK1/2 as well as p38 MAPK phosphorylate and activate PLA2. MAPK, PLA2, Ca2+, and PKC all play an important role in mast cell degranulation, cytokine secretion, and eicosanoid generation. Simultaneous cross-linking of FceRI and FcgRIIb leads to recruitment of SHIP and SHP-1 to the ITIM (immunoreceptor tyrosine-based inhibitory motif ) residue of FcgRIIb. SHP-1 and SHIP block signal generation by dephosphorylating protein and lipid kinases. GADS, Grb2-like adaptor downstream of Shc; Grb2, growth factor receptor-bound 2; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; SHIP, SH2 domain-containing inositol phosphatase; SHP, SH2 domain-containing phosphatase; SLP76, SH2-containing leukocyte-specific protein of 76 kDa. (See CD-ROM for color version.)

et al. 1991), and PIR-B (Uehara et al. 2001). The ligand for gp49B1 is integrin αvβ3. The ligands for MAFA and PIR-B are unknown. As mentioned previously, FcγRIIb, gp49B1, MAFA, and PIR-B contain ITIM sequences in their cytosolic domain, which recruit phosphatases such as SHP-1, SHP-2, SHIP, and the adapter protein DOK. The phosphatases dephosphorylate receptors and kinases such as Lyn, Syk, and PI3K. Simultaneous activation of FcεRI and the inhibitory receptors FcγRIIb, gp49B1, MAFA or PIR-B leads to attenuation and/or termination of the activation signal.

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Mast Cells: Biological Properties and Role in Health and Allergic Diseases Peter Bradding and Glenn Cruse

Summary Mast cells are derived from progenitor cells in the bone marrow, which circulate as undifferentiated CD34+ mononuclear cells in the peripheral circulation, and subsequently mature following migration into tissue. Stem cell factor (SCF) is the critical growth and differentiation factor for human mast cells. In health, mast cells are widely distributed throughout the body in both connective tissue and at mucosal surfaces, and form a heterogeneous population of cells with differences apparent in their mediator content, ultrastructure, and functional behavior. Human mast cells can be divided into two clear phenotypes based on their content of the neutral serine proteases tryptase and chymase. Mast cells have been implicated in many diverse biological processes in both health and disease, with an important role in host defense against invading pathogens. It is likely that their primary role is to sense the external environment, ready to respond to a variety of diverse tissue insults with an early and appropriate program of gene expression and mediator release aimed at initiating inflammation and then repair. When the insult becomes chronic, then it is our view that their continuing activation contributes to tissue dysfunction and remodeling. In addition, through the misguided generation of allergen-specific IgE by host B cells, they have the potential to induce acute, sometimes life-threatening symptoms on exposure to allergen. There is overwhelming evidence that mast cells play a central role in the pathophysiology of asthma, allergic rhinoconjunctivitis, urticaria, and anaphylaxis through their activation by both allergen and other nonimmunologic stimuli. Histamine, prostaglandin (PG)D2 and leukotriene (LT)C4 contribute to mucosal symptoms through the induction of mucus secretion and mucosal edema, and in the case of asthma bronchoconstriction. Mast cells also synthesize and secrete a number of proinflammatory chemokines and cytokines including interleukin (IL)-4, IL-5, and IL-13, which regulate both IgE synthesis and the development of eosinophilic inflammation. In addition, the mast cell proteases tryptase and chymase have the potential to cause tissue damage as well as contributing to the inflammatory response. Importantly, mast

cells are present in an activated secretory state in these diseases, with evidence of ongoing mediator release, degranulation visible with electron microscopy, and cytokine mRNA synthesis. Mast cells also relocate to specific structures within diseased tissues. This is particularly evident in asthma where they migrate into the airway epithelium, mucous glands and airway smooth muscle, placing them in direct contact with these dysfunctional airway elements. Developing a better understanding of the mechanisms driving chronic mast cell activation and migration may uncover truly novel therapies for the treatment of mast cell-mediated disease.

Introduction Since their discovery over a century ago, the role of mast cells in human pathophysiology has been the subject of much debate. Mast cells are ubiquitous throughout connective tissues and mucosal surfaces, particularly at the interface with the external environment such as the skin, respiratory tract, and gastrointestinal tract. At these sites they are well placed and well equipped to deal with a multitude of tissue insults. Mast cells contribute to the maintenance of tissue homeostasis, with examples including roles in wound repair (Iba et al. 2004; Weller et al. 2006) and revascularization (Heissig et al. 2005), and protective roles in acquired and innate immune responses to bacterial infection (Echtenacher et al. 1996). They are also implicated in many diverse diseases such as asthma and related allergies, pulmonary fibrosis, connective tissue disease, multiple sclerosis, and atherosclerosis. Mast cells can therefore be considered to represent a double-edged sword. This chapter focuses on the biological properties of mast cells, and how these fascinating cells contribute to both health and allergen-related disease with a fine balance between protection and destruction.

Mast cell development Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Mast cells derive from pluripotent hematopoietic stem cells in the bone marrow (Fig. 11.1). Mast cell-committed progenitors

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Bone Marrow

Pluripotent stem cell Myelocyte precursor

Mast cell precursor

Blood Vessel

Proliferation

Rolling

Adhesion

Lamina Propria

Chemotaxis Cell to cell contact with exchange of growth factors

Transendothelial migration (diapedesis)

Maturation

SCF

Epithelium

are released into the systemic circulation as agranular, undifferentiated, CD34+ mononuclear cells that then migrate into their destination tissue where they terminally differentiate (mature) under the influence of the local cytokine milieu (for review of mast cell ontogeny see Gurish & Boyce 2006). In addition, interactions with the cell matrix and resident cells such as fibroblasts profoundly alters their phenotype (LeviSchaffer et al. 1986; Rothenberg & Austen 1989; Swieter et al. 1993; Rubinchik & Levi-Schaffer 1994; Ogasawara et al. 1997; Hsieh et al. 2005). The vital growth factor for mast cells is the stromal cell and fibroblast-derived cytokine, SCF (Valent et al. 1992). Previously known as steel factor, SCF is the ligand for the receptor tyrosine kinase CD117, encoded by the protooncogene c-kit (Williams et al. 1990). SCF exists as both a cell membrane-bound protein and a soluble protein that can be detected in the blood (Langley et al. 1993). The activation of

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Fig. 11.1 Ontogeny of mast cells. Mast cells originate from pluripotent hematopoietic stem cells in the bone marrow. They enter the bloodstream as mast cell-committed CD34+ progenitor cells. Mast cells differentiate after tissue recruitment within the tissue itself, which accounts for much of their heterogeneity. For more details, see text. (See CD-ROM for color version.)

c-kit by SCF is vital for the growth and survival of mast cells, which undergo apoptosis on SCF withdrawal (Iemura et al. 1994). Mast cells can also undergo apoptosis in the presence of SCF since they have been found to have tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptors that initiate apoptosis (Berent-Maoz et al. 2006). Activation of c-kit on mast cells regulates expression levels of c-kit (Tsujimura et al. 1996), induces immature cell proliferation, promotes chemotaxis, and suppresses apoptosis, thus enhancing survival and promoting recruitment and growth (Li & Krilis 1999). The activation of c-kit also has a regulatory role in mast cell activation. At 10 ng/mL it potentiates IgE-dependent mast cell mediator and cytokine release, while at 100 ng/mL it directly activates the cells (Columbo et al. 1992; Takaishi et al. 1994; Taylor et al. 1995; Lin et al. 1996; Lukacs et al. 1996; Petersen et al. 1996; Frenz et al. 1997; Cruse et al. 2005).

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Mast Cells: Biological Properties and Role in Health and Allergic Diseases

SCF is the critical growth factor responsible for mast cell growth and differentiation in humans. Mast cells will grow in vitro with SCF as the only exogenous factor, although the cells remain immature and are predominantly of the tryptase-only (MCT) phenotype (Mierke et al. 2000). However, cells that are grown in suspension culture in a medium supplemented with serum, SCF, and IL-6 are more mature in terms of their nuclear morphology and granular structure but retain the MCT phenotype (Saito et al. 1995; Yanagida et al. 1995; Saito et al. 1996; Bradding & Holgate 1999). Cells grown on a fibroblast or endothelial cell monolayer have a tryptase- and chymase-positive (MCTC) phenotype and resemble skin mast cells (Levi-Schaffer et al. 1987; Furitsu et al. 1989; Bradding & Holgate 1999; Mierke et al. 2000). In addition, bone marrow-derived mast cells (BMMC) and umbilical cord blood-derived mast cells (CBMC) grown in conditioned medium from a cell strain derived from a patient with systemic mastocytosis together with SCF yielded fully mature cells containing chymase only (MCC phenotype) (Li et al. 1996). These observations suggest that factors other than SCF are required for complete mast cell maturation. The phenotypic differences could be partly due to the presence of IL-4, which dramatically increases chymase expression in

CBMC (Toru et al. 1998). It is also possible that the expression of chymase is part of a maturation pathway, with MCT phenotypes maturing into MCTC phenotypes (Li & Krilis 1999). There are cofactors that can enhance or inhibit the effects of SCF, which appear to be dependent on the origin of the mast cells (Okayama 2000). Nerve growth factor (Kanbe et al. 2000), IL-3, IL-6, IL-9, IL-10 (Yanagida et al. 1995; Norrby 2002), and thrombopoietin enhance SCF-dependent mast cell growth. Conversely, granulocyte–macrophage colonystimulating factor (GM-CSF) (Welker et al. 2001), retinoids, interferon (IFN)-γ, and transforming growth factor (TGF)-β inhibit the growth and differentiation of mast cells (Ishida et al. 2003).

Mast cell heterogeneity Tissue-dependent influences result in marked heterogeneity of mast cells across species, between different organs, and even within the same organ. This heterogeneity is evident in terms of their structure, mediator content, immunologic and nonimmunologic activation, and pharmacologic responsiveness (Table 11.1) (reviewed by Church et al. 1994). Rodent mast

Table 11.1 Mast cell subtype characteristics. Rodent mast cells

Human mast cells

MMC

CTMC

MC T

MC TC

Protease content

Rat mast cell protease 2

Rat mast cell protease 1

Tryptase

Tryptase, chymase, carboxypeptidase, cathepsin G

Proteoglycan content

Chondroitin sulfate

Heparin

Heparin

Heparin

Scroll

Lattice

Predominant granule patterning evident with electron microscopy Common location

Mucosa

Submucosal tissues

Epithelium

Lamina propria, connective tissue and skin

Staining characteristics

Alcian Blue +

Safranin +

Safranin +

Safranin +

Suggested primary role

Host defense

Tissue repair

Host defense

Tissue repair

Relative histamine content

Low

High

Low

High

Relative LTC4 release

High

Absent

High

Skin low

Relative PGD2 release

Low

Low

High

Skin high

IL-4 (low) IL-5 (high) IL-6 (high) IL-13 (low)

IL-4 (high) IL-13 (high)

Cytokine profile

Activated by antigen

Yes

Yes

Yes

Yes

Activated by substance P

No

Yes

No

Yes

No

Yes

Yes (weak effect)

No

Responds to C5a Inhibited by sodium cromoglycate

No

Yes

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(a)

(b)


Chymase

Chymase

IL-4

IL-6

cells were originally classified according to the histochemical staining properties of their granules and their most common location (Enerback 1966). Using this nomenclature, safraninpositive, alcian blue-negative rodent mast cells are mostly associated with the submucosal tissues and are thus called connective tissue mast cells (CTMC); safranin-negative, alcian blue-positive rodent mast cells are predominantly mucosal and termed mucosal mast cells (MMC) (Table 11.1). This differential staining is due to the proteoglycan content of the granules. Rodent CTMC produce the highly sulfated glycosaminoglycan heparin, whereas rodent MMC produce chondroitin sulfate (Gurish et al. 1992). This classification does not work in humans because all human mast cells contain heparin (Craig et al. 1993), which is essential for the stabilization of the β-tryptase tetramer (Pereira et al. 1998). Thus human mast cells are classified according to their protease content. Mast cells that contain only tryptase (MCT) are usually situated in mucosal tissue, and may be functionally associated with the immune system and host defense (Irani et al. 1987). There are also mast cells containing tryptase, chymase, carboxypeptidase A, and cathepsin G (MCTC) (Table

220

Tryptase

Tryptase

Fig. 11.2 Mast cell heterogeneity in terms of cytokine content. Three sequential 2-mm sections of nasal mucosa from a patient with allergic rhinitis stained for (a) tryptase, IL-4 and chymase, demonstrating that nearly all chymase-positive mast cells contain IL-4, and (b) tryptase, IL-6 and chymase, demonstrating colocalization of IL-6 to a tryptase-positive, chymase-negative mast cell (arrows). A chymase-positive mast cell does not contain IL-6 (arrowheads). (From Bradding et al. 1995a, with permission.) (See CD-ROM for color version.)

11.1), which are normally situated in the skin and submucosal connective tissue, and which are proposed to be involved in tissue repair, fibrotic reactions, and angiogenesis (Artuc et al. 1999; Trautmann et al. 2000). There are also reports of mast cells containing chymase and carboxypeptidase (MCC) without tryptase (Bradding et al. 1995a; Yamada et al. 2001; Horny et al. 2003) which vary in location and whose function has yet to be elucidated. The heterogeneity of mast cells also extends to their cytokine content (Fig. 11.2). MCTC predominantly express IL-4 and IL-13, whereas MCT express IL-5 and IL-6 almost exclusively (Bradding et al. 1995a; Anderson et al. 2001), again suggesting distinct roles for these phenotypes.

General morphology and biology Despite the heterogeneity of mast cells, their general morphology is similar regardless of the tissue site they reside in. Ultrastructurally, the cell membrane contains finger-like projections, and while immature mast cells may have a multilobed nucleus, mature cells have a monolobed nucleus

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with no apparent nucleoli and little condensed chromatin. They have few mitochondria and ribosomes, as well as an inconspicuous Golgi apparatus, scant rough endoplasmic reticulum (RER) and, in contrast to basophils, a lack of cytoplasmic glycogen aggregates (Fig. 11.3) (reviewed by Dvorak 2005). In fact, the only prominent cytoplasmic structures are the electron-dense granules (Fig. 11.3). Unusually, mature human mast cells have ribosomes closely associated with these secretory granules, and little association between ribosomes and RER (Dvorak & Morgan 2001). This suggests that the secretory granules play a significant role in RNA metabolism in human mast cells. The granules are membrane bound and contain preformed mediators, while dense lipid bodies are a store of arachidonic acid. The membrane-bound secretory granules contain crystalline structures that resemble scrolls, lattices, crystals, or whorls (Dvorak 2005). These

(a)

(b) Fig. 11.3 Scanning electron micrographs of mast cells. (a) Normal mast cell at rest showing electron-dense granules and a monolobed nucleus with little condensed chromatin. (b) Mast cell showing evidence of piecemeal degranulation with variable, selective loss of granule contents (arrows).

structures are more visible in MCT subtypes as the sheer volume of protease in the MCTC subtype masks their appearance. Despite this, electron microscopy shows that MCT granules contain predominantly scroll patterns and MCTC granules contain predominantly lattice patterns (Table 11.1) (Craig et al. 1988). Mast cells have basophilic cytoplasm that stains pink with Wright’s or May–Grünwald’s Giemsa, with a purple/blue nucleus and blackish granules. Mast cells can have an irregular shape in tissues but can be identified by selective staining using cationic dyes (such as aniline dyes) that bind to sulfated glycosaminoglycans specific to the mast cell granules. Using stains that utilize the anionic property of the mast cell granules to identify their presence is a useful tool. However, in humans the most effective way to identify the location and subtype of mast cells histologically is to use immunohistochemistry with antibodies raised against the mast cellspecific enzymes tryptase and chymase (Walls et al. 1990). The proteoglycans of the granules are the backbone of the granule matrix. They are a long single peptide with glycosaminoglycans attached. In human mast cells, the proteoglycan content of the granules is mainly heparin and chondroitin E. Neutral proteases, acid hydrolases, and histamine molecules are attached to the proteoglycans by ionic linkage to the sulfate groups on the glycosaminoglycans (Aborg et al. 1967). The sulfate groups generate an acidic environment within the granules that maintains the mediators in an inactive state (Humphries et al. 1999). IgE-dependent activation of the mast cell induces granule swelling, crystal dissolution, and granule fusion, both with surrounding granules as well as the cell membrane, followed by exocytosis with release of mediators into the extracellular space (Caulfield et al. 1980). This process is termed “compound exocytosis” or “anaphylactic degranulation.” Once in the extracellular space, the neutral pH activates the mediators (Caulfield et al. 1980, 1990). However, in many diseased tissues including asthmatic bronchial mucosa, the ultrastructural appearance of mast cells typically demonstrates piecemeal degranulation (Djukanovic et al. 1992a; Aoki & Kawana 1999; Begueret et al. 2007), in which there is variable loss of granule contents although the granules and their membranes remain intact (Fig. 11.3). The mechanisms leading to piecemeal degranulation in mast cells are poorly understood and require further research. The effects of preformed mediators often remain localized as histamine is short-lived in vivo, being broken down by histamine N-methyltransferase, and the active tetramer of tryptase rapidly dissociates into inactive monomers in the absence of heparin (Schwartz & Bradford 1986). In addition to stored granule-derived mediators, newly formed metabolites of arachidonic acid are also released from the cell following IgE-dependent activation (Table 11.2). This phospholipid is liberated from the cell membrane, nuclear envelope, or lipid bodies during immunologic activation and is rapidly oxidized by either the cyclooxygenase (COX) or

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lipoxygenase pathways to form the eicosanoids PGD2 and LTC4 respectively (Bradding et al. 1995b; Boyce 2003). In addition, the mast cell is capable of synthesizing and secreting numerous cytokines and chemokines depending on the stimulus (see below). The principal biological properties of mast cell autacoids (histamine, PGD2, LTC4), proteases, and cytokines are summarized in Tables 11.2–11.4.

Mechanisms of mast cell activation IgE-dependent activation The best-studied mechanism of mast cell activation, and that considered most relevant to allergic disease is activation through the high-affinity IgE receptor FcεRI (reviewed by Gilfillan & Tkaczyk 2006; Rivera & Gilfillan 2006). Most of the downstream signaling events identified following FcεRI engagement have been defined in rodent models, with relatively little known about events in human mast cells. Where human cells have been investigated, some important differences in signaling have been observed (Duffy et al. 2001a). FcεRI is a tetrameric structure that belongs to the multichain immune recognition receptor (MIRR) family. It comprises an α chain (FcεRIα) that binds IgE, a β-chain signaling subunit (FcεRIβ), and two γ subunits that exist as a homodimer signaling subunit (FcεRIγ) (Fig. 11.4). A detailed description of the multiple signaling cascades activated following receptor activation is beyond the scope of this chapter but is summarized in Fig. 11.4 (for reviews see Gilfillan & Tkaczyk 2006; Rivera & Gilfillan 2006). In terms of the proximal signaling pathways, the γ signaling subunits contain an immunoreceptor tyrosine-based activation motif (ITAM) (Cambier 1995) within their cytoplasmic C-terminal domains, which bind to Syk tyrosine kinases initiating phosphorylation. The β chain of FcεRI also contains an ITAM. However, the FcεRIβ ITAM contains a noncanonical tyrosine residue that prevents binding of Syk kinase. Instead, FcεRIβ signals through activation of the lipid raft-associated Lyn tyrosine kinase, which in turn activates Syk kinase (Fig. 11.4) (Rivera & Gilfillan 2006). Signaling in vivo is initiated when multivalent allergen binds to allergen-specific IgE bound to the FcεRIα chain. This promotes FcεRI aggregation and can be mimicked in vitro by the use of anti-IgE antibodies. Receptor aggregation initiates association of the receptor with lipid rafts containing Lyn, a membrane-anchored member of the Src family of protein tyrosine kinases (reviewed by Dykstra et al. 2003). Lyn kinase transphosphorylates tyrosine residues in the ITAMs before binding strongly to the phosphorylated FcεRIβ ITAM through the SH2 (Src homology 2) domain (reviewed by Siraganian 2003). Syk protein tyrosine kinases are recruited to the rafts and bind the doubly phosphorylated ITAMs. They are themselves phosphorylated by recruited Lyn and Syk kinases, promoting the Syk activation loop which results in a fully activated Syk with adjacent tyrosine phosphorylation that

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Table 11.2 Classical human mast cell mediators and their biological effects. Preformed Histamine Bronchoconstriction Tissue edema Mucus secretion Fibroblast proliferation Collagen synthesis Endothelial proliferation, dendritic cell activation Heparin Anticoagulant Storage matrix for mast cell mediators Fibroblast activation Protects growth factors from degradation and potentiates their action Endothelial cell migration Tryptase Generates C3a and bradykinin Degrades neuropeptides Increases bronchial hyperresponsiveness Indirectly activates collagenase Fibroblast proliferation and collagen synthesis Bone remodeling and epithelial activation Potentiates mast cell histamine release Chymase Mucus secretion Extracellular matrix degradation Type I procollagen processing Converts angiotensin I to angiotensin II Inhibits T-cell adhesion to airway smooth muscle Activates IL-1b, degrades IL-4 and releases membrane-bound SCF Newly generated PGD2 Bronchoconstriction Tissue edema Mucus secretion Dendritic cell activation Chemotaxis of eosinophils, Th2 T cells and basophils via the CRTH2 receptor LTC4 /LTD4 Bronchoconstriction Tissue edema Mucus secretion Enhances IL-13-dependent airway smooth muscle proliferation Dendritic cell maturation and migration Eosinophil IL-4 secretion and mast cell IL-5, IL-8 and TNF-a secretion Tissue fibrosis

begins a cascade of events leading to the activation of inositol trisphosphate (IP3). The generation of IP3 induces calcium mobilization from intracellular RER stores, which initiates the influx of extracellular calcium via store-operated calcium

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Table 11.3 In vitro biological effects of human mast cell-derived cytokines. Cytokine

Target cells

Biological effects

IL-4

B cells

Eosinophils Fibroblasts Mast cells

IgE production; proliferation; MHC class II, CD40 and CD25 expression; IL-6 production Proliferation; induction of Th2 phenotype ↑ VCAM-1 and ↓ ICAM-1 expression; proliferation ↓ generation of H2O2 and O2−, ↓ parasite killing and ↓ tumoricidal activity; ↓ IL-1, IL-6, IL-8, and TNF-a production ↑ MHC class II and CD23 expression; monocyte/macrophage differentiation; 15-lipoxygenase expression Transendothelial migration Proliferation, chemotaxis and matrix protein secretion; ↑ ICAM-1 expression ↑ FceRI expression, ↑ ICAM-1 expression

IL-3, IL-5

Eosinophils and prolonged survival

Growth, adhesion, transendothelial migration, chemotaxis, activation, GM-CSF

IL-6

B cells T cells Airway glandular cells Mast cells

Immunoglobulin secretion including ↑ IgE synthesis Growth, differentiation and activation Mucus secretion Survival

IL-13

B cells Monocyte/macrophages Eosinophils Vascular endothelial cells

IgE synthesis As for IL-4 Activation, ↑ survival ↑ VCAM-1

IL-16

T cells

Chemotaxis

TNF-a

Monocyte/macrophages T cells Neutrophils Eosinophils Mast cells Vascular endothelial cells

Airway glandular cells

Enhanced cytotoxicity; chemotaxis and prolonged survival Class II antigen and IL-2R expression; proliferation Chemotaxis, enhanced cytotoxicity Enhanced cytotoxicity and oxidant production Histamine and tryptase secretion E-selectin, ICAM-1 and VCAM-1 expression. Adhesion and transendothelial migration of most leukocytes Growth and chemotaxis; ↓ collagen synthesis but ↑ collagenase production; IL-6 and IL-8 synthesis Mucus secretion

SCF

Mast cells

Growth, differentiation, survival, chemotaxis

NGF

Mast cells B cells T cells Eosinophils Basophils Neutrophils Monocyte/macrophages Fibroblasts Smooth muscle cells

Differentiation, survival, activation, mediator release Differentiation, proliferation, immunoglobulin synthesis Differentiation, proliferation Proliferation Activation, mediator release Chemotaxis, survival, mediator release Proliferation, mediator release Migration, contraction Migration, contraction, proliferation

TGF-b

Smooth muscle cells Epithelial cells Endothelial cells

Differentiation, activation Inhibition of proliferation Induces angiogenesis

bFGF

Fibroblasts Vascular endothelial

Proliferation Stimulates angiogenesis

IFN-a

NK cells Macrophages Dendritic cells T cells

Increased cytotoxicity Development, maturation Activation and maturation, upregulation of IFN-g production Promotes survival of activated T cells, induction of Th1 phenotype

T cells Vascular endothelial cells Monocyte/macrophages

Fibroblasts

NK, natural killer; VCAM, vascular cell adhesion molecule. See text for definition of other abbreviations.

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Table 11.4 In vitro biological effects of human mast cell-derived chemokines. Chemokine

Target cells

Biological effects

CCL1

T cells

Chemotaxis (selective for Th2), survival?

CCL2

T cells Mast cells Eosinophils Monocytes Fibrocytes Epithelial cells Basophils

T-cell polarization toward Th2 phenotype Chemotaxis Chemotaxis Chemotaxis Chemotaxis Proliferation, chemotaxis Activation, mediator release

CCL3

Macrophages Neutrophils Eosinophils Monocytes Mast cells Basophils T cells

Differentiation Chemotaxis (in vivo) and cytotoxicity Chemotaxis Chemotaxis Activation, mediator release Activation, mediator release Chemotaxis (selective for Th1), polarization toward Th1 phenotype

CCL4

Eosinophils Neutrophils T cells

Chemotaxis Chemotaxis (in vivo) Chemotaxis (selective for Th1), polarization toward Th1 phenotype

CCL5

Mast cells Eosinophils Monocytes T cells

Chemotaxis? Chemotaxis Chemotaxis Chemotaxis (selective for Th1), polarization toward Th1 phenotype

CCL7

Eosinophils Monocytes Basophils

Chemotaxis Chemotaxis Activation, mediator release

CCL12

Fibrocytes Monocytes Eosinophils Lymphocytes

Chemotaxis Chemotaxis Chemotaxis Chemotaxis

CCL17

T cells

Chemotaxis (selective for Th2)

CCL19

Airway smooth muscle

Chemotaxis

CCL20

Dendritic cells T cells

Chemotaxis Chemotaxis

CCL22

T cells

Chemotaxis (selective for Th2)

CXCL5

Neutrophils

Chemotaxis

CXCL8

Neutrophils Mast cells Endothelial cells Eosinophils

Chemotaxis Inhibition of chemotaxis, inhibition of mediator release Proliferation, survival, chemotaxis, angiogenesis Chemotaxis after priming with IL-3, IL-5 or GM-CSF

channels (Fig. 11.4) (Faeder et al. 2003). In terms of IgEdependent mediator release in both rodents and humans, influx of extracellular Ca2+ is a critical requirement for the release of both preformed and newly generated mediators (Church et al. 1982; Kim et al. 1997).

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The process of Ca2+ entry through the plasma membrane as a result of the depletion of Ca2+ from internal stores is termed capacitive Ca2+ entry. The Ca2+ current passing through the plasma membrane is known as the Ca2+ release activated Ca2+ current (ICRAC), and is believed to play a central role in

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Allergen

g

b

a

ICRAC

LAT

Syk

Syk Lyn

Lyn

PLC-g

Syk

Syk

DAG

PKC

Ca2+

RER

Ca2+

Sos

Ras

Grb2

+ KCa3.1

+

K+

Raf-1

IP3

IP3R

K+

Ca2+

IgE

Mediator release Cytokine production

MEK

Ca2+

ERK PLA2

Arachidonic acid metabolism

Mediator release Cytokine production Arachidonic acid metabolism

Fig. 11.4 Simplified schematic of mast cell signaling events leading to degranulation and mediator production. Cross-linking of IgE molecules bound to FceRI initiates ITAM phosphorylation at the cytoplasmic termini of the dimeric g signaling subunits of the receptor (depicted in green). Syk kinases are recruited to the activated ITAMs, which autophosphorylate and recruit more Syk kinases (along with other kinases) leading to a cascade of signaling events. The b chain of the receptor (depicted in blue) also contains an ITAM, which binds to the lipid raft-associated Lyn kinase which in turn recruits, to the lipid rafts, and activates Syk kinases, thus amplifying receptor signaling. Syk kinases activate the membrane-associated LAT, which activates phospholipase C (PLC)-g leading to the release of intracellular calcium stores from the rough endoplasmic reticulum via inositol trisphosphate (IP3) and its receptor. PLC-g also leads to activation of protein

kinase C (PKC), which induces mediator release and cytokine production. In addition, PLC-g initiates the Grb2/Sos/Ras pathway, which leads to extracellular regulated kinase (ERK) activation that initiates arachidonic acid metabolism via activation of phospholipase A2 (PLA2) and subsequent eicosanoid production and release. Mast cell degranulation is dependent on influx of extracellular calcium through store-operated calcium channels (SOCC) such as ICRAC, which is initiated by the release of calcium from internal stores (depicted by red dashed line). This influx of calcium activates the intermediate conductance Ca2+-activated K+ channel, KCa3.1 (depicted by blue arrow), leading to an efflux of K+ which counteracts the membrane depolarization induced by Ca2+ influx, thus increasing the driving force for Ca2+ entry. (See CD-ROM for color version.)

many physiologic processes such as gene transcription, proliferation and cytokine release (Lewis 2003). ICRAC has been well characterized electrophysiologically in several cells including rodent mast cells (Hoth & Penner 1992), but the channel carrying it has only recently been identified. Studies from separate groups using an RNAi screen in Drosophila cells have identified the gene product of FLJ 14466 as an essential component of ICRAC (Feske et al. 2006; Vig et al. 2006), and two further mutagenesis studies suggest this is indeed the poreforming protein (Prakriya et al. 2006; Yeromin et al. 2006). The gene product has been given different names (Orai1, CRACM1). We will refer to it as CRACM1. Although it is generally accepted that Ca2+ influx following store release is required for mediator release from human and rodent mast cells following immunologic activation, whether CRACM1 channels alone control granule exocytosis is not clear, and has not been investigated in human mast cells. One study in rat basophil leukemia (RBL) cells, which combined measurements of ICRAC with membrane capacitance measurements to monitor exocytosis, found that ICRAC did not provide suf-

ficient Ca2+ to support granule fusion (Artalejo et al. 1998), although it may well contribute to the production of lipid mediators in these cells (Chang & Parekh 2004). Human mast cells also express several members of the transient receptor potential family of ion channels that also have the potential to contribute to Ca2+ influx following immunologic activation (Bradding et al. 2003). K+ channels have the potential to modulate Ca2+ influx and hence mediator release due to their profound effects on the cell membrane potential. K+ channels hyperpolarize the plasma membrane when open and thus increase the electrical driving force for Ca2+ entry (Panyi et al. 2004), but perhaps more importantly, CRACM1 conducts larger currents at negative membrane potentials (Hoth & Penner 1992). In both RBL cells and rat IL-3-dependent BMMC, an inwardlyrectifying K+ channel (Kir2.1) is open when the cells are at rest (Lindau & Fernandez 1986; McCloskey & Qian 1994). However, the K+ channels present in human mast cells differ to those in rodents, highlighting an example of speciesdependent heterogeneity. Of note we have never seen a Kir

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indirectly enhances Ca2+ influx (Fig. 11.5c) and histamine release (Fig. 11.5d), but is not critical for secretion, and can thus be considered as increasing the gain of an immunologic stimulus (Duffy et al. 2004). This channel is closed by compounds that inhibit mast cell secretion such as β2-adrenoceptor agonists, providing a mechanism for the coupling of receptor activation and impaired secretion (Fig. 11.5e). Interestingly, this effect appears to act through a cAMP-independent mechanism (Duffy et al. 2005).

current in any human mast cell (Duffy et al. 2001a,b, 2003, 2004, 2005; Cruse et al. 2006). Thus the majority of human lung mast cells (HLMC) and human peripheral blood-derived mast cells are electrically “silent” at rest, with a resting membrane potential of around 0 mV (Fig. 11.5a,b). Following IgE-dependent activation, human mast cells rapidly open the intermediate conductance Ca2+-activated K+ channel KCa3.1 (Fig. 11.5a) (Duffy et al. 2001a; Kaur et al. 2005), which has not been described in rodent mast cells. The KCa3.1 channel

80 100

Membrane voltage (mV)

Anti-IgE

Current (pA)

80 60 40 20

Baseline

0 –20 –40 –150 –100 –50

0

50

0

–80

100 150

Command potential (mV)

(a)

Anti-IgE added

(b)

1:30000 Anti-IgE

500 300 100

1-EBIO 0

12

200 300 Time (sec)

Anti-IgE 1:30000 n=6

*

1:30000 300 Anti-IgE 200 100

1-EBIO 200 300 Time (sec)

500

1:30000 Anti-IgE

300 100

400

1-EBIO 0

100

200 300 Time (sec)

1-EBIO 100 mM

300

8 6 4

Wash

100 0

Salbutamol 1 mM Salbutamol 3 mM Baseline

–100 –200

2 SO .1% mM mM mM mM DM O 0 10 30 100 300 S IO IO IO O DM 1-EB 1-EB -EB -EBI 1 1

–300 –150 (e)

Fig. 11.5 (a) Whole-cell patch-clamp recording of a human peripheral blood-derived mast cell at baseline and following addition of anti-IgE. Current–voltage curve demonstrating a characteristic KCa3.1 current. (b) Current clamp trace demonstrating acute change in membrane potential as KCa3.1 opens. (c) Enhancement of Ca2+ influx by the KCa3.1 opener 1-EBIO following submaximal IgE-dependent activation of human

226

100

700

200

10

No

400

0

400

Current (pA)

Net IgE-dependent histamine release (% of cell total)

14

100

500

Cytosolic free Ca2+ (nM)

700

(c)

(d)



100 sec

–100

–50 0 50 Command potential (mV)

100

150

lung mast cells. (d) Enchancement of histamine release by the KCa3.1 opener 1-EBIO following submaximal IgE-dependent activation of human lung mast cells. (e) Reversible closure of KCa3.1 by the b2adrenoceptor agonist salbutamol. (a) and (b) from Duffy et al. 2001a, (c) and (d) from Duffy et al. 2004 and (e) from Duffy et al. 2005, with permission.

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Monomeric IgE

Allergen Tetrameric FceRI

Y

Y Stem cell factor

a Ig free light chains

c-kit +

gb

Protease-activated receptors

↓ SHIP (?) Chemokines

Protein tyrosine kinases Serine/threonine kinases

TLRs (viruses bacteria)

Second messengers IP3 DAG Cytokines (TNF-a)

Ca2+ release from stores

Adenosine Neuropeptides C5a

PKC

Further protein kinases

Hyperosmolality

Cell–cell signals Fig. 11.6 IgE-dependent and -independent mechanisms of mast cell activation. See text for definition of abbreviations. (See CD-ROM for color version.)

Cl− Ca2+

Monomeric IgE-dependent mast cell activation In addition to the cross-linking of FcεRI by allergen, the binding of monomeric IgE alone to FcεRI can initiate intracellular signaling events and Ca2+ influx (Fig. 11.6) (Kalesnikoff et al. 2001; Huber et al. 2002; Kitaura et al. 2003; Oka et al. 2004; Pandey et al. 2004; Cruse et al. 2005; Kitaura et al. 2005; Liu et al. 2005; Matsuda et al. 2005; Nunomura et al. 2005; Tanaka et al. 2005). In rodents this results in the release of granulederived mediators and the secretion of cytokines including IL-6 (Kitaura et al. 2003; Oka et al. 2004; Tanaka et al. 2005). This IL-6 acts in an autocrine manner and prolongs mast cell survival following growth factor withdrawal (Kitaura et al. 2003). In human CBMC, monomeric IgE alone induces the release of the cytokines I-309, GM-CSF, and MIP-1α without histamine release (Gilchrest et al. 2003). However, in HLMC that have been maintained in culture, IgE induces the secretion of histamine, LTC4, and IL-8, which is markedly enhanced in the presence of SCF (Cruse et al. 2005). Interestingly, in both rodent mast cells and HLMC, ongoing signaling is dependent on the presence of “free” IgE, and this ceases immediately when free IgE is removed, suggesting that these findings are physiologically relevant (Pandey et al. 2004; Cruse et al. 2005). The mechanisms behind this are uncertain but in part are thought to involve FcεRI aggregation. These observations

K+

are of great interest because SCF and free IgE concentrations are elevated in asthmatic airways, and there is a robust correlation between total serum IgE and the presence of asthma and bronchial hyperresponsiveness (Burrows et al. 1989; Sears et al. 1991; Sunyer et al. 1995, 1996). This provides a mechanism for the ongoing activation of mast cells through FcεRI in the absence of allergen, and could partly explain the efficacy of anti-IgE therapy in chronic allergic disease (D’Amato et al. 2004; Djukanovic et al. 2004; Holgate et al. 2005a; Ong et al. 2005).

Nonimmunologic stimuli Mast cells may also be activated through a plethora of non-IgE-dependent mechanisms (Fig. 11.6). These include proteases (including tryptase) (He et al. 2004; Moormann et al. 2006), cytokines (e.g., SCF, TNF-α, IFN-γ) (Columbo et al. 1992; Sperr et al. 1993; Yanagida et al. 1996; BrzezinskaBlaszczyk & Pietrzak 1997), complement (Nilsson et al. 1996; Ahamed et al. 2004), adenosine (Forsythe et al. 1999), Tolllike receptor ligands (Varadaradjalou et al. 2003; Kulka et al. 2004), neuropeptides (particularly skin mast cells) (Columbo et al. 1992; Heaney et al. 1995), and hyperosmolality (Eggleston et al. 1987; Genovese et al. 1996a,b; Peachell & Morcos 1998). For example, the C5a receptor CD88 was not thought to be

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expressed on HLMC, but recent work demonstrates that it is in fact expressed on the MCTC subset of HLMC (Oskeritzian et al. 2005). Elevated C5a concentrations have been identified in the induced sputum of asthmatic subjects (Marc et al. 2004), thus providing an alternative means of mast cell activation, and of particular relevance to those mast cells (MCTC) within the airway smooth muscle bundles (see below). Human progenitor-derived mast cells and mouse mast cells express Toll-like receptor (TLR)1–7 and TLR9 (Applequist et al. 2002; Matsushima et al. 2004). These play an important role in the innate host response to pathogens, activating diverse programs of gene expression depending on the stimulus. For example, in mouse mast cells functional responses to TLR2 (the receptor for bacterial peptidoglycan) results in production of TNF-α, IL-4, IL-5, IL-6, IL-13, and IL-1β, while activation of TLR4 (the receptor for lipopolysaccharide, LPS) induces production of TNF-α, IL-1β, IL-6, and IL-13 but not IL-4 or IL-5. In addition, activation of TLR2 but not TLR4 induces Ca2+ mobilization, degranulation, and LTC4 production (Supajatura et al. 2001, 2002; Ikeda & Funaba 2003). Examination of the gene expression profile from human CBMC using high-density oligonucleotide probe arrays following activation with LPS compared with anti-IgE demonstrates that both induce a core response, plus an LPS or anti-IgE specific program of gene expression (Okumura et al. 2003). Perhaps of more relevance to asthma is mast cell activation via TLR3, the ligand for which is double-stranded viral RNA (Kulka et al. 2004). Poly:IC, a synthetic activator of TLR3, induces the specific release of IFN-α as does exposure to respiratory syncytial virus (RSV) and influenza virus. Since viruses are a common cause for asthma exacerbations, the mast cell antiviral response may be an important contributor to the deteriorating airway physiology. A further interesting area of study is the role of immunoglobulin free light chains. These are present in serum in normal subjects and their production is augmented in inflammatory diseases such as rheumatoid disease. In mice, immunoglobulin free light chains can confer mast cell-dependent hypersensitivity through an unknown mechanism, and antigen-specific light chains can mediate mast cell-dependent bronchoconstriction following antigen challenge (Kraneveld et al. 2005). Concentrations of immunoglobulin free light chains are elevated in the sera of asthmatic compared with normal subjects, suggesting they may be relevant to the pathophysiology of human asthma (Kraneveld et al. 2005). Finally, there are likely to be genetic factors that lower the mast cell threshold for activation in asthma. For example, an important negative regulator of mast cell activation is SH2containing inositol phosphatase (SHIP) (Huber et al. 2002). SHIP-deficient rodent mast cells exhibit markedly enhanced secretory responses and, with respect to human basophils at least, cells that are “hyperreleasable” demonstrate a relative deficiency of this molecule (MacDonald & Vonakis 2002).

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Role of mast cells in health Interest in the function of mast cells in disease often takes precedence over their role in health. However, mast cells may play a significant role in the healing of wounds and defense against bacterial and parasitic infection, participating in both innate and adaptive immunity. They are also major effector cells in inflammatory processes, attracting leukocytes to the area of insult, which contributes to host defense and repair. It is likely therefore that their primary role is to sense the external environment, ready to respond to a variety of diverse tissue insults with an early and appropriate program of gene expression and mediator release aimed at initiating inflammation and then repair. When the insult becomes chronic, then it is our view that their continuing activation contributes to tissue dysfunction and remodeling. In addition, through the misguided generation of allergen-specific IgE by host B cells, they have the potential to induce acute, sometimes life-threatening symptoms on exposure to allergen.

Mast cells in wound repair and angiogenesis Because of the biological actions of their mediators (Tables 11.2–11.4), mast cells have been thought to be involved in the healing of wounds. Early studies using metachromatic staining showed that mast cells “disappear” at the wound edge in the first few days, possibly due to degranulation, then increase twofold over baseline by 10 days before returning to normal (Persinger et al. 1983). However, studies using mast cell-deficient mice have provided conflicting results. Egozi et al. (2003) found that mast cells modulated the early inflammatory response to wound healing and angiogenesis, but were not required for the late-phase proliferative response to injury and had no effect on collagen deposition and reepithelialization. In contrast, Iba et al. (2004) showed that mast cells contributed to the late-phase remodeling in wound healing and that collagen fibrils were more interwoven in wild-type mice than mast cell-deficient mice (Kit w/kit w-v). The differing results between these studies may be due to the experimental models used. However, neither study showed any real convincing evidence of a major role for mast cells in wound repair. In contrast, a study by Weller et al. (2006) suggested that mast cell deficient (Kit w/kit w-v) mice have significant retardation of wound closure compared with wild type, and that mast cells are required for normal wound healing. Mast cells may potentially inhibit thrombosis within damaged tissues through the release of heparin, tryptase, chymase, and tissue plasminogen activator, allowing perfusion and nutrition to the site of injury (Thomas et al. 1998; Valent et al. 2002). In support of this, mast cell-deficient mice are more susceptible to lethal thrombogenic stimuli than wild-type mice. Neovascularization occurs in a number of physiologic and pathologic situations including wound healing and tumor

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Mast Cells: Biological Properties and Role in Health and Allergic Diseases reproduced by exposure of vascular cells to purified human tryptase, and inhibited approximately 80% following pretreatment of HMC-1 supernatants with the specific tryptase inhibitor BABIM.

growth. Mast cells are usually found at sites of neovascularization such as around the periphery of solid tumors (Westphal 1891; Hartveit 1981; Welsh et al. 2005). Rodent mast cells and the human mast cell line HMC-1 induce proliferation of microvessels (Rizzo & DeFouw 1996). Several cytokines identified in human mast cells have potential roles in angiogenesis, including TNF-α, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). Supernatants from unstimulated HMC-1 cells induce proliferation of human microvascular endothelial cells, which is largely mediated by VEGF (Feoktistov et al. 2003). In a model of vascular tube formation, human dermal microvascular endothelial cells exposed to HMC-1 cell supernatants or cocultured with HMC-1 cells rapidly differentiate and mature into vascular tubes (Blair et al. 1997). This effect is

Mast cells and inflammation The mechanism by which leukocytes are recruited to sites of inflammation includes a series of steps, namely leukocyte– endothelial cell adherence, diapedesis or transendothelial migration, chemotaxis and, at various stages during these processes, cell activation for mediator secretion. A number of mast cell mediators and cytokines have the potential to orchestrate each of these events (Fig. 11.7; Tables 11.3 & 11.4), and robust evidence suggests that this is indeed the case (reviewed by Bradding & Holgate 1999).

Capillary leakage

Histamine LTC4 PGD2

Rolling adhesion Firm adhesion Transendothelial migration PSGL-1 E-selectin

PSGL-1 P-selectin

Histamine LTC4

TNF-a

VLA-4 VCAM-1

TNF-a IL-4 IL-13

CD11/18 ICAM-1

TNF-a

TNF-a IL-4 Chemotaxis

MC

IL-5 IL-8 IL-16 GM-CSF CCL2

Mast cell activation

Complement Antigen Proteases Bacteria

LPS

IL-4 TNF-a IL-5 SCF IL-6 CCL3 IL-13 Tryptase GM-CSF

Activation and mediator release

Neuropeptides

Fig. 11.7 The contribution of mast cell mediators to inflammatory cell recruitment. See text for definition of abbreviations. (See CD-ROM for color version.)

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Parasite infection Mast cells have been considered to represent the primary defense against parasitic infections and are thought to be important for the expulsion of parasites (reviewed by Finkelman et al. 1997). However, inoculation of mice with neutralizing antibodies to both IL-3 and IL-4 abrogates the mast cell hyperplasia that occurs in the gut of Nippostrongylus brasiliensis-infected mice and the associated IgE synthesis, but does not alter the clinical course (Madden et al. 1991). Experiments using parasite-infected mast cell-deficient mice have been contradictory, and so the role of mast cells in immunologic responses to parasites seems far from clear. It has been suggested that the inappropriate activation of mast cells by otherwise innocuous antigens, which is fundamental in allergic reactions, is a maladaption of their antiparasitic role. This “maladaption hypothesis” stems from the observation that the immunopathologic response to parasitic infection and strong allergens is remarkably similar (Finkelman et al. 1997).

Defense against bacterial and viral infection In mice, mast cells have a critical role in the host response to acute bacterial infection. A series of experiments in different laboratories have shown that the release of TNF-α from resident mast cells at various tissue sites is an essential prerequisite for the recruitment and activation of neutrophils required to control the infection. This has been demonstrated with a diverse range of bacteria and experimental models (Malaviya et al. 1996; Supajatura et al. 2001; Jippo et al. 2003). Mast cells can be activated through a variety of mechanisms, including bacterial peptidoglycan (binds to TLR2), LPS (binds to TLR4) and the type 1 fimbrial subunit (binds to CD48) (Malaviya et al. 1994, 1999). Mast cells can also ingest opsonized bacteria and potentially kill them following oxidative burst (Malaviya et al. 1999), and are activated following ligation of TLR3, indicating a likely role in the host response to viral infection (Kulka et al. 2004). These studies provide a clear example of where mast cells, in mice at least, provide an important protective role for the host. This role in the defense against local infection perhaps explains why mast cells are so widely distributed throughout the human body, particularly at mucosal sites and within the skin, which provide a ready portal of entry for foreign organisms.

Mast cell interactions with the specific immune system Mast cells, antigen presentation, and differentiation of Th2 T cells In addition to the innate mechanisms of mast cell activation, both rodent and human mast cells express class II major histocompatibility complex (MHC) antigens; additionally, in both mice and rats, bone marrow-derived and peritoneal mast cells, respectively, are capable of presenting soluble antigens to T cells with subsequent T-cell proliferation (Fox et al. 1994; Frandji et al. 1995). This antigen-presenting activ-

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ity of rat mast cells is enhanced by IL-4 and GM-CSF, and inhibited by IFN-γ (Frandji et al. 1995). IL-4 is required for the differentiation of the Th2 subset of T cells, which themselves produce IL-4 and IL-5. It has been hypothesized that the release of IL-4 from mast cells could provide the right microenvironment for such T-cell polarization (Bradding et al. 1992). One study has reported that rat mast cells can indeed shift the differentiation of T cells toward the Th2 phenotype (Huels et al. 1995). It is therefore plausible that antigen presentation by mast cells in concert with mast cell IL-4 secretion could contribute to Th2 skewing at the onset of an immune response. Mast cells also influence dendritic cell development and their ability to activate T cells. For example, histamine increases IL-10 and decreases IL-12 production by mature dendritic cells, with the result that naive T cells become polarized toward a Th2 phenotype (Idzko et al. 2002). Similar effects have been observed with PGD2 (Theiner et al. 2006), and mast cell dependence for the generation of Th2-promoting dendritic cells is evident in mice in vivo (Mazzoni et al. 2006). Mast cell exosomes induce immature dendritic cells to upregulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent antigen-presenting capacity to T cells (Skokos et al. 2003). Mast cell TNF-α is important for dendritic cell migration during immune responses (Suto et al. 2006), and of particular relevance to allergy, a population of FcεRI+ dendritic-like cells also emerge in parallel with developing mast cells from human hip bone marrow cultures (Kaur et al. 2005).

Mast cells as initiators of allergic inflammatory responses in the absence of IgE A number of diverse allergens, including bee venom phospholipase (PL)A2, Der p I and III, and schistosomal protease, induce the release of histamine and IL-4 from HLMC in the absence of cell-bound IgE (Dudler et al. 1995; Machado et al. 1996). These allergens are all enzymes, and the response to house-dust mite antigens requires the presence of catalytic activity. A rat basophilic leukemia cell line (RBL-2/2/C) also degranulates and secretes IL-4 in response to trypsin and Aspergillus protease. PLA2, PLC, thrombin, trypsin, and papain also induce mediator release from RBL cells, and all depend on the presence of an active catalytic site. Phospholipases, proteases, or both are associated with many allergens including those originating from house-dust mites, cockroaches, fungal spores, grain dust, plant pollens, cats, and insect venoms. The molecular mechanism by which proteases release mediators from mast cells independently of IgE cross-linking involves in part the protease-activated receptor PAR2 (He et al. 2004; Moormann et al. 2006). It is therefore plausible that activation of mast cells by allergens through an IgE-independent mechanism provides an initial stimulus in the immune response to these molecules and may promote the generation of Th2 cells. The reason why this does not occur in everyone can still be explained by

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environmental factors (e.g., level of allergen exposure) and genetic factors (e.g., mast cell releasability, epithelial integrity/ permeability, local antiprotease activity, regulation of cytokine production).

Role of mast cells in allergic diseases Despite the potential physiologic roles that mast cells perform, they are still synonymous with allergy. IgE-mediated allergic diseases include asthma, allergic rhinitis and conjunctivitis, eczema, urticaria, and systemic anaphylaxis. The incidence of these is increasing (Lack et al. 2002; Madsen 2005; Burr et al. 2006; Galassi et al. 2006), but the cause for this remains uncertain. It is evident from Tables 11.2 and 11.3 and the above discussion that mast cells secrete a plethora of autacoids, proteases and cytokines relevant to the pathophysiology of allergic diseases, and many of these are released via both IgE-dependent and -nondependent mechanisms. Depending on the site of mediator release, symptoms manifest clinically as rhinitis, conjunctivitis, urticaria, angioedema, erythema, bronchospasm, diarrhoea, vomiting, and hypotension, which can be fatal in severe reactions (such as anaphylactic shock). In some of these diseases, such as seasonal allergic rhinoconjunctivitis and anaphylaxis, the role of IgE and allergen are relatively clear-cut, while in chronic asthma and atopic eczema the involvement of IgE and mast cells is probably one of many factors contributing to disease expression. We initially focus in detail on the immunopathology of asthma: many of the principles discussed are also applicable to the immunopathology of related allergic diseases.

Mast cells in asthma Asthma is a complex disease characterized by the presence of airway obstruction. This obstruction is potentially reversible, at least in part, either spontaneously or with pharmacologic intervention, and is characterized by the symptoms of wheeze, dyspnea, cough, and chest tightness. Exacerbations may be triggered by a number of different stimuli, one or more of which may predominate in any individual. The major pathologic processes by which airflow obstruction occurs are smooth muscle contraction, mucosal edema due to increased vascular permeability, excessive mucus secretion, airway inflammation, and various structural changes referred to as airway wall remodeling. In this section we concentrate on the evidence that places mast cells as central effector cells in asthma pathophysiology as determined by the profile of mediators they release, their ongoing release of these mediators, and their relocation to key structures within the airways.

Evidence of mast cell activation in asthma Experimental allergen-induced asthma Approximately 90% of subjects with asthma under the age of 30 are atopic (Smith 1974), most frequently with reactions to the house-dust mite Dermatophagoides pteronyssinus or farinae, whereas in subjects developing asthma for the first time over the age of 40, the prevalence of atopy is no greater than in the general population. Allergen exposure clearly plays a role in asthma pathophysiology but it is not the whole story. The level of dust mite exposure in early life is a strong risk factor for the development of asthma, and it is clear that certain pollenallergic individuals only develop symptoms during the pollen season. Clusters of acute asthma exacerbations occur following the acute release of pollen particles into the atmosphere after thunderstorms (Newson et al. 1997, 1998). In addition, the clear clinical efficacy of the anti-human IgE monoclonal antibody omalizumab in severe allergic asthma and its ability to markedly reduce airway inflammation (Djukanovic et al. 2004) points to the importance of IgE in ongoing disease. However, omalizumab does not cure the disease, and symptoms often persist albeit at a lower level. Furthermore, allergen avoidance usually has only a minor effect on the state of established disease, which therefore appears to become “self-perpetuating.” This is typified in cases of occupational asthma, where asthma will persist on removal of the sensitizing agent if exposure is not prevented early in the course of the disease. Because of the evidence that asthma is at least partly an IgE-dependent disease, the method of acute bronchial challenge with a relatively large dose of allergen in the laboratory has provided a useful model for studying asthma pathophysiology. Following bronchial allergen challenge in nearly all atopic asthmatics and many atopic nonasthmatics, there is a rapid fall in pulmonary function (e.g., forced expiratory volume in 1 s or FEV1) at 10–20 min that gradually recovers over the following 2 hours, defined as the early asthmatic reaction (EAR). In about 50% of subjects, between 4 and 6 hours there is a further fall in FEV1, the late asthmatic reaction (LAR). This may last up to 12 hours and in some individuals may be followed by recurring airway obstruction for several days or even weeks (Booij-Noord et al. 1972).

Early asthmatic reaction During the EAR several vasoactive and spasmogenic mediators are released, most of which originate from mast cells resident in the airway mucosa (see Table 11.2). The relative rate of mediator release from HLMC in vitro is histamine > PGD2 > LTC4, with half-maximal release occurring at 2, 5, and 10 min respectively (Schleimer et al. 1986). This is reflected in vivo by the recovery of these mediators in bronchoalveolar lavage (BAL) fluid within 5–10 min following local bronchial allergen challenge (Fig. 11.8) (Murray et al. 1986; Casale et al. 1987a,b; Wenzel et al. 1988, 1990, 1991; Liu et al. 1991; Sedgwick et al. 1991).

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8

20

6

15

4

10

2

5

Tryptase (ng/mL)

Histamine (ng/mL)

PART 2

0

0 Pre

Post Histamine

Pre

Post

Tryptase

Fig. 11.8 Concentrations of histamine and tryptase in bronchoalveolar lavage fluid before and 5 min after local bronchial allergen challenge in a group of mild allergic asthmatic subjects. Medians are denoted by horizontal bars. (Data from Wenzel et al. 1988.)

The ability of histamine, PGD2 and LTC4 to produce bronchoconstriction, mucosal edema, and mucus secretion suggests that they are responsible for the acute airway narrowing following allergen challenge (Fig. 11.9). Calculations indicate that the concentrations of mediators generated are similar to those required to produce bronchoconstriction in vitro (Liu et al. 1991) or when delivered by nebulizer (Wenzel et al. 1990). Better evidence for their role is provided by studies using potent and selective receptor antagonists, which demonstrate that the EAR is significantly attenuated by antagonists of histamine (Curzen et al. 1987; Rafferty et al. 1987), LTC4 (Taylor et al. 1991; Findlay et al. 1992), and to a lesser extent PGD2 (Beasley et al. 1989a). The mast cell as a source of these mediators is supported by three lines of evidence. Firstly, the kinetics of IgE-dependent mediator release in vivo parallels that of purified mast cells in vitro. Secondly, the presence of mast cell activation during the EAR is confirmed by the rapid increase in concentrations of the preformed mast cell-specific protease tryptase, recovered by BAL within minutes following local bronchial allergen challenge (Wenzel et al. 1988; Sedgwick et al. 1991). Thirdly, β-agonists such as salbutamol, known inhibitors of mast cell degranulation when applied acutely in vitro (Church & Hiroi 1987), completely abolish the early reaction and the associated increase in plasma histamine levels (Pepys et al. 1968; Howarth et al. 1985). In contrast, corticosteroids administered just prior to allergen challenge do not inhibit the EAR (Booij-Noord et al. 1971) and do not inhibit mast cell degranulation (Schleimer et al. 1983), although repeated treatment for several days may lead to attenuation of the EAR (Burge et al. 1982), possibly as a result of a reduction in the number of mast cells in the bronchial mucosa (Djukanovic et al. 1992b). The EAR is markedly attenuated after pretreatment with anti-IgE (Boulet et al. 1997; Fahy et al. 1997), confirming that IgE-dependent signaling is critical.

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Late asthmatic reaction In contrast to the acute mediator-induced bronchoconstriction and mucosal edema characteristic of the EAR, the LAR is associated with inflammatory cell accumulation and activation. Subsequent mediator release and tissue damage following these events is thought to account for the ensuing airway obstruction and associated increase in bronchial hyperresponsiveness that accompanies the LAR. This situation has been considered to be analogous to that seen in chronic airway inflammation, although some caution is needed in extrapolating the results of applying a single large dose of allergen to the airways, in the presence of natural disease, to the natural disease itself. The role of the mast cell as a source of bronchospastic mediators during the LAR is more difficult to define than during the EAR due to the recruitment and activation of many cell types, including eosinophils (de Monchy et al. 1985; Metzger et al. 1987; Bentley et al. 1993; Montefort et al. 1994), activated CD4+ T cells (Bentley et al. 1993), activated macrophages (Tonnel et al. 1983; Diaz et al. 1989), and small numbers of basophils (Liu et al. 1991; Braunstahl et al. 2001), which are not usually present in stable asthma (Wardlaw et al. 1988; Beasley et al. 1989b). Increased concentrations of histamine, PGD2 and LTC4 are present in the LAR, but in different ratios than during the EAR, raising the possibility that a contribution comes from sources other than mast cells, such as macrophages and eosinophils (Liu et al. 1991; Sedgwick et al. 1991). Tryptase levels fall during the LAR (Sedgwick et al. 1991), which might indicate an absence of mast cell activity. However, mast cell degranulation is not “all or nothing,” with clear examples available of differential mediator release under various conditions (Theoharides et al. 1982; Benyon et al. 1989; Stellato et al. 1991; Leal-Berumen et al. 1994). Interestingly GM-CSF, which is released following allergen provocation (Broide & Firestein 1991), inhibits expression of tryptase in the immature mast cell line HMC-1 but does not attenuate histamine release (Finotto et al. 1996), and in HLMC may in fact potentiate IgE-dependent histamine release (Louis et al. 1995). Furthermore, in rodent mast cells, protease and proteoglycan peptide core mRNA transcripts remain low after the activation/ secretion response, suggesting that regranulation of mast cells is a slow process (Gurish et al. 1991). Thus plausible mechanisms exist to explain the disparity between tryptase and histamine levels during the LAR. Mast cell-derived mediators may therefore contribute directly to the symptomatology of the LAR, although their precise role is uncertain. However, the LAR is also attenuated markedly by anti-IgE therapy (Fahy et al. 1997), indicating that mast cell activation during the EAR initiates events leading to the LAR. It is therefore likely that the secretion of mast cell mediators and cytokines orchestrates the development of the late inflammatory response (Fig. 11.9).

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Mast Cells: Biological Properties and Role in Health and Allergic Diseases Epithelial denudation, activation, permeability

Tryptase, Chymase, IL-4, IL-13

Airway Lumen

Epithelium MC

MC

MC

Histamine, Tryptase, PGD2, LTC4, IL-4, IL-6, IL-13, TNF-a and amphiregulin

ASM

CXCL8 CCL11 CCL5

Submucosal gland

Subepithelial fibrosis Mucus hypersecretion

MC

MC

TGF-b bFGF Tryptase

CXCL8 CXCL10 CXCL12 Histamine, Tryptase, PGD2, LTC4, IL-4, IL-13

ASM hypertrophy and hyperplasia

Bronchoconstriction Airway hyperresponsiveness

ASM

ASM mast cell recruitment CXCL10 SCF

MC Mast cell differentiation, survival and activation

MC

Differentiation

SCF MC

MC

Rolling and adhesion

Blood Vessel MC

Fig. 11.9 Mast cell interactions with structural airway cells in the pathogenesis of asthma. See text for definition of abbreviations. (See CD-ROM for color version.)

Mast cell progenitors circulate in the blood

Chronic allergic asthma If mast cell activation is relevant to chronic everyday asthma, it would be expected that bronchial mucosal mast cells in asthmatic subjects would be in an activated state, which is indeed the case. Morphologic assessment using electron microscopy indicates that in “stable” atopic asthma there is continuous ongoing degranulation in both the airway epithelium and submucosa (Beasley et al. 1989b; Djukanovic et al. 1992a) (see Fig. 11.3). Several studies have also shown

MC

Precursor recruitment (CXCL12?)

increased numbers of mast cells in BAL fluid from stable asthmatics compared with normal controls (Flint et al. 1985; Casale et al. 1987b; Kirby et al. 1987), together with increased concentrations of histamine and tryptase, providing further evidence of ongoing mast cell degranulation (Casale et al. 1987b; Kirby et al. 1987; Wenzel et al. 1988) (Fig. 11.10). Mast cells within the bronchial mucosa in asthma express several cytokines, including IL-4, IL-5, IL-6, TNF-α, and IL-13 (Bradding et al. 1992, 1993, 1994; Ying et al. 1997; Berry et al.

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Inflammatory Cells and Mediators 7

5 Mediator concentration

** 4

*

1800 **

6

1500

5 3

4

2

3

1200 900 600

2 1

300

1

0

0

0 Histamine (ng/mL)

Tryptase (ng/mL)

PGD2 (pg/mL)

Fig. 11.10 Concentrations of mast cell-derived histamine, tryptase, and PGD2 in bronchoalveolar lavage fluid recovered from airways of individuals with (solid bars) and without (shaded bars) asthma. The bars represent 1 standard deviation from the mean of 12 observations. **, P < 0.01; *, P < 0.05.

2004). When asthmatic airways are compared with normal airways, there is evidence of increased expression of IL-4 and IL-5 mRNA in mast cells (Ying et al. 1995), and increased expression of mast cell-associated IL-4 and TNF-α protein (Bradding et al. 1994). Strong correlations have been described between numbers of eosinophils and IL-4-, IL-5- and TNFα-positive mast cell densities, although correlations do not necessarily imply cause and effect. However it seems likely that mast cell-derived cytokines make a major contribution to the pathophysiology of asthma. BAL mast cells from symptomatic asthmatic subjects probably differ from normal BAL mast cells by exhibiting greater spontaneous histamine release (Flint et al. 1985; Broide et al. 1991). In addition, the secretory response of asthmatic BAL mast cells to IgE-dependent activation is altered. Broide et al. (1991) found that IgE-dependent activation of mast cells from symptomatic asthmatics did not produce any significant increase in histamine release compared with the already high spontaneous release, in contrast to the findings in asymptomatic asthmatic subjects, perhaps supporting the idea that high spontaneous release in the presence of symptoms is related to IgE-dependent activation in vivo. Both Flint et al. (1985) and Casolaro et al. (1989) reported increased IgEdependent histamine release from atopic asthmatic BAL mast cells compared with nonatopic normal controls. This may have resulted from greater mast cell sensitization by IgE in the atopic subjects, which is known to augment IgEdependent histamine release in a dose-dependent manner (Tunon de Lara et al. 1995). Increased FcεRI occupation by IgE not only enhances receptor aggregation by anti-IgE/ allergen, but also increases surface FcεRI expression (Saito et al. 1997; Welker et al. 1997; Yamaguchi et al. 1997, 1999), and activates HLMC in the absence of allergen (Cruse et al. 2005). Enhanced mediator releasability in asthma may also

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stem from in vivo activation by other inflammatory stimuli described above, but could also represent a fundamental functional difference predisposing to asthma, perhaps as a result of genetic factors. It is often suggested that mast cells cannot be important in asthma because so-called mast cell “stabilizing” drugs are not effective at preventing symptoms or attenuating the underlying inflammation. However, although several commonly used drugs are able to inhibit mast cell secretion, they either lack potency or are ineffective at inhibiting mast cell activation in chronic asthma. For example, sodium cromoglycate is only a weak inhibitor of IgE-dependent HLMC secretion, with maximal inhibition of histamine release in vitro of 10 –20% when used in the high micromolar range (Church & Hiroi 1987), and it also exhibits rapid tachyphylaxis. The β2-adrenoceptor agonists such as salbutamol are more potent inhibitors of HLMC mediator release in vitro (Church & Hiroi 1987), but again there is rapid tachyphylaxis so that with chronic administration the clinical evidence is that they do not attenuate mast cell secretion in the asthmatic airway and may even enhance it (Chong et al. 1995, 2003; Swystun et al. 2000; Scola et al. 2004). Furthermore, the morphology of mast cell degranulation in chronic asthma is predominantly piecemeal (Djukanovic et al. 1992a; Begueret et al. 2007), the mechanism of which is unknown, and this may simply not be susceptible to inhibition by drugs that attenuate classical IgE-dependent anaphylactic degranulation. Mast cells are therefore in a state of continuous activation in chronic allergic asthma, with some evidence to suggest that they may exhibit increased mediator releasability, both spontaneously and in response to IgE-dependent activation. When this information is coupled with the epidemiologic data implicating allergen exposure in the development of asthma and the efficacy of omalizumab, it becomes attractive to hypothesize that the atopic asthmatic phenotype results in part from the everyday interaction between allergens, IgE, and hyperreactive mast cells.

Nonallergic (“intrinsic”) asthma A subgroup of asthmatic patients have traditionally been described as having so-called “intrinsic” or nonallergic asthma, where no sensitivity to environmental allergens or occupational agents can be identified. This form of the disease is often of later onset, more severe and persistent, and more often associated with nasal polyposis and aspirin sensitivity. However, in spite of the different clinical picture, a pattern of inflammation more or less identical to that found in extrinsic asthma is present in the bronchial mucosa of intrinsic asthmatics (Bentley et al. 1992a). This suggests that a common mechanism may in fact be operating in the development of both phenotypes of asthma. Although the serum concentration of total IgE falls within the “normal range” in nonallergic asthma, it is still higher than in nonatopic nonasthmatic subjects (Bentley et al.

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1992a). Furthermore, it has been demonstrated in epidemiologic studies that total serum concentrations of IgE correlate with indices of asthma at all age groups, irrespective of atopic status, raising the possibility that IgE-mediated mechanisms are also relevant to intrinsic asthma (Burrows et al. 1989). This hypothesis is supported by several pieces of evidence that also implicate a role for mast cells. Firstly, FcεRI+ cells are present in increased numbers in the bronchial mucosa of both atopic and nonatopic asthmatic subjects compared with normal controls (Humbert et al. 1996a). Although total mast cell counts are the same in these three groups, the majority of the FcεRI+ cells are mast cells, indicating that FcεRI density is greatly increased on mast cells in both intrinsic and extrinsic asthma. Secondly, and of equal interest, there is increased expression of the Th2 cytokines IL-4 and IL-5 at both the mRNA and protein level in intrinsic asthma (Humbert et al. 1996b). As in atopic asthma, mast cells account for a significant proportion of the cells expressing mRNA and protein for both these cytokines (Ying et al. 1997), suggesting there is ongoing mast cell activation and cytokine secretion in intrinsic asthma. Both IL-4 and IgE are powerful inducers of FcεRI expression on mast cells (Saito et al. 1997; Toru et al. 1997; Welker et al. 1997; Xia et al. 1997; Yamaguchi et al. 1999), and IL-4 is of course a key cytokine involved in the promotion of IgE synthesis. There are increases in ε germline gene (Iε) and mature ε heavy chain (Cε) RNA-positive B cells in the bronchial mucosa in both atopic and nonatopic asthma, suggesting that there is local IgE synthesis (Ying et al. 2001). This is supported by the observation that the concentration of IgE in BAL fluid is increased 24 hours after bronchial allergen challenge (Wilson et al. 2002). Thus it is plausible that IgE production occurs locally within the bronchial mucosa in intrinsic asthma, promoting the increased expression of FcεRI, and perhaps explaining the absence of high serum concentrations. Since nasal mast cells can induce IgE synthesis by B cells in an IL-4 and IL-13 dependent manner (Pawankar et al. 1997), lung mast cells might contribute to local bronchial IgE production.

Virus-induced asthma Viral infections induce exacerbations of asthma in many individuals, particularly those caused by the rhinovirus (RV) (Minor et al. 1974; Johnston et al. 1995). Several mechanisms may be active including direct airway damage with loss of epithelial structure and function, and the presence of virus-specific IgE. The loss of epithelium might allow greater penetration of allergens into the bronchial mucosa, so it is interesting that experimental nasal infection with live RV16 increases the frequency of bronchial LAR after inhaled allergen provocation (Lemanske et al. 1989). Nasal RV16 infection also enhances both the immediate release of histamine into BAL fluid (presumably mast cell-derived) and the later recruitment of eosinophils into the airway after local bronchial allergen challenge (Calhoun et al. 1994).

Whether this is actually due to increased allergen availability or other mechanisms that amplify the inflammatory response is uncertain. Viral infection can also result in the production of virusspecific IgE, and the intensity of this antibody response has been correlated with changes in airway function during the acute infection (Welliver et al. 1981, 1986a). In addition, children who develop higher levels of virus-specific IgE during infection with RSV are more prone to later wheezing than children who do not (Welliver et al. 1986b). An in vivo animal model of asthma and RSV infection has shown that RSV can induce mast cell degranulation through the crosslinking of virus-specific IgE on mast cells, with associated increases in bronchial hyperresponsiveness (BHR) (Dakhama et al. 2004). In addition it is apparent that human mast cells can be activated via TLR3, a receptor for double-stranded viral RNA, secreting both IFN-α and IFN-β (Kulka et al. 2004). This provides a further mechanism through which viruses might directly interact with mast cells.

Occupational asthma Occupational asthma is defined as asthma that develops, or is exacerbated, following specific exposure in the workplace and should exclude nonspecific stimuli that will produce bronchoconstriction in any asthmatic subject. The prevalence of occupational asthma varies, but in some areas may account for 15–20% of adult asthma (Blanc 1987). Over 300 agents are recognized as inducers of occupational asthma, and common to all these compounds is that they are inhaled (which probably informs us about the induction of atopic and intrinsic asthma). They fall into three main groups, the first of which is associated with the synthesis of specific IgE antibodies. Compounds in this IgE-dependent group are either directly immunogenic proteins, such as high-molecularweight enzymes, or low-molecular-weight compounds that bind to body proteins and act as haptens, for example the acid anhydrides and complex halogenated platinum salts. The second group is thought to produce sensitization through as yet undefined immunologic mechanisms, with specific IgE antibodies usually absent. Examples from this group include the low-molecular-weight chemicals plicatic acid (present in the dust of western red cedar) and the isocyanates. The third group consists of agents that are mostly irritant gases, fumes, or chemicals, and which are capable of producing asthma after a single large exposure (reactive airways dysfunction syndrome or irritant-induced asthma). Interestingly, the pathology of occupational asthma (with the exception of irritant-induced asthma) is virtually identical to that seen in atopic and intrinsic asthma, including those cases where IgE-independent mechanisms have been advocated such as with western red cedar asthma (WRCA) and toluene diisocyanate (TDI) asthma (Frew et al. 1995; Saetta et al. 1992). Thus in TDI asthma, numbers of mast cells are increased in the bronchial epithelium compared with

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Histamine

LTE4

*

*

Mediator concentration (mg/mL)

7 6 5 4 3 2 1 0 Pre Post WRCA

Pre Post NOA

Pre Post WRCA

Pre

Post NOA

Fig. 11.11 Histamine and LTE4 concentrations in bronchoalveolar lavage fluid before and 10 min after bronchial provocation with plicatic acid in patients with western red cedar asthma (WRCA) and nonoccupational asthma (NOA). Medians denoted by horizontal bars. *, P < 0.01 (Mann–Whitney U test) for comparison of change between the two groups. (Data from Chan-Yeung et al. 1989.)

normal controls, and electron microscopy demonstrates that the majority of mast cells are degranulated (Saetta et al. 1992). Subjects who develop occupational asthma after a short period of exposure to TDI (2 years) have more mast cells in their airway mucosa than subjects who develop asthma after a long period of exposure (22 years) (Di Stefano et al. 1993). Whether this is a predisposing factor for the early onset of TDI asthma or a secondary effect due to different levels of exposure awaits clarification. Bronchial provocation with plicatic acid results in the rapid release of histamine into the BAL fluid of patients with WRCA but not normal subjects (Fig. 11.11) (Chan-Yeung et al. 1989). Similarly, in vitro, plicatic acid releases histamine from mast cells in both BAL fluid and bronchial mucosal biopsies obtained from patients with WRCA but not those with atopic asthma (Frew et al. 1993). This occurs via an undefined IgE-independent mechanism, which is in keeping with the observation that patients with WRCA do not usually have specific IgE to plicatic acid (Frew et al. 1993).

Exercise-induced asthma Of asthmatic subjects, 80% develop airway narrowing with exercise or hyperventilation of cold dry air. Typically bronchoconstriction occurs 5–10 min after exercise and usually recovers within 30 min. Approximately half these patients exhibit a refractory period for about 1 hour following exercise, during which additional exercise does not induce further bronchoconstriction. The mechanisms behind exercise-induced asthma probably relate to the effects of airway cooling and water loss during exercise and may be mimicked by hyperventilation of cold dry air (reviewed by Anderson 2006).

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Some but not all studies have identified increased concentrations of circulating histamine in the serum of asthmatic subjects following exercise, which has been taken to indicate a possible role for mast cell degranulation (Anderson et al. 1981; Barnes & Brown 1981; Lee et al. 1982). Both mast cells and basophils release histamine in a hyperosmolar environment (Eggleston et al. 1984, 1987), and this release is attenuated by sodium cromoglycate, providing evidence that could link changes in airway osmolarity to bronchoconstriction. Analysis of induced sputum following exercise has demonstrated increased concentrations of histamine, cysteinyl leukotrienes, and tryptase indicative of mast cell activation (Hallstrand et al. 2005). In support of this, terfenadine and clemastine (histamine H1 receptor antagonists), flurbiprofen (potent COX inhibitor), and the LTD4 receptor antagonists MK 571 and ICI 204219 all significantly attenuate exercise-induced bronchoconstriction (Hartley & Nogrady 1980; Finnerty & Holgate 1990; Manning et al. 1990; Finnerty et al. 1992). The refractory period following exercise remains unexplained. Interestingly, challenge of the airways with adenosine 5-monophosphate (AMP), which induces bronchoconstriction indirectly via activation of mast cells, exhibits crossrefractoriness with exercise, implicating a similar mechanism (Finnerty et al. 1990). Depletion of mast cell mediators has also been put forward as an explanation, but this seems unlikely. To summarize then, there is strong evidence implicating mast cell activation as an important component of the bronchoconstrictor response to exercise.

Aspirin-induced asthma Asthma is exacerbated by aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) in about 10% of all asthmatics. Classically these are nonatopic female patients with late-onset asthma who have associated nasal polyps, vasomotor rhinitis, and moderate blood eosinophilia. Asthmatic symptoms usually start 1–2 hours after drug ingestion and may be severe. The observation that this occurs with all NSAIDs suggests that manipulation of arachidonic acid metabolism is the cause. One hypothesis is that a conformational change in COX-2 induced by nonselective NSAIDS favors the formation of hydroxyeicosatetraenoic acids and leukotrienes via the lipooxygenase pathway, resulting in bronchospasm, increased mucus production, and airway edema (Mitchell & Belvisi 1997). This is supported by the finding of increased LTC4 in nasal secretions (Ferreri et al. 1988), increased LTE4 in the urine (Christie et al. 1992), and the protection afforded by LTC4 receptor antagonists (Dahlen et al. 1993) following ingestion of aspirin in aspirin-sensitive asthmatics. This could therefore involve mast cell leukotriene generation. In support of this, a recent study by Machado et al. (1996) reported that aspirin induces mediator release from RBL cells, a cell line with phenotypic features typical of rodent mucosal mast cells. A further clue that mast cells may be dysfunctional in

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aspirin-sensitive asthma comes from observations that there are increased numbers of mast cells in the airways of these patients, and that an increased proportion of these mast cells express COX-2 (Sousa et al. 1997). In addition, mast cells are the predominant cells expressing LTC4 synthase in the airways of aspirin-sensitive asthmatics (Cai et al. 2003).

Mast cell microlocalization in asthmatic airways Mast cells are present in normal airways adjacent to blood vessels, and scattered throughout the lamina propria (Pesci et al. 1993; Carroll et al. 2002a; de Magalhaes et al. 2005). The number of mast cells in the lamina propria is not increased in asthmatic compared with normal airways, but in asthma mast cells infiltrate three key sites: the airway epithelium (Pesci et al. 1993; Bradding et al. 1994; Amin et al. 2005), airway mucosal glands (Brightling et al. 2002a; Carroll et al. 2002b; Chen et al. 2004), and airway smooth muscle (Brightling et al. 2002a, 2005a; Berger et al. 2003; Chen et al. 2004; Amin et al. 2005; El-Shazly et al. 2006; Begueret et al. 2007). This anatomic relocation places the mast cell at the site of several dysfunctional airway elements and, as shown below and alluded to above, the local delivery of their mediators is likely to be central to the disordered airway physiology.

100 Mast cells/mm2 smooth muscle

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P < 0.0001

30 10 3 1

0 Normal (a)

Asthma

Eosinophilic bronchitis

Mast cell infiltration of airway smooth muscle as a key determinant of the asthmatic phenotype It is often stated that the disordered airway physiology and airway wall remodeling characteristic of asthma are consequences of Th2 lymphocyte-driven eosinophilic inflammation. However, there are many examples where the relationship between airflow obstruction and inflammation is weak. This is particularly evident in patients with eosinophilic bronchitis (EB). This disease is responsible for approximately 15% of cases of cough referred to respiratory specialists (Brightling et al. 1999). It is characterized by the presence of sputum eosinophilia occurring in the absence of variable airflow obstruction or BHR (Brightling et al. 1999), and usually responds to treatment with inhaled corticosteroids. An immunopathologic comparison of asthma and EB has revealed that these two conditions are identical in terms of the mucosal inflammatory cell infiltrate, mucosal IL-4 and IL-5 expression, and the degree of subbasement membrane collagen deposition (Brightling et al. 2000, 2002b, 2003a; Berry et al. 2004). Furthermore in BAL fluid and/or induced sputum, mediator concentrations including histamine and PGD2, and numbers of IL-4-expressing T cells are almost identical (Brightling et al. 2000, 2002b). This raises the possibility that many of the Th2-related changes in asthmatic airways considered fundamental in disease pathogenesis may not be so important for the development of airflow obstruction and BHR after all. The really striking difference between the pathology of asthma and EB lies within the airway smooth muscle (ASM). Although ASM function is a disease-defining feature of asthma, it has been largely ignored in previous immunopathologic

(b) Fig. 11.12 (a) Mast cell numbers in the airway smooth muscle of patients with asthma, eosinophilic bronchitis, and normal control subjects. (From Brightling et al. 2002a, with permission.) (b) Light micrograph of tryptasepositive mast cells within the airway smooth muscle bundle (arrows) of a patient with asthma. (See CD-ROM for color version.)

studies of the bronchial mucosa. In asthmatic bronchial biopsies, there are numerous mast cells present within the ASM bundles, but these are virtually absent in the ASM of normal subjects and patients with EB (Fig. 11.12) (Brightling et al. 2002a). In contrast, there are no T cells or eosinophils in the ASM in any of these subject groups. This suggests that mast cell infiltration of the ASM in asthma may be important for the development of BHR and variable airflow obstruction (see Fig. 11.9) (Brightling et al. 2002a). This hypothesis is supported by the finding of a significant correlation between the number of ASM mast cells and the severity of BHR (Brightling et al. 2002a). This increase in ASM mast cells in asthmatic compared with normal control subjects has been confirmed in several further studies (Berger et al. 2003; Amin et al. 2005; Brightling et al. 2005a; El-Shazly et al. 2006; Begueret et al. 2007). It is not linked to atopic status, and so to some extent can be said to extend across asthma phenotypes

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although further work is needed. The majority of mast cells in the ASM are of the MCTC subset (i.e., containing both tryptase and chymase) and express both IL-4 and IL-13 but not IL-5 (Brightling et al. 2002a, 2003b). Furthermore, the mast cells within the ASM demonstrate ultrastructural evidence of activation (Begueret et al. 2007). Taken together these observations suggest that ASM infiltration by mast cells is a key determinant of the asthmatic phenotype. There are also further implications. For example, not only might it clarify why many atopic patients do not have asthma but it could also explain why the presence of asthma is such a strong risk factor for death from anaphylaxis and allergen desensitization (Bock et al. 2001). A second pathologic difference between asthma and EB is that the concentration of IL-13 is elevated in the induced sputum supernatant of asthmatic compared with normal and EB subjects (Berry et al. 2004). Associated with this, the number of IL-13-positive cells in the airway mucosa in asthma is elevated when compared with EB, although nevertheless relatively low (Berry et al. 2004). Interestingly, most of the IL-13-positive cells in asthmatic biopsies in this study are in fact eosinophils, suggesting that there may be dysregulation of IL-13 expression in asthmatic as opposed to EB eosinophils.

Putative mast cell–airway smooth muscle interactions The localization of mast cells within the ASM in asthma is likely to facilitate specific interactions between these two cell types through the specific targeting of both soluble mediators and signals delivered via direct cell–cell contact. We would therefore predict that the microlocalization of mast cells within the ASM will contribute to the development of ASM hypertrophy and hyperplasia, and smooth muscle dysfunction expressed as BHR and variable airflow obstruction. In turn, we would predict that the ASM provides a suitable environment for mast cell growth and survival.

Mechanisms of mast cell recruitment by asthmatic airway smooth muscle The mechanism by which mast cells are recruited to the ASM is important because inhibiting this might offer a novel approach to the treatment of asthma. A likely source for the primary stimulus for mast cell recruitment is the ASM itself, and might involve the recruitment of mast cell progenitors or mature tissue-resident cells (see Fig. 11.9). The ASM secretes many chemokines and growth factors known to exhibit mast cell chemotactic activity, including CCL11, CXCL8, CXCL12, SCF, and TGF-β (John et al. 1998; McKay et al. 1998; Ghaffar et al. 1999; Kassel et al. 1999; Berger et al. 2003; Brightling et al. 2005a; Sutcliffe et al. 2006). A detailed characterization of the chemokine receptor profile of HLMC revealed that they express the relevant receptors for the above chemokines, namely CCR3, CXCR1, and CXCR4 respectively (Fig. 11.13a) (Brightling et al. 2005b). Of great interest, however, was the

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observation that they also express CXCR3 and that this was the most highly expressed receptor (Fig. 11.13a) (Brightling et al. 2005b). Furthermore, almost 100% of mast cells within asthmatic ASM bundles express CXCR3 compared with about 50% in the surrounding mucosa, suggesting that there is selective recruitment of CXCR3-positive mast cells to the ASM in asthma (Brightling et al. 2005a). ASM secretes the three CXCR3 ligands CXCL9, CXCL10, and CXCL11 (Hardaker et al. 2004; Brightling et al. 2005a) and, interestingly, cultured human ASM from asthmatic subjects preferentially secretes CXCL10 after cytokine activation. This CXCL10 accounts for greatly enhanced HLMC chemotaxis mediated by conditioned medium from asthmatic compared with normal cultured ASM (Fig. 11.13b) (Brightling et al. 2005a). The relevance of this to mast cell recruitment by the asthmatic ASM in vivo is further demonstrated by the increased expression of CXCL10 by the ASM in bronchial biopsies from asthmatic compared with normal subjects (Fig. 11.13c) (Brightling et al. 2005a). Other chemoattractants may also contribute to the ASM mast cell myositis. SCF (c-kit ligand) is produced by both ASM and mast cells and is both a chemoattractant and an essential survival factor for mast cells (Iemura et al. 1994; Nilsson et al. 1994; Kassel et al. 1999). In addition, TGF-β is another mast cell chemoattractant released by ASM following exposure to tryptase, providing a mechanism by which mast cells might contribute to further mast cell recruitment via autocrine pathways (Berger et al. 2003). However, there appears to be an added level of complexity in the regulation of mast cell migration by ASM in that normal ASM in vitro also appears to secrete an inhibitor of HLMC migration (Sutcliffe et al. 2006).

Mast cell adhesion to airway smooth muscle Cell–cell adhesion is a fundamental mechanism by which cells communicate, facilitating the targeting of specific signals. It is also important for the retention of cells at particular locations. Resting nonactivated HLMC adhere avidly to resting ASM cells in culture (Yang et al. 2006a). This alone is interesting because T cell and eosinophils, which are not found in the ASM bundles in asthma, adhere poorly unless activated (Lazaar et al. 1994; Hughes et al. 2000). This suggests that adhesion may be important for retaining HLMC within the ASM bundles. This adhesive process is mediated partly via an undefined Ca2+-dependent pathway, and partly through a Ca2+-independent pathway that utilizes a molecule known as TSLC-1 (tumor suppressor in lung cancer 1, also known as SgIGSF, IGSF4, RA175, Necl2, SynCAM). TSLC-1 is highly expressed by HLMC and mediates HLMC adhesion to ASM through a heterophilic mechanism (Yang et al. 2006a). TSLC-1 also mediates the adhesion of mouse mast cells to fibroblasts and nerves (Ito et al. 2003; Furuno et al. 2005; Koma et al. 2005), and may therefore represent a particularly important molecule facilitating mast cell interactions with various cell types.

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% of human lung mast cells expressing chemokine receptors

50 P < 0.05

40

30

20

10

CC C C R1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CC R9 R1 0 CX C CX R1 CXCR2 CXCR3 CXCR4 CXCR5 CX CR 6 3C R1

0 (a)

Fig. 11.13 (a) Chemokine receptor expression on ex vivo human lung mast cells. (b) Mast cell chemotaxis to conditioned media from cytokine-activated normal and asthmatic airway smooth muscle (ASM) cultures and its inhibition by chemokine receptor blockade. (c) CXCL10 protein expression in asthmatic but normal airway smooth muscle in situ. (a) from Brightling et al. 2005b, (b) and (c) from Brightling et al. 2005a, with permission. (See CD-ROM for color version.)

Fold difference in migration compared with control

6

*P < 0.05 compared with control ***P < 0.01 compared with control **P < 0.05 compared with asthmatic ASM alone

5

Normal

***

4

3

2

**

**

** *

1

(b)

CXCR3 CCR3 CXCR1 CXCR4 Normal ASM blocking antibody Supernatant Asthmatic ASM Supernatant

Biological effects of mast cell mediators on airway smooth muscle Immunohistochemical and electron microscopical analyses suggest that mast cells in the asthmatic ASM form intimate contact with ASM cells and are present in an activated state, with evidence of ongoing mediator release (Begueret et al. 2007). The effects of mast cell mediators on ASM are therefore likely to be profound. The classical mast cell autacoid mediators histamine, PGD2 and LTC4 are all potent agonists for ASM contraction. Exogenously administered tryptase induces bronchoconstriction and the development of BHR in dogs and sheep (Sekizawa et al. 1989), and tryptase can potentiate the contractile response of sensitized bronchi to histamine in vitro (Berger et al. 1999). In addition to its ability to stimulate cytokine release from ASM, tryptase can also act as a potent ASM mitogen in vitro (Berger et al. 2001; Brown et al. 2002). The precise mechanism whereby tryptase may interact with these cells is unclear because although several studies have suggested the need for an intact catalytic site

(c)

Asthma

(Brown et al. 1995; Berger et al. 2001, 2003), there is a report that nonproteolytic actions may be involved in mitogenesis (Brown et al. 2002). The recently identified transmembrane form of tryptase, but not the secreted form of tryptase (which is about 50% identical), also induces BHR in mice, an effect dependent on expression of the IL-4Rα subunit and STAT6 (Wong et al. 2002). Transmembrane tryptase increases expression of IL-13 in the airways of these mice and also stimulates IL-13 release from T cells, so it would be of great interest to know if it has a similar effect on mast cells, leading to autocrine amplification of mast cell cytokine release and the subsequent development of BHR. Chymase is a mast cell neutral protease that has been less extensively studied than tryptase, but is expressed by those mast cells infiltrating the ASM in asthma (Brightling et al. 2002a). Interestingly, chymase degrades human ASM pericellular matrix and inhibits T-cell adhesion to ASM, which might explain the paucity of T cells within this structure in asthma (Lazaar et al. 2002). However, unlike tryptase,

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chymase inhibits human ASM proliferation. The effects of the joint expression of these proteases and other mast cell mediators in vivo therefore remain unclear, but it is noteworthy that ASM proliferation in asthma in situ has never been observed (Benayoun et al. 2003; Woodruff et al. 2004). It has therefore been proposed that the ASM hyperplasia in asthma may be due to the recruitment of ASM precursors known as fibrocytes from the peripheral circulation (Kaur et al. 2006). In support of this, both ASM and myofibroblasts in tissue express the chemokine receptor CCR7, while one of the relevant ligands, CCL19, is expressed both by the ASM and by mast cells within the ASM bundles (Kaur et al. 2006). This suggests that mast cells and ASM could recruit ASM progenitors to the ASM bundle, a supposition supported by the ability of these cells to induce CCL19-dependent ASM migration in vitro (Kaur et al. 2006). Potential interactions between ASM and infiltrating mast cells are summarized in Fig. 11.9. IL-4 and IL-13 are believed to be key in the development of BHR. This is supported by an in vivo study in mice where instillation of Th2 cell-conditioned medium to the airways of naive mice induced BHR within 6 hours. This required expression of the IL-4Rα subunit and STAT6, suggesting a critical role for IL-4 and/or IL-13, and both of these cytokines produced similar effects when administered individually (Venkayya et al. 2002). IL-4 and IL-13 also enhance the magnitiude of agonist-induced intracellular Ca2+ responses in cultured human ASM. Since mast cells within the asthmatic ASM express both IL-4 and IL-13 (Brightling et al. 2003b), this may represent a further important pathway by which mast cells could contribute to the development of BHR.

Mast cells, airway epithelial dysfunction, and subepithelial fibrosis The epithelium is fragile and denuded even in mild asthmatics. Often the basal cell layer is left intact, suggesting the point of weakness is between this layer and the surface epithelium (Montefort et al. 1992). Whether this abnormality is primary or secondary is unclear, although evidence suggests the latter may be the case. Mast cells infiltrate the bronchial epithelium in asthma (Pesci et al. 1993; Bradding et al. 1994; Amin et al. 2005). This is of potential importance because it places mast cells at the portal of entry of noxious stimuli such as aeroallergens and viruses, which could facilitate an effector role in the ongoing immunologic response (antigen presentation, Th2 cell differentiation, IgE synthesis). Furthermore, there are likely to be important consequences of mast cell degranulation on epithelial function. For example, mast cells adhere avidly to bronchial epithelial cells (Sanmugalingam et al. 2000; Yang et al. 2006b) and tryptase stimulates airway epithelial cell IL-8 release and upregulates intercellular adhesion molecule (ICAM)-1 expression (Cairns & Walls 1996), a receptor for RV. That mast cell activation can actually damage epithelium is supported by a study investigating the

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mechanisms of ozone-induced epithelial damage in mast cell-deficient mice. This demonstrated that ozone-induced epithelial sloughing is entirely mast cell dependent (Longphre et al. 1996). The mechanisms behind this and the effects of mast cell activation on human airway epithelial function deserve further investigation. A characteristic histologic feature of asthma is thickening of the subbasement membrane due to deposition of type III and V collagen in the lamina reticularis (Roche et al. 1989). The most likely origin for this collagen is proliferating myofibroblasts whose number correlates with collagen thickness (Brewster et al. 1990). Mast cells in or adjacent to the bronchial epithelium also have the potential to activate subepithelial myofibroblasts as there is longstanding evidence that mast cells and fibroblasts interact intimately through several mechanisms. For example, cultured rat embryonic skin fibroblasts phagocytose rat mast cell granules, and this is followed by secretion of collagenase and β-hexosaminidase (Subba Rao et al. 1983). Histamine, TNF-α, bFGF, and IL-4 promote fibroblast proliferation in humans (Boucek & Noble 1973; Sugarman et al. 1985; Jordana et al. 1988; Feghali et al. 1992). IL-4 is a chemoattractant for human fibroblasts (Postlethwaite & Seyer 1991) and also induces human fibroblasts to secrete collagen types I and III and fibronectin (Postlethwaite et al. 1992). bFGF is a potent fibrogenic cytokine (Qu et al. 1995; Reed et al. 1995), and heparin stabilizes bFGF structurally and preserves its bioactivity by protecting it from degradation (Gospodarowicz & Cheng 1986). Furthermore, heparin and/or heparan sulfate are required for binding of bFGF to its receptors (Yayon et al. 1991), and also release it from basement membranes where it is stored (Folkman et al. 1988). Heparin may thus potentiate fibroblast activation and proliferation indirectly through the regulation of bFGF bioactivity. In coculture, human mast cells adhere avidly to human fibroblasts through an as yet undefined mechanism, which does not involve known integrin or cadherin receptors (Trautmann et al. 1997). Both HMC-1 cells and human skin mast cells augment proliferation of human skin fibroblasts, which is dependent on this heterotypic cell–cell contact (Trautmann et al. 1998). This proliferative response was dependent on the expression of mast cell-associated IL-4, although IL-4 could not be detected in the supernatant of the coculture system (but was present in lysates of HMC-1 cells), suggesting that IL-4 was secreted by mast cells in low amounts and strictly limited to cell–cell contacts with fibroblasts. Interestingly, we had suggested previously that mast cells may present IL-4 on their surface in order to confer local cytokine specificity (Bradding et al. 1993). This was based on the observation that in mucosal biopsies from patients with asthma and allergic rhinitis (where mast cells are chronically activated), but not normal subjects, we observed a ring-staining pattern of IL-4 immunoreactivity around mast cells (Bradding et al. 1992, 1993, 1994).

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Mast cell microlocalization within airway submucosal glands Severe mucus plugging is an established feature of severe fatal asthma but is also present in milder disease (Carroll et al. 2002b; Kuyper et al. 2003). This results from mucus hypersecretion by hyperplastic submucosal glands and epithelial goblet cells. Carroll et al. (1993) performed a detailed analysis of cartilaginous airways in postmortem lung specimens from patients with fatal asthma, patients with asthma who died from other causes (nonfatal asthma), and subjects without asthma who died of nonpulmonary causes. Immunohistochemistry for mast cell tryptase revealed a significant increase in the number of mast cells within the mucosal gland stroma in nonfatal asthma, and a marked increase in the number of degranulated mast cells in both fatal and nonfatal asthma compared with normal controls. There were significant correlations between the density of both intact and degranulated mast cells within the mucous glands and the degree of mucus obstruction in the airways. Numerous products released by these activated mast cells have the potential to contribute to both mucous gland hyperplasia and mucus secretion. In terms of the autacoids, their potency for stimulating mucus secretion is in the order LTD4 > LTC4 > prostanoids > histamine (Shelhamer et al. 1980; Marom et al. 1981, 1982). Canine mast cell chymase is also a potent mucus secretagogue when added to cultures of bovine airway glands (Sommerhoff et al. 1989). IL-6 and TNF-α induce mucous glycoprotein secretion and MUC-2 gene expression by both human bronchial organ explant cultures and airway epithelial cells obtained by bronchial brushing (Levine et al. 1994, 1995). IL-6 also induces expression of MUC5B and MUC5AC (Chen et al. 2003). This is relevant because mast cells within the bronchial mucous glands in asthma express IL-6 (Bradding et al. 1994). Animal models also indicate roles for tryptase and IL-13 in mucus hypersecretion (Zhu et al. 1999; Oh et al. 2002). A further molecule of interest and of relevance to both the airway epithelium and mucosal glands is amphiregulin, a member of the epidermal growth factor family. Amphiregullin expression is induced in human progenitor-derived mast cells following activation through FcεRI (Okumura et al. 2005; Wang et al. 2005), an effect which is not suppressed by dexamethasone. Amphiregulin expression by mast cells in the asthmatic bronchial mucosa is increased compared with normal controls, and mast-cell derived amphiregulin increases mucin gene expression in the NCI-H292 epithelial cell line in vitro (Okumura et al. 2005). Thus mast cell-derived amphiregulin may contribute to epithelial goblet cell metaplasia and mucus hypersecretion in asthma, a process refractory to corticosteroids clinically. In addition, recombinant amphiregulin induces the proliferation of human airway fibroblasts but not ASM cells, suggesting a further mechanism whereby mast cells might contribute to subepithelial fibrosis.

Mast cells in animal models of asthma Several models have been developed that aim to induce the airway features of asthma. The most widely reported is the mouse model using intraperitoneal antigen sensitization followed by antigen challenge of the airways. This most closely resembles the model of acute allergen challenge in the airways, although the route of sensitization is obviously different. The dependence on mast cells with regard to the development of airway hyperresponsiveness and inflammatory changes in the airways is highly contingent on the model studied and the mode of antigen sensitization. Thus sensitization without adjuvant generates a mast cell-dependent model, while sensitization with adjuvant creates a mast cell-independent model (Williams & Galli 2000). An alternative model uses airway sensitization without adjuvant from the outset and to some extent is more physiologic. In this setting, mast cells are again an essential component required for the development of airway hyperresponsiveness, inflammation, and remodeling (collagen deposition, goblet cell hyperplasia) (Taube et al. 2004; Yu et al. 2006). However, there are inevitably a number of problems in relating these models to the human disease. For example, mouse airways contain very few mast cells at baseline, so the changes seen following antigen challenge rely heavily on the recruitment of mast cell progenitors rather than the activity of resident cells, and it is perhaps not surprising that subsequent short-term studies using intraperitoneal sensitization did not find a role for mast cells in the outcomes commonly measured. In addition, mice have relatively little smooth muscle in their airways, and so there is no model described to date in mice that has recapitulated the infiltration of ASM by mast cells, a feature that may be key to the development of asthma in humans. So while mouse models are useful for generating hypotheses regarding the pathogenesis of asthma, their findings may also be potentially misleading.

Possible targets for novel therapies in asthma Asthma remains a considerable cause of morbidity and occasionally death, and constitutes a major economic burden: in consequence novel approaches to treatment are urgently required. It is self-evident from the information provided above that successfully inhibiting the release of various mediators from mast cells in asthma could be particularly effective for its treatment. A possible target for treatment has already been tested clinically. The nonanaphylactogenic humanized monomeric antibody omalizumab, which prevents IgE from binding to its high-affinity (FcεRI) and lowaffinity (CD23) receptors by binding to an epitope on the CH3 domain of IgE (Holgate et al. 1998), is partially effective and now licensed for the treatment of asthma in many countries. There are obviously many possible targets, but we discuss two in further detail.

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α as a pivotal Mast cell-derived TNF-α cytokine in asthma

Inhibiting mast cell migration as novel approach to asthma therapy

Several cytokines expressed by mast cells are attractive targets, but targeting IL-4 with soluble receptors or IL-5 with neutralizing monoclonal antibodies has met with little success (Leckie et al. 2000; O’Byrne 2006). In contrast, preliminary evidence suggests that neutralizing TNF-α in severe asthma may be more effective. TNF-α is a proinflammatory cytokine strongly implicated in the pathogenesis of asthma (Bradding et al. 1994). When administered by inhalation to animals and humans it induces both BHR and sputum neutrophilia and exacerbates BHR in patients with asthma (Thomas et al. 1995; Thomas & Heywood 2002). TNF-α immunoreactivity is increased in the airways of mild asthmatics, largely due to increased expression by mucosal mast cells (Bradding et al. 1994). Two studies have demonstrated that TNF-α expression is increased markedly in severe asthma, as shown by increased TNF-α protein in BAL fluid, increased protein expression on peripheral blood mononuclear cells, and both increased protein and a 30-fold increase in mRNA in the bronchial mucosa (Howarth et al. 2005; Berry et al. 2006). This is remarkable because these patients were receiving high-dose inhaled and oral corticosteroid therapy. Interestingly, the increased protein expression in endobronchial biopsies was accounted for by increased numbers of TNF-α-positive mast cells (Howarth et al. 2005). An uncontrolled proof-of-concept study demonstrated that administration of the soluble TNF-α P75 receptor–IgG1Fc fusion protein etanercept (Enbrel) for 12 weeks significantly improved quality of life, lung function, and BHR by 2.5 doubling dilutions of methacholine (Howarth et al. 2005). Interestingly, there was no change in sputum inflammatory cells. A further small, double-blind, placebo-controlled, crossover study administered etanercept for 10 weeks to patients with severe refractory asthma (Berry et al. 2006), leading to marked improvements in quality of life, lung function, and methacholine-induced BHR (3.5 doubling dilution improvement). Again there were no significant changes in sputum inflammatory cells but there was a marked reduction in sputum histamine concentration. This latter observation is compatible with the inhibition of lung mast cell activation. In vitro, TNF-α not only induces skin mast cell histamine and tryptase release (van Overveld et al. 1991) but, following IgE-dependent activation of HLMC, the release of preformed mast cell-associated TNF-α has been shown to serve as a positive autocrine feedback signal to augment NF-κB activation and further production of TNF-α and other cytokines, including GM-CSF and IL-8 (Coward et al. 2002). Taken together, these studies provide strong evidence that mast cell-derived TNF-α plays a major role in the pathophysiology of severe asthma, and that antagonizing TNF-α is useful in this group of patients who are otherwise largely resistant to treatment.

The reason for studying the mechanisms of mast cell migration is that if the relocalization of mast cells in asthmatic airways can be prevented, this may offer a truly novel means of treating the disease. As discussed above, the CXCL10/CXCR3 axis appears important for the recruitment of mast cells by the asthmatic ASM. This therefore represents an interesting target, but other chemoattractants may be involved under various conditions, and may be involved in the recruitment of mast cells to other sites such as the airway mucosal glands. A more generalized approach to inhibiting migration may therefore be more effective than targeting individual chemoattractants. Ion channels are emerging as potential therapeutic targets in inflammatory and structural cells. The intermediate conductance calcium-activated potassium channel KCa3.1 (also known as IKCa1) is expressed by human mast cells and is particularly interesting (Duffy et al. 2001a, 2004; Kaur et al. 2005; Cruse et al. 2006). We have recently demonstrated that blockade of KCa3.1 using the highly specific small-molecule blocker TRAM-34, and the peptide charybdotoxin (derived from scorpion venom), not only markedly attenuated HLMC migration toward CXCL10 and SCF but also stimulated asthmatic ASM supernatants (Fig. 11.14) (Cruse et al. 2006). Furthermore, this channel potentiates human mast cell and T-cell mediator release (Ghanshani et al. 2000; Fanger et al. 2001; Duffy et al. 2004), and may contribute to the pathologic function of other cell types in the airways. Since KCa3.1 can be blocked effectively in both animals and humans without causing undue toxicity (Kohler et al. 2003), blockers of this channel such as TRAM-34 represent a real

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100 90 ASM-induced migration (%)

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10 0 Control

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200 nM

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ChTX

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Fig. 11.14 Inhibition of human lung mast cell migration toward TNF-a-activated airway smooth muscle (ASM) supernatants following KCa3.1 blockade with TRAM-34 and charybdotoxin. *P < 0.05; ** P < 0.01. (From Cruse et al. 2006, with permission.)

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therapeutic possibility for mast cell-mediated inflammatory disease.

Mast cells in allergic rhinitis Allergic rhinitis is characterized by the symptoms of nasal and palatal itch, rhinorrhea, sneezing, nasal blockage and, in severe cases, anosmia. Symptoms may be either seasonal (SAR) due to sensitivity to seasonal allergens such as grass, tree or shrub pollens, or perennial (PAR), often due to sensitivity to house-dust mite. No firm definition of allergic rhinitis exists, and patients often present with symptoms when no identifiable allergy is present (nonallergic rhinitis).

Seasonal and perennial allergic rhinitis Allergic rhinitis is characterized by the presence of a mucosal inflammatory response, similar in many respects to that seen in asthma. In both SAR and PAR there are increased numbers of eosinophils in the lamina propria and epithelium, increased mast cell numbers in the epithelium, and increased expression of Th2 cytokines in mast cells, eosinophils and T lymphocytes (Bentley et al. 1992b; Durham et al. 1992; Bradding et al. 1993, 1995c; Ying et al. 1993, 1994). Increased numbers of CD34+ tryptase-negative cells have been seen in the nasal epithelium, suggesting that there is recruitment of mast cell progenitors to this site (Kawabori et al. 1997). These intraepithelial mast cells are predominantly of the MCT phenotype, whereas those in the lamina propria are about 60% MCTC (Bentley et al. 1992a; Otsuka et al. 1995). Mast cells in the airway epithelium and lamina propria are activated in that there is ultrastructural evidence of mast cell degranulation (Howarth et al. 1991), and increased concentrations of LTC4 but not histamine are present in nasal lavage (Volovitz et al. 1988). In addition, mast cells express IL-4, IL-5, IL-6, and TNF-α in the nasal mucosa and demonstrate increased expression of IL-4, a feature reversed by the application of topical corticosteroids (Bradding et al. 1995c). Although histamine concentrations are not elevated, in contrast to asthma, antihistamine therapy is highly effective at ameliorating symptoms (Bachert et al. 2004). Topical corticosteroids are very effective at reversing the inflammatory changes (Bradding et al. 1995c; Foresi et al. 1996; Holm & Fokkens 2001). Anti-IgE therapy is also effective at treating both SAR and PAR when serum IgE levels are suppressed effectively, supporting the view that this is an IgE-driven disease (Casale et al. 2001; Holgate et al. 2005a,b). Mast cells recovered from the nasal mucosa of patients with allergic rhinitis have been studied in vitro. Interestingly these cells are able to induce B-cell IgE synthesis through the IgE-dependent release of IL-4 and IL-13 and the interaction of CD40 (expressed by B cells) with its ligand (CD40L expressed by mast cells) suggesting that mast cells could

contribute to the local production of IgE in the nasal mucosa (Pawankar et al. 1997). This is relevant because increased numbers of IgE-secreting B cells are present in the allergic nasal mucosa (Coker et al. 2005). It is therefore evident from biological effects of mast cell products (see Tables 11.2 and 11.3) that ongoing mast cell activation in the allergic nasal mucosa can explain much of the symptomatology and pathology of allergic rhinitis.

Experimental allergen-induced rhinitis Nasal allergen challenge has been used extensively to explore the immunopathology of allergic rhinitis and provides further evidence that mast cells are important effectors and orchestrators of the disease. Nearly all patients with a history of allergic rhinitis develop an early-phase response (EPR) following appropriate allergen challenge, characterized by the acute development of rhinitic symptoms. In about 40% of subjects, a late-phase response (LPR) follows after about 6 hours with recurrence of symptoms. During the EPR there is the release of a spectrum of inflammatory mediators, including histamine (Juliusson et al. 1991; Proud et al. 1992), tryptase (Juliusson et al. 1991; Proud et al. 1992), and LTC4 (Wang et al. 1997; Zweiman et al. 1997), supportive of mast cell activation. These symptoms are attenuated by antihistamines (Klementsson et al. 1990) and markedly inhibited by anti-IgE therapy (Hanf et al. 2004). During the LPR there is tissue infiltration by eosinophils and CD4+ T cells. Again this phase is markedly attenuated by anti-IgE therapy (Djukanovic et al. 2004), suggesting that mast cell-driven events at the time of challenge, such as adhesion molecule upregulation, chemoattractant release and enhanced vascular permeability, are largely responsible. A model using low-dose repeat allergen challenge more closely mimics events during the pollen season (Pipkorn et al. 1989). After repeated daily challenges, mast cell numbers start to increase in the nasal epithelium at day 6, indicating that if this involves recruitment and differentiation of progenitors, then this occurs relatively quickly.

Mast cells in allergic conjunctivitis Allergic conjunctivitis commonly accompanies allergic rhinitis and the term “rhinoconjunctivitis” is commonly used. In mild allergic eye disease, patients complain of variable itch, tearing, and swelling that is uncomfortable but does not threaten sight. Chronic forms of the disease can give rise to more severe symptoms including pain, corneal scarring, cataract or glaucoma with the potential to threaten sight. The most common form is seasonal allergic conjunctivitis (SAC), with perennial allergic conjunctivitis (PAC), atopic keratoconjunctivitis (AKC), atopic blepharoconjunctivitis (ABC), and vernal conjunctivitis (VC) less common (for detailed

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review see McGill et al. 1998). Mast cells are recognized as central effector cells in all types of allergic eye disease, demonstrating increased numbers and morphologic evidence of degranulation (Cook et al. 2001; Graziano et al. 2001; Leonardi 2002). Following conjunctival allergen challenge, elevated levels of the mast cell products histamine, tryptase, and LTC4 have also been detected in tears (Margrini et al. 1996; Bacon et al. 2000). The MCTC phenotype predominates in normal conjunctiva but in PAC, SAC, and VC the number of MCT cells increases in both the conjunctival epithelium and subepithelial layers (Irani et al. 1990; Baddeley et al. 1995). In contrast, in AKC and ABC, the numbers of MCTC increase (Yao et al. 2003). It has been proposed that MCTC contribute to tissue fibrosis in various diseases (Gotis-Graham & McNeil 1997; Mitani et al. 1999; Yamada et al. 2001; Andoh et al. 2006), and their increased numbers may therefore contribute to the excess fibrosis evident in AKC and ABC. As in the nose and bronchi (Bradding et al. 1995a), the mast cells in the allergic conjunctiva in SAC demonstrate heterogeneity in terms of cytokine expression, with the MCTC subset expressing predominantly IL-4 and IL-13, while the MCT subset expresses IL-5 and IL-6 (Anderson et al. 2001). Allergen challenge again produces early and late-phase responses, suggesting similar mechanisms to those in the nose and lower airway. Antihistamines are useful for the treatment of symptoms, suggesting that mast cell-derived histamine contributes to the symptomatology (Yanni et al. 1994; Cook et al. 2002; Abelson 2004; Abelson et al. 2004).

assumed to relate to the systemic spread of allergen, but it also needs to be considered that amplification mechanisms might exist, for example neurologic reflexes. The best evidence that there is systemic mast cell activation in anaphylaxis is from studies demonstrating increased concentrations of tryptase, particularly β-tryptase, in the peripheral circulation (Schwartz et al. 1987; Schwartz 2006). Unlike α-tryptase, which is released by mast cells constitutively, β-tryptase is stored in mast cell granules and released following IgE-dependent activation and therefore represents a more specific marker of activation than total tryptase. Histamine and β-tryptase are released from mast cells together, but while histamine concentrations in the plasma peak within 5 min, the peak of tryptase is delayed due to its slower diffusion from the tissue. This is because it is a larger molecule than histamine but also remains bound to heparin longer. Thus serum histamine concentrations peak at about 5 min while those of β-tryptase levels are maximal 15–120 min after the onset of symptoms (Schwartz et al. 1989). Tryptase is therefore not only a more specific marker for mast cell activation than histamine, which is also expressed by basophils, but also more convenient to measure after a suspected anaphylactic event. Interestingly, patients with systemic mastocytosis are at increased risk of anaphylactic and anaphylactoid reactions (Biedermann et al. 1999; Ludolph-Hauser et al. 2001), and often demonstrate increased baseline concentrations of plasma tryptase (predominantly α-tryptase) (Schwartz et al. 1995).

Mast cells in anaphylaxis

Mast cells in atopic dermatitis (eczema) and urticaria

The most striking and immediately life-threatening IgEdependent reaction is that manifesting as anaphylaxis. Food allergies are the most common cause of anaphylaxis, with 1–2% of adults having a food allergy (Jansen et al. 1994; Young et al. 1994) and as many as 8% of children (Bock 1987; Lack et al. 2002). Peanut allergy is the most common cause of fatal food-induced anaphylaxis (Bock et al. 2001), although these statistics vary between countries (Shimamoto & Bock 2002). Anaphylaxis can also result from drug allergies, in particular to penicillin, and insect venom such as bee stings. Reactions clinically indistinguishable from anaphylactic reactions but which are not IgE-dependent are termed “anaphylactoid reactions.” It is generally agreed that anaphylaxis is a syndrome with varied mechanisms and clinical presentations, mediated predominantly by mast cells and basophils. The key difference between anaphylaxis and other mast cell-associated disease is that anaphylaxis involves the systemic activation of mast cells and basophils leading to cardiovascular collapse and respiratory embarrassment due to either bronchospasm or laryngeal edema. This is often fatal if left untreated. The reason for the systemic spread of mast cell activation is often

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While atopic dermatitis is strongly associated with atopy, as indicated by its name, the role of IgE in its pathogenesis is not well defined (Williams & Flohr 2006). In addition, there are no studies examining the effects of omalizumab in atopic dermatitis that might support or refute a pathogenic role for IgE. Unlike the other “allergic” diseases described above, evidence for a role for mast cells in atopic dermatitis is relatively sparse. However, bearing in mind their wide distribution in the skin and the multiple modes they exhibit for sensing the surrounding environment, it seems unlikely that they are not involved in the immunopathology. Studies have shown that the number of the MCT phenotype of mast cells increases in the skin of patients with atopic dermatitis (Irani et al. 1989; Jarvikallio et al. 1997). In addition, skin mast cells demonstrate increased expression of IL-4 in atopic dermatitis, suggesting this may have a pathogenic role. It is evident from the other sections in this chapter that a number of mast cell mediators have the potential to contribute to disease pathogenesis, but evidence for this is currently lacking. Urticaria occurs in acute and chronic forms, with a complex classification and multiple etiologies. This is covered in

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more detail in Chapter 93. In various forms of acute urticaria, antihistamines provide a useful means of treatment and support the view that the skin lesions occur predominantly as a result of mast cell activation. In chronic urticaria, mast cell activation is also a factor, although some patients require treatment with oral steroids to control the disease, indicating a more complex immunopathology. In about 30% of patients with chronic uritcaria, circulating autoantibodies to FcεRI or IgE have been identified, suggesting a pathogenic role involving mast cells (Hide et al. 1993; Niimi et al. 1996; Sabroe et al. 1999, 2002; Sabroe & Greaves 2006). However, these patients do not usually have evidence of mast cell activation elsewhere in the body, which is unexplained, although it has been suggested that these autoantibodies might actually activate skin mast cells through complement-dependent receptors such as the C5a receptor in addition to FcεRI-dependent mechanisms (Ferrer et al. 1999).

Concluding remarks In summary, mast cells are multifaceted tissue-resident cells capable of responding to a variety of noxious stimuli with the secretion of numerous multifunctional autacoids, proteases, and cytokines. Current evidence indicates roles in host defense and repair, as well as many diverse diseases. As evident from this chapter, they play a central role in many aspects of allergic disease, although it needs to be appreciated that their activity in these and other disorders will occur through complex interactions with other immunologic and structural cells. Developing drugs that inhibit pathologic mast cell secretion when administered regularly should improve the treatment of many patients with asthma and related allergic diseases.

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Eosinophils: Biological Properties and Role in Health and Disease Simon P. Hogan, Helene F. Rosenberg, Redwan Moqbel, Simon Phipps, Paul S. Foster, Paige Lacy, A. Barry Kay and Marc E. Rothenberg

Summary Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the emerging properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil-mediated mucosal diseases are conceptualized.

Introduction Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory processes including parasitic helminth, bacterial and viral infections, tissue injury, tumor immunity, and allergic diseases (Gleich & Loegering 1984; Weller 1994; Rothenberg 1998). In response to diverse stimuli, eosinophils are recruited from the circulation into inflammatory foci where they modulate immune responses through an array of mechanisms. Triggering of eosinophils by engagement of receptors for cytokines, immunoglobulins, and complement can lead to the secretion of an array of proinflammatory cytokines, such as interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-16, IL-18, and transforming growth factor (TGF)-α/β, chemokines such as CCL5/RANTES and


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CCL11/eotaxin-1, and lipid mediators such as plateletactivating factor (PAF) and leukotriene (LT)C4 (Kita 1996). These molecules have proinflammatory effects that include upregulation of adhesion systems, modulation of cellular trafficking and activation and regulation of vascular permeability, mucus secretion, and smooth muscle constriction. Eosinophils can initiate antigen-specific immune responses by acting as antigen-presenting cells. Furthermore, eosinophils can serve as major effector cells inducing tissue damage and dysfunction by releasing toxic granule proteins and lipid mediators (Gleich & Adolphson 1986). In this chapter, we summarize eosinophil surface marker expression and the growing number of properties defined for eosinophil degranulation. We review the molecular mechanisms involved in eosinophil development and trafficking, including the role of the transcription factors GATA-1, PU.1, and c/EBP members and the eosinophil selective cytokine IL-5 and the eotaxin subfamily of chemokines. Furthermore, we discuss the views on the role of eosinophils in homeostatic function, including developmental biology and innate and adaptive immunity, and in disease processes including infections, asthma, and gastrointestinal disorders.

Eosinophil granule proteins Eosinophils contain up to four different populations of secretory organelles: crystalloid granules, primary granules, small granules, and secretory vesicles. The largest of the secretory organelles are the crystalloid granules (0.5–0.8 μm in diameter), which store the majority of granule proteins in eosinophils. The unique crystalloid granules are so called because they contain an intensely staining electron-dense crystalline core surrounded by an electron-lucent matrix when cells are stained and imaged by electron microscopy. Most of the granule proteins packaged into the crystalloid granules are composed of four highly basic proteins. Major basic protein (MBP) is crystallized in the core of the crystalloid granule, where it accounts for virtually all the protein (Gleich et al. 1973; Lewis et al. 1978). Eosinophil peroxidase (EPO),

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Eosinophils: Biological Properties and Role in Health and Disease

eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) reside in the granule matrix (Egesten et al. 1986; Peters et al. 1986). The primary granules appear during the promyelocytic stage of eosinophil development and are enriched in Charcot–Leyden crystal (CLC) protein. Small secretory vesicles have also been identified that overlap in their contents with those of small granules, and are packed densely in the cytoplasm of eosinophils. The biology of the major cationic proteins in eosinophils has been reviewed in

detail (Gleich & Adolphson 1986; Walsh 2001); their functions are summarized in Table 12.1.

Table 12.1 Functions of eosinophil cationic granule proteins. MBP Toxicity toward helminthic parasites such as schistosomulae of Schistosoma mansoni (Butterworth 1984; Ackerman et al. 1985; Gleich & Adolphson 1986) Cytotoxicity toward airway epithelium (Frigas et al. 1980; Hastie et al. 1987; Hisamatsu et al. 1990; Furuta et al. 2005) Bronchoconstriction and hyperresponsiveness on aerosolization in rats and monkeys (Gundel et al. 1991; Coyle et al. 1993; Uchida et al. 1993) Platelet agonist (Rohrbach et al. 1990) Activation of complement via classical and alternative pathways (Weiler et al. 1992, 1995) Antibacterial properties (Lehrer et al. 1989) Activation of remodeling factors from epithelial cells (Pégorier et al. 2006) Increased cutaneous vasopermeability (Davis et al. 2003) Stimulation of signaling pathways and mediator release from mast cells, neutrophils and basophils (Zheutlin et al. 1984; Haskell et al. 1995; Page et al. 1999; Shenoy et al. 2003) ECP Ribonuclease activity (100 times less potent than EDN) (Slifman et al. 1986) Toxicity toward helminthic parasites and mammalian epithelial cells (McLaren et al. 1981; Ackerman et al. 1985) Bactericidal properties (Lehrer et al. 1989) Induction of Gordon phenomenon (Durack et al. 1979; Fredens et al. 1982) Promotion of mast cell degranulation (Zheutlin et al. 1984) EDN Weakly toxic for parasites and mammalian cells (Ackerman et al. 1985) Induction of Gordon phenomenon (Durack et al. 1979) Antiviral activity in respiratory infection (Rosenberg & Domachowske 2001) EPO Toxic for mammalian cells and degradative toward connective tissue via ability to form hypohalous acids (Slungaard & Mahoney 1991; Wang & Slungaard 2006) Cytotoxicity toward airway epithelium (Brottman et al. 1996; Pégorier et al. 2006) Bactericidal, membrane lysis, and signaling pathway effects (Wang & Slungaard 2006) Induction of oxidative damage and mutagenesis of DNA and RNA (Shen et al. 2000; Henderson et al. 2001)

Major basic protein As one of the most highly cationic proteins synthesized by eosinophils, MBP is expressed as two different homologs (MBP1 and MBP2). MBP is a small protein that consists of a single polypeptide chain of 117 amino acids, with a molecular mass of 13.8 kDa and a high isoelectric point (>11), which cannot be measured accurately due to its extremely basic nature (Hamann et al. 1991). Its basicity is due to the presence of 17 arginine residues, and it also contains nine cysteine residues that enable it to form disulfide bonds. The cDNA for MBP encodes a pre-prosequence that includes a putative signal peptide and an acidic 90-amino acid prosequence that may serve to neutralize MBP’s highly basic charge as it is processed through the Golgi and transported to the granule, where the prosequence is cleaved (Barker et al. 1988; McGrogan et al. 1988; Popken-Harris et al. 1998). MBP is among the most abundant proteins in eosinophils, with as much as 250 pg/cell detected in guinea-pig eosinophils, while comparatively less is found in human eosinophils (5–10 pg/cell). MBP1 can also be detected in basophil granules, although there is considerably less expressed than in eosinophils (Ackerman et al. 1983). Mature eosinophils lose the ability to transcribe mRNA encoding MBP, indicating that all the MBP stored in crystalloid granules is synthesized during early eosinophil development prior to maturation (Popken-Harris et al. 1998). Plasma concentrations of MBP are elevated in the sera of pregnant women, with a peak 2–3 weeks before parturition. Placental eosinophils are few in numbers, and MBP1 has been shown to be synthesized by placental X cells and placentalsite giant cells (Maddox et al. 1984). MBP2 is exclusively expressed by eosinophils, and may be a more specific marker for elevated eosinophils in patients with eosinophilia than MBP1 (Plager et al. 2006). The classical role of eosinophils in protection against parasitic infections has been supported by the toxicity of MBP against helminthic worms (O’Donnell et al. 1983; Butterworth 1984; Ackerman et al. 1985; Gleich & Adolphson 1986). MBP has also been shown to be cytotoxic to airways and may be at least partly responsible for tissue damage associated with eosinophil infiltration in bronchial mucosa in asthma (Frigas et al. 1980; Hisamatsu et al. 1990; Furuta et al. 2005). The toxic effect of MBP is thought to result from increased membrane permeability through surface charge interactions leading to perturbation of the cell-surface lipid bilayer (Wasmoen et al. 1988).

Eosinophil cationic protein ECP is a member of a subfamily of ribonuclease (RNase)A multigenes expressed in eosinophils, with approximately 15–25 pg synthesized per cell in human eosinophils. Similarly to MBP, ECP is a single-chain cationic polypeptide with

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a pI > 11. On molecular sizing, ECP displays marked heterogeneity, as a result of differential glycosylation, with a molecular mass ranging between 16 and 21.4 kDa. Two isoforms, ECP-1 and ECP-2, have been identified using heparin Sepharose chromatography (Gleich et al. 1986). The cDNA for ECP encodes a leader sequence of 27 amino acids and a mature protein of 133 amino acids with a calculated molecular mass of 15.6 kDa (Barker et al. 1989; Rosenberg et al. 1989). The amino acid sequence is 66% homologous to EDN and 31% homologous to human pancreatic ribonuclease. ECP does have ribonuclease activity but is 100 times less potent than EDN (Slifman et al. 1986). ECP is bactericidal, promotes degranulation from mast cells, and is toxic to helminthic parasites on its own (Gleich et al. 1986; Lehrer et al. 1989). The mechanism of action of ECP is thought to involve pore formation in target membranes, which is apparently not dependent on its RNase activity (Young et al. 1986). ECP was originally characterized for its ability to elicit the Gordon phenomenon (neurotoxicity causing stiffness, ataxia and paralysis) when injected into the cranial ventricles of rabbits (Durack et al. 1979).

Eosinophil-derived neurotoxin A second member of the RNase A multigene family, EDN, also called EPX (Slifman et al. 1986), is expressed in eosinophils and is less basic than ECP or MBP, with a pI of 8.9 due to a smaller number of arginine residues in its sequence. EDN is a single-chain polypeptide with an observed molecular mass of 18.6 kDa. EDN expression is not restricted to eosinophils, as it is also detected in mononuclear cells and possibly neutrophils. ECP and EDN share high sequence homology of 70% at the amino acid level for the pre-form of both proteins, suggesting that these proteins derived from the same gene during evolutionary development (Hamann et al. 1990). Eosinophils express approximately 10 pg of EDN per cell, with marked variation between individuals. EDN similarly induces the Gordon phenomenon when injected intracranially in laboratory animals (Durack et al. 1979). EDN is also implicated in antiviral activity against respiratory infections (Rosenberg & Domachowske 2001). The gene family expressing ECP and EDN has one of the highest rates of mutation in the primate genome, ranking with those of immunoglobulins, T-cell receptors, and major histocompatibility complex (MHC) classes (Rosenberg et al. 1995). This extreme rate of mutation suggests that evolutionary constraints acting on the ECP/EDN subfamily have promoted the acquisition of a specialized antiviral activity, inferred by the high mutation rates of other genes commonly associated with host protection against viral infection.

neutrophil myeloperoxidase, suggesting that a peroxidase multigene family may have developed through gene duplication (Ten et al. 1989; Hamann et al. 1991). Eosinophils store approximately 15 pg/cell of EPO. The functional role of EPO is associated with bacterial killing. EPO catalyzes the peroxidative oxidation of halides (such as bromide, chloride, and iodide) and pseudohalides (thiocyanate) present in the plasma together with hydrogen peroxide generated by dismutation of superoxide produced during respiratory burst (Weiss et al. 1986; Mayeno et al. 1989; Thomas et al. 1995). This reaction leads to the formation of bactericidal hypohalous acids, particularly hypobromous acid, under physiologic conditions. Eosinophils are robust producers of extracellular superoxide due to expression of high levels of the enzyme complex that generates superoxide (NADPH oxidase) (Someya et al. 1997) and preferential assembly of the enzyme complex at the cell surface (Lacy et al. 2003).

Cytokines Eosinophils can synthesize and secrete at least 35 important inflammatory and regulatory cytokines, chemokines, and growth factors (Table 12.2). Many of these cytokines are potent inducers of immune responses in asthma, eczema, rhinitis, and other inflammatory diseases. Those eosinophilderived cytokines that have been quantified generally appear to be generated in relatively small amounts, suggesting an autocrine, paracrine, or juxtacrine role in regulating the function of the microenvironment. However, in some circumstances, eosinophils are the chief producers of cytokines such as TGF-β, which is linked with tissue remodeling in a variety of eosinophil-associated diseases, such as asthma (Kay et al. 2004). A major distinction in cytokine production between eosinophils and T cells, which generate much larger quantities of cytokines, is that eosinophils store their cytokines intracellularly as preformed mediators. Several eosinophil cytokines have been shown to be stored in crystalloid granules and small secretory vesicles, and possess bioactivity on their release (Lacy & Moqbel 2000). This allows the immediate release of cytokines on eosinophil activation, instead of the several hours or days required to generate cytokines from T cells. For example, release of the chemokine RANTES was shown to occur within 60–120 min of eosinophil stimulation by interferon (IFN)-γ. This was related to rapid mobilization (within 10 min) of RANTES in small secretory vesicles that translocated this chemokine to the cell membrane prior to its release (Fig. 12.1). Cytokines generated by eosinophils are discussed in more detail in several comprehensive reviews (Lacy & Moqbel 1997, 2000; Moqbel & Lacy 1998).

Other eosinophil-derived mediators Eosinophil peroxidase EPO is a heme-containing haloperoxidase with a high pI (> 11) composed of two subunits: a heavy chain of 50–57 kDa and a light chain of 11–15 kDa. EPO has 68% sequence identity to

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A major constituent of the human eosinophil is CLC protein, also known as galectin-10 (Ackerman et al. 2002). CLC is a hydrophobic protein of molecular mass 17.4 kDa that was thought to possess a weak lysophospholipase activity,

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Table 12.2 Cytokine generation by eosinophils.

Cytokine

Products

Stored protein in resting cells (/106 cells)

Interleukins Interleukin-1a Interleukin-2 Interleukin-3 Interleukin-4 Interleukin-5

mRNA, protein mRNA, protein mRNA, protein mRNA, protein mRNA, protein

– 6 ± 2 pg – ∼75 ± 20 pg –

mRNA, protein mRNA, protein mRNA, protein mRNA mRNA, protein mRNA, protein mRNA, protein

25 ± 6 pg – ∼25 pg – – – 1.6 ± 0.8 ng

– Crystalloid granules (core) – Crystalloid granules (core) Crystalloid granules (core/matrix?) Crystalloid granules (matrix) – – – – – –

mRNA, protein

–

–

mRNA, protein mRNA, protein mRNA, protein

– – 15.1 ± 0.3 pg

– Crystalloid granules (matrix) Crystalloid granules (core)

mRNA, protein

12 ± 2 pg

–

mRNA, protein mRNA, protein mRNA, protein mRNA, protein mRNA

19 ± 4 pg – 140 pg – –

Crystalloid granules – Cytoplasmic – –

mRNA, protein Protein

– –

– –

mRNA, protein mRNA mRNA mRNA, protein

– – – 72 ± 15 pg

– – – Crystalloid granules (matrix) and small secretory vesicles

mRNA

–

–

mRNA, protein mRNA mRNA, protein mRNA, protein

4 ± 2 pg – – 22 ± 6 pg

mRNA, protein

–

– – Membrane, cytoplasm Crystalloid granules (matrix) and small secretory vesicles –

Interleukin-6 Interleukin-9 Interleukin-10 Interleukin-11 Interleukin-12 Interleukin-13 Interleukin-16 Interleukin-17 Leukemia inhibitory factor Interferons and others Interferon (IFN)-g Tumor necrosis factor a Granulocyte–macrophage colony-stimulating factor Chemokines Epithelial cell-derived neutrophil activating peptide (ENA-78/CXCL5) Eotaxin (CCL11) Growth-related oncogene (GROa/CXCL1) Interleukin-8 (CXCL8) IFN-g-inducible protein (IP-10/CXCL10) IFN-inducible T-cell alpha chemoattractant (I-TAC/CXCL11) Macrophage inflammatory protein 1a Monocyte chemoattractant protein 1 (MCP-1/CCL3) Monokine induced by IFN-g (MIG/CXCL9) MCP-3 (CCL7) MCP-4 (CCL13) RANTES (CCL5)

Growth factors Heparin-binding epidermal growth factor-like binding protein (HB-EGF-LBP) Nerve growth factor Platelet-derived growth factor, B chain Stem cell factor Transforming growth factor a Transforming growth factor b1

Intracellular site of storage

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Fig. 12.1 Human eosinophils stimulated with interferon (IFN)-g to induce rapid release of RANTES. Cells were stained with antibody to major basic protein (MBP) (red) and antibody to RANTES (green) in a time course study of RANTES mobilization. Yellow indicates colocalization of MBP and RANTES to similar granule compartments, which become distinct as early as 10 min following stimulation with IFN-g (500 U/mL). Unstimulated cells (a) were compared with cells stimulated for (b) 5 min, (c) 10 min, (d) 30 min, (e) 60 min, and (f) 16 hours. Original magnification ×1000. (See CD-ROM for color version.)

but instead modulates this by interacting with eosinophil lysophospholipases. It is synthesized at very high levels by eosinophils, and is produced at lesser levels in basophils. CLC possesses strong sequence homology to the carbohydratebinding galectin family of proteins, hence its designation as galectin-10. CLC was first characterized by Charcot and Robin in 1853 for its abundance in sputum and fecal samples from patients with severe respiratory and gastrointestinal eosinophilia. Its release results in the formation of distinct, needle-shaped structures that are colorless, measuring 20– 40 μm in length and 2– 4 μm across. However, the function of CLC remains obscure. In addition, the eosinophil contains a number of other granule-stored enzymes whose exact role in eosinophil function has not been defined (Spry 1988). They include acid phosphatase (large amounts of which have been isolated from eosinophils), collagenase, arylsulfatase B, histaminase, phospholipase D, catalase, nonspecific esterases, and vitamin B12-binding protein. Eosinophils are also a source of matrix metalloproteases, which have an important role in cell transmigration and inflammation (Ohno et al. 1997; Okada et al. 1997; Schwingshackl et al. 1999; Gauthier et al. 2003; Wiehler et al. 2004), although much less is produced than from monocytes, macrophages, and neutrophils. The intracellular location of matrix metalloprotease-9 has been localized to perinuclear regions and not the crystalloid granules (Ohno et al. 1997).

Eosinophils express SNARE isoforms Secretory cells from diverse biological systems express components of a fusion complex of membrane-bound proteins known as the SNARE (SNAP Receptor) complex, which is essential for vesicular docking and fusion (Sollner et al. 1993; Sutton et al. 1998). This complex, originally characterized in

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neuronal cells, is composed of VAMP-1 (vesicle-associated membrane protein, also known as synaptobrevin-1), syntaxin1, and SNAP-25 (synaptosome-associated protein of 25 kDa). These molecules are categorized into two groups, namely vesicular SNAREs (v-SNAREs), which bind to plasma membrane target SNAREs (t-SNAREs). The SNARE molecules form a coiled-coil structure with four separate α-helices contributed by three different molecules during vesicle docking with the plasma membrane. The binding region associated with the four α-helices is known as the SNARE motif. Fusion of the granule membrane with the plasma membrane is dependent on cytosolic NSF (N-ethylmaleimide-sensitive factor) and α, β, or γ-SNAP (soluble NSF-attachment protein)mediated disassembly of the SNARE complex (Sollner et al. 1993). Cleavage of SNAREs can occur via clostridial neurotoxins containing zinc endopeptidase activity, particularly tetanus toxin and botulinum toxin serotypes (BoNT/A, B, C, D, E, F, and G). These toxins have been used to characterize the dependency of secretion on SNARE complex formation. Many SNARE proteins have also been identified in nonneuronal secretory cells including syntaxin-4 and SNAP-23 (Ravichandran et al. 1996), while VAMP-2 expression is distributed between neuronal and nonneuronal tissues (Rossetto et al. 1996). In addition, VAMP-4 (Steegmaier et al. 1999), VAMP-5 (Zeng et al. 1998), the tetanus toxin-insensitive proteins VAMP-7 (formerly known as tetanus toxin-insensitive VAMP or TI-VAMP) (Galli et al. 1998; Advani et al. 1999; Hibi et al. 2000; Ward et al. 2000) and VAMP-8 have been characterized in nonneuronal tissues (Mullock et al. 2000; Paumet et al. 2000; Polgar et al. 2002). Eosinophils express VAMP-2, VAMP-7, VAMP-8, syntaxin4, and SNAP-23 (Feng et al. 2001; Lacy et al. 2001; Logan et al. 2002, 2003, 2006), whereas they do not contain detectable levels of the classical neuronal SNARE proteins (syntaxin-1,

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(a)


(b)

Fig. 12.2 Translocation of VAMP-2 during interferon (IFN)-g stimulation of human eosinophils. Immunolabeling for VAMP-2 (green) showed moderate colocalization with RANTES (red) in (a) unstimulated eosinophils, but these were strongly colocalized at the cell membrane (arrow) following 5 min of stimulation with IFN-g 500 U/mL (b). Original magnification ×1000. (See CD-ROM for color version.)

SNAP-25, and VAMP-1) (Lacy et al. 1995). Eosinophil VAMP-2 is expressed in a population of small secretory vesicles that store the chemokine RANTES, which translocates to the cell membrane during IFN-γ stimulation (Fig. 12.2) (Lacy et al. 1999, 2001). Immunofluorescence staining showed that syntaxin-4 and SNAP-23 were localized to the cell membrane in eosinophils, where they may function as cognate intracellular receptors for VAMP-2 (Fig. 12.3) (Logan et al. 2002). Inhibition of VAMP-2 binding led to the loss of IgE-induced ECP release in permeabilized eosinophils (Hoffmann et al. 2001). A novel isoform of VAMP, known as tetanus-insensitive VAMP (TI-VAMP, also known as VAMP-7), is a putative vesicular SNARE isoform for regulation of lysosomal fusion (Advani et al. 1999; Ward et al. 2000; Rao et al. 2004). VAMP7 and VAMP-8 are abundantly expressed in eosinophil crystalloid granules. VAMP-7 has been shown to be required for

Fig. 12.3 Localization of SNAP-23 and syntaxin-4 in eosinophils. (Left panel ) Distribution of SNAP-23 at cell membranes (arrow) as well as at an intracellular site that colocalizes with the Golgi apparatus (arrowhead). (Right panel) Antibody to syntaxin-4 shows expression in the cell membrane (arrow) and endoplasmic reticulum (arrowhead). Lower panels indicate differential interference contrast images. Original magnification ×630. (See CD-ROM for color version.)

exocytosis of crystalloid granule as well as small secretory vesicles (Logan et al. 2006). This finding suggests that VAMP2 and VAMP-7 may play overlapping roles in the release of small secretory vesicles from eosinophils, although their release may be more dependent on VAMP-7 (Fig. 12.4). In

Resting

Stimulated

Plasma membrane VAMP-7

Fig. 12.4 Scheme showing SNARE-dependent exocytotic pathways for crystalloid granules and secretory vesicles in eosinophils. (See CD-ROM for color version.)

Syntaxin-4

SNAP-23 VAMP-2 or VAMP-7

SNARE complex

EPO, EDN, RANTES

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summary, SNARE isoforms may play a crucial role in the regulation of granule fusion in eosinophils.

Eosinophil surface markers The first characterization of eosinophil surface molecules demonstrated that eosinophils express a large number of cell-surface markers including adhesion molecules, apoptotic signaling molecules, chemokine, complement and chemotactic factor receptors, cytokine receptors, and immunoglobulin receptors (Gupta et al. 1976; Ebisawa et al. 1995). Since these studies and the discovery of new immune receptors (Toll-like receptors, inhibitory receptors and Siglecs) and development of new reagents, the list has been extended revealing that eosinophils express an array of surface structures that were previously thought to be exclusively expressed by other cell types (Tachimoto & Bochner 2000; Rothenberg & Hogan 2006).

Adhesion molecules Transmigration of the eosinophil through the vascular endothelium is a multistep process involving rolling, tethering, firm adhesion, and transendothelial migration (Wardlaw et al. 1994; Wardlaw 2000). The initial steps of eosinophil rolling and tethering are regulated by selectins and their counterligands expressed on the endothelium (Ebnet et al. 1996; Wardlaw 1999). Eosinophils have been shown to constitutively express L-selectin, which regulates eosinophil rolling on the endothelium in vivo (Georas et al. 1992; Sriramarao et al. 1994). Ligands for L-selectin include CD34 and MAdCAM-1 which are expressed endothelium (Berg et al. 1993). Eosinophils also express CD162 (P-selectin glycoprotein ligand-1 or PSGL-1) and sialyl-Lewis X (CD15s), which interact with Eselectin and P-selectins and regulate eosinophil tethering to endothelium (Symon et al. 1996). The firm adhesion of the eosinophil and transmigration across the vascular epithelium into tissues is regulated by coordinated interaction between networks involving chemokine and cytokine signaling, eosinophil adhesion molecules (e.g., selectins and integrins), and integrin receptors such as vascular cell adhesion molecule (VCAM)-1, mucosal addressin cell adhesion molecule (MAdCAM)-1 and intercellular adhesion molecule (ICAM)-1 expressed on vascular endothelial cells (Kunkel & Butcher 2002; Hogan et al. 2004). Integrins are heterodimeric surface molecules consisting of an α and β chain and eosinophils express members of the β1 (α4β1 and α6β1), β2 (αLβ2, αMβ2, αXβ2, and αDβ2) and β7 (α4β7) integrin families (Georas et al. 1993; Grayson et al. 1998; Tachimoto & Bochner 2000; Bochner & Schleimer 2001; Tachimoto et al. 2002). These various integrin molecules selectively interact with adhesion receptors (VCAM-1, MAdCAM-1, ICAM-1, -2 and -3, and fibrinogen) expressed on the vascular endothelium. The specific interaction of cell-surface integrins with ad-

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hesion receptors (VCAM-1, MAdCAM-1, ICAM-1, ICAM-2, ICAM-3, and fibrinogen) facilitates eosinophil migration into various tissue compartments during inflammation. For example, eosinophil recruitment to the site of allergic inflammation in the lung and skin is regulated by VLA-4 (α4β1 integrin)/VCAM-1-dependent processes (Weg et al. 1993; Abraham et al. 1994; Nakajima et al. 1994; Pretolani et al. 1994; Gonzalo et al. 1996). Pretreatment of mice with neutralizing monoclonal antibodies against α4 or β1 integrin or genetic deletion of VCAM-1 attenuates eosinophil accumulation in the lung during allergic airways disease (Weg et al. 1993; Abraham et al. 1994; Nakajima et al. 1994; Pretolani et al. 1994; Gonzalo et al. 1996). Recent experimental studies have demonstrated that eosinophil recruitment into different tissue compartments (gastrointestinal tract) is regulated by differential adhesion pathways. For example, eotaxin-1dependent eosinophil recruitment to the small intestine is MAdCAM-1/α4β7 integrin dependent (Mishra et al. 2002), whereas eosinophil accumulation in the colon is regulated by a β2 integrin pathway (ICAM-1) and can occur independently of α4 and β7 integrin pathways (Forbes et al. 2006).

Chemokine, complement and other chemotactic factor receptors Experimental investigations have shown eosinophils to constitutively express the chemokine receptors CCR3 and CCR1 (Ponath et al. 1996; Phillips et al. 2003; Elsner et al. 2005). Consistent with this observation, eosinophils respond to CCR1 and CCR3 ligands including macrophage inflammatory protein (MIP)-1α/CCL3, RANTES/CCL5, macrophage chemotactic protein (MCP)-2/CCL8, MCP-3/CCL7 and MCP-4/CCL-13, eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26, and mucosa-associated epithelial chemokine (MEC)/CCL28. Eosinophils have also been shown to express a number of other chemokine receptors including CXCR3, CXCR4, CCR5, CCR6, and CCR8 following activation by IL-5 (Sullivan et al. 1999; Nagase et al. 2000; Oliveira et al. 2002). While chemokines are thought primarily to regulate the migration pattern of eosinophils, they have also been shown to promote eosinophil activation and function (Zimmermann et al. 2003). For example, RANTES/CCL5 and eotaxin-1/CCL11 have been shown to promote cellular activation and modulate respiratory burst in eosinophils (Elsner et al. 1997, 1999).

Cytokine receptors Three cytokines, IL-3, IL-5, and granulocyte–macrophage colony-stimulating factor (GM-CSF), are particularly important in regulating eosinophil development, and eosinophils have been shown to express the specific cytokine receptor subunit for IL-3 (IL-3Rα, CD123), IL-5 (IL-5Rα, CD125) and GM-CSF (GM-CSFRα, CD116) as well as the shared β chain (CD131) (Lopez et al. 1986, 1988; Rothenberg et al. 1988; Takatsu et al. 1994). Functional studies have demonstrated that cytokines including stem cell factor (SCF), IFN-γ, tumor

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necrosis factor (TNF)-α, IL-4, and IL-9 activate eosinophil functions, suggesting that eosinophils express the c-kit receptor (CD117), IFN-γR α-chain (CDw119), TNF-α receptor types 1 and 2 (CD120a, CD120b), type 1 IL-4 receptor [IL-4R α-chain (CD124) and the common γ chain (CD132)], and the IL-9 receptor [IL-9R α-chain (CD129)/CD132] (Wallen et al. 1991; Yuan et al. 1997; Dubois et al. 1998; Nutku et al. 1999; Hauber et al. 2004). Consistent with these obervations, both the type 1 (CD120a) and type 2 (CD120b) TNF receptors have been identified on human eosinophils by fluorescence-activated cells sorting analysis and immune electron microscopy. Activation of these receptors are thought to promote eosinophil apoptosis (Zeck-Kapp et al. 1994; Zeck-Kapp & Kapp 1995). While eosinophils have been shown to express a number of IFN receptor superfamily members, including receptors for IFN-α, IFN-β, IFN-γ, and IL-10 (Giembycz & Lindsay 1999), only the receptor for IFN-γ (type 2 IFN) has been convincingly identified (Aldebert et al. 1996; Ishihara et al. 1997; Matsuyama et al. 1998; Ochiai et al. 1999). The IL-2 receptor is composed of three polypeptide chains, an α chain (p55) (CD25), β chain (p75) (CD122), and a γ chain (CD132) that is common to several other cytokine receptors. Experimental investigations have demonstrated that IL-2 induces eosinophil chemotaxis, suggesting that the cognate receptor for the cytokine was expressed on the eosinophils. This was confirmed by the observation that the IL-2-mediated effects could be blocked by antibodies against IL-2R α chain (p55) and β chain (p75) (Rand et al. 1991).

phils and immature eosinophil progenitors express CysLT1R, whereas CysLT2R has only been identified on mature eosinophils. Expression of these receptors has been shown to be upregulated on eosinophils from asthmatics during excerbations (Fujii et al. 2005). Notably, CysLT2R expression on eosinophils was selectively greater in nonatopic asthmatics (Fujii et al. 2005). The function of these receptors on eosinophils has not yet been fully defined. However, leukotrienes (LTB4, LTD4, LTE4), PAF, and 5-oxo-6,8,11,14-eicosatetraenoic acid induce eosinophil recruitment, suggesting that they may regulate eosinophil transmigration (Powell et al. 1995; Bandeira-Melo et al. 2000; Ohshima et al. 2002; Shiraishi et al. 2005). Previous reports have also demonstrated that in vitro suppression of cysteinyl leukotriene activity by a CysLT1R antagonist blocks eosinophil differentiation and/or maturation, suggesting that cysteinyl leukotrienes may play a role in eosinophil lineage commitment and maturation (Thivierge et al. 2000). Cysteinyl leukotrienes have also been shown to promote eosinophil release of cytokines including IL-4 (Bandeira-Melo et al. 2002a,b). Eosinophils have also been shown to express high levels of the histamine H4 receptor that mediate eosinophil chemoattraction and activation in vitro (O’Reilly et al. 2002).

Complement receptors Initial studies suggested that eosinophils express complement receptors for C3a and C5a (Daffern et al. 1995; DiScipio et al. 1999); however, more recent studies have revealed that eosinophils also express CR1 (CD35), CR3 (CD11b/CD18), CR4 (CD11c), CD103, and receptors for C1q (Walsh et al. 1990; Giembycz & Lindsay 1999). CR1 is recognized by the complement fragments C3b, C4b, iC3b, and C1q. The expression of CR1 on eosinophils is regulated by certain stimuli, including LTB4, 5-HETE, and 5-HPETE (Fischer et al. 1986). CR3 has also been shown to be expressed on eosinophils: CR3 interacts with a number of ligands including iC3b and ICAM-1, all of which could activate eosinophils resulting in eosinophil priming and degranulation (Koenderman et al. 1991).

Prostaglandin and leukotriene receptors Clinical and experimental studies have demonstrated that eosinophils express both cysteinyl leukotriene receptors (CysLT1R and CysLT2R), high-affinity prostaglandin (PG)D2 type 2 receptor, and the PAF receptor (Wang et al. 1999; Fujii et al. 2005; Zinchuk et al. 2005). Interestingly, the PGE2 receptor is also expressed by basophils and Th2 cells (and is now designated “chemoattractant receptor Th2 cells” or CRTH2) and appears to co-mediate Th2 cell and eosinophil/ basophil recruitment (Hirai et al. 2001). Both mature eosino-

Immunoglobulin receptors Eosinophils express Fc receptors for IgA, IgD, IgG, and IgM (Giembycz & Lindsay 1999). CD32 (FcγRII) is constitutively expressed on resting human eosinophils (Hartnell et al. 1990) and is upregulated by IFN-γ (Hartnell et al. 1992). These receptors not only function as IgG receptors but also appear to have a role in stimulating eosinophil survival, degranulation, and generation of leukotrienes (Cromwell et al. 1988, 1990; Kita et al. 1991; Kim et al. 1999). Eosinophils do not constitutively express FcγRI (CD64) or low-affinity FcγRIII (CD16), although expression can be upregulated by cytokines, IFN-γ, complement (C5a), and PAF (Hartnell et al. 1992). Eosinophils do appear to express IgA receptors (CD89) (Monteiro et al. 1993) and exvivo studies have demonstrated that eosinophil degranulation can be induced by IgA-coated particles, suggesting that IgA–receptor interaction induces eosinophil degranulation (Abu Ghazaleh et al. 1989). The expression or presence of the low-affinity IgE receptor (CD23) or the high-affinity IgE receptor on eosinophils remains controversial (Kita & Gleich 1997). Some studies suggest that eosinophils bind to IgE (Capron et al. 1985, 1995), although more recent investigations suggest that eosinophils express little if any α or β chains for the high-affinity receptor or the low-affinity CD23 IgE receptor (Ying et al. 1998; Kita et al. 1999; Seminario et al. 1999).

Other cell-surface structures Eosinophil apoptosis has been shown to be induced by two surface structures, CD95 (first) and CD69 (Matsumoto et al. 1995; Walsh et al. 1996). Eosinophils also express CD9,

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CD37, CD52, CD63, CD81, CD82, and CD151 (Ebisawa et al. 1995). Experimental investigations have demonstrated that eosinophils can express antigen to naive CD4+ T cells and promote T-cell proliferation and polarization (Shi et al. 2000, 2004; Shi 2004). Consistent with this observation, eosinophils have also been shown to express MHC class II and the necessary costimulatory signals for T-cell activation and proliferation, including the type 1 interval surface membrane glycoprotein CD40 as well as CD80 and CD86 (Ohkawara et al. 1996; Woerly et al. 1999). The expression of CD80 and CD86 appears to be independently regulated by IL-3 and GM-CSF (Tamura et al. 1996). Human eosinophils have also been shown to express the MHC class II protein human leukocyte antigen (HLA)-DR (Shi 2004). Interestingly, peripheral eosinophils of most normal eosinophilic donors do not express HLA-DR proteins, although sputum eosinophils and bronchoalveolar lavage (BAL) eosinophils from asthmatics have been shown to express HLA-DR (Hansel et al. 1991; Sedgwick et al. 1992). Notably, levels of HLA-DR expression on BAL eosinophils from allergic subjects following segmental challenge were elevated compared with peripheral blood eosinophils, suggesting that recruitment and activation of eosinophils following allergen challenge promotes HLA-DR expression (Sedgwick et al. 1992). HLA-DR expression on eosinophils has been shown to be regulated by IL-3, IL-4, GM-CSF, and IFN-γ (Lucey et al. 1989; Weller et al. 1993).

Inhibitory receptors The CD2 subfamily of the IgE superfamily includes CD2, CD48 (BLAST1) and BTM1 (CD58), LFA3, CD84, IL-9b, CD150, CD229, and 2B4 (CD244). Recent studies have demonstrated that eosinophils express the CD2 subfamily of receptors, namely CD48 and 2B4 (CD244) (Munitz et al. 2005). CD48 is a glycosylphosphatidylinositol (GPI)-anchored protein involved in cellular activation, costimulation, and adhesion. CD48 expression is elevated on human eosinophils from atopic asthmatics and is upregulated by IL-3 (Munitz et al. 2006). Cross-linking of CD48 on eosinophils triggers eosinophil degranulation (Munitz et al. 2005, 2006). Notably, CD48 is the high-affinity ligand for 2B4. Eosinophils also express inhibitory receptor IRp60/CD300a, p140, Siglec-8, Siglec-10, ILT5/LIR3, CD33, and p75/adhesion inhibitory receptor molecules (Munitz & Levi-Schaffer 2007). IRp60 activation has been shown to be involved in the suppression of eosinophil activation (Munitz et al. 2005). Interestingly, CCR3 has been shown to induce negative signaling in murine eosinophils following receptor engagement with the Th1 chemokine CXCL9 (MIg) (Fulkerson et al. 2005).

Toll-like receptors Eosinophils express mRNA for a number of Toll-like receptors (TLR) including TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 (Plotz et al. 2001; Sabroe et al. 2002; Nagase et al. 2003). The level of TLR expression on eosinophils is low

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relative to other granulocytes such as neutrophils, except for relatively elevated levels of TLR7/TLR8 (Nagase et al. 2003). The natural ligands for TLR7/TLR8 are currently not clear, although significant evidence suggests that guanosine- and uridine-rch ssRNA are physiologic ligands for these TLRs (Heil et al. 2004). Functional analysis using TLR-specific ligands reveals that TLR7/TLR8 ligands (R-848) induced eosinophil activation (superoxide production) and prolonged eosinophil survival. The expression of TLR7/TLR8 has been shown to be regulated by cytokines including IFN-γ (Nagase et al. 2003).

Eosinophils and immune regulation In recent years, eosinophils have been shown to possess the ability to perform numerous immune functions, including antigen presentation (Shi et al. 2000; MacKenzie et al. 2001) and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators (Gleich & Adolphson 1986; Weller 1994).

Thymic eosinophils Eosinophils transmigrate into the thymus during the neonatal period, reaching maximum levels by 2 weeks of age. Interestingly, their absolute levels are approximately equivalent to that of thymic dendritic cells (Throsby et al. 2000). Eosinophils primarily localize to the corticomedullary region of the thymus and reach basal levels by 28 days of age. Subsequently, an increase in thymic eosinophil levels at 16 weeks of age corresponds to the commencement of thymic involution. Notably, eosinophils at this stage localize to the medullary region. Thymic eosinophils express high levels of MHC class II molecules and moderate levels of MHC class I and the costimulatory molecules CD86 (B7.2) and CD30L (CD153) (Fig. 12.5). Furthermore, thymic eosinophils are CD11b/CD11c double-positive and appear to be activated, as they lose expression of GL-1 and CD62L and upregulate CD25 and CD69 surface expression. Analysis of thymic eosinophil cytokine production reveals that eosinophils express mRNA for the proinflammatory cytokines TNF-α, TGF-β, IL-1α and IL-6 and the Th2 cytokines IL-4 and IL-13 (Throsby et al. 2000). Notably, the recruitment of eosinophils into the thymus is regulated by eotaxin-1, which is constitutively expressed in the thymus (Matthews et al. 1998). It has been postulated that eosinophils are associated with MHC class I-restricted thymocyte deletion. Consistent with this notion, the biphasic recruitment of eosinophils and their anatomic localization within discrete compartments of the thymus coincides with negative selection of double-positive thymocytes (Throsby et al. 2000). Employing an experimental model of acute negative selection, increased thymic eosinophil levels have been demonstrated in MHC class I-restricted female H-Y T-cell receptor transgenic mice

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Immunoglobulin receptors and members of the immunoglobulin superfamily CD4 CD16 CD32 CD33 CD47 CD48 CD50 CD54

CD58 CD66 CD89 CD100 CD101 HLA class I HLA-DR FceRI

Chemokine, complement and other chemotactic factor receptors CD35 CD88 CD182 CD183 CD191 CD192 CD193 CD196

Enzymes

Cytokine receptors CD25 CD116 CD117 CD119 CD120 CD123 CD124

PAFR LTB4R C3aR CystLT1R CystLT2R fMLPR CRTH2 Histamine 4R

CD13 CD45 CD45RB CD45RO CD46

CD125 CD131 CD213 IL-9R IL-13Ra1 TGF-bR

CD55 CD59 CD87 PAR-2

Apoptosis, signaling and others Adhesion molecules Fig. 12.5 Schematic diagram showing surface molecules expressed by human eosinophils. Molecules have been listed based on convincing evidence for their expression as assessed by flow cytometry or inferred by cellular responsiveness to specific stimuli. Cluster designation (CD) for particular molecules is indicated based on the most recent classification (www.ncbi.nlm.nih.gov/prow/). (See CD-ROM for color version.)

CD11a CD11b CD11c CD15 CD18 CD29 CD44 CD49d

following cognate peptide injection. In addition, eosinophils are associated with clusters of apoptotic bodies, suggesting eosinophil-mediated MHC class I-restricted thymocyte deletion. Thymic eosinophils have the capacity to promote thymocyte apoptosis as they express costimulatory molecules involved in clonal deletions, such as CD30L (CD153) and CD66 (Throsby et al. 2000). Additionally, eosinophils may induce thymocyte apoptosis via free radicals, as thymic eosinophils express high levels of NADPH oxidase activity; notably, developing thymocytes have increased sensitivity to free radicals due to downregulation of Cu2+/Zn2+ superoxide dismutase.

Antigen presentation Recent clinical and experimental investigations have shown that eosinophils can function as antigen-presenting cells (Fig. 12.6). Eosinophils are capable of processing and presenting a variety of microbial, viral, and parasitic antigens, as well as superantigens (Shi 2004). GM-CSF-treated eosinophils promote T-cell proliferation in response to staphylococcal superantigen (Staphylococcus enterotoxins A, B and E) stimulation (Mawhorter et al. 1994). Furthermore, eosinophils incubated with human rhinovirus-16 promote rhinovirus-16-specific

CD49f CD62L CD156 CD162 CD174 ad integrin b7 integrin

CD9 CD17 CD24 CD28 CD37 CD39 CD43 CD48 CD52 CD53 CD63 CD65 CD69

CD71 CD76 CD81 CD82 CD86 CD92 CD95 CD97 CD97 CD98 CD99 CD137 CD139

CD148 CD149 CD151 CD153 CD161 CD165 Siglec-8 Siglec-10 LIR1 LIR2 LIR3 LIR7 TLR7 TLR8

T-cell proliferation and IFN-γ secretion (Handzel et al. 1998). Eosinophils can also effectively present soluble antigens to CD4+ T cells, thereby promoting T-cell proliferation and polarization. Adoptive transfer of antigen-pulsed eosinophils results in eosinophil-dependent T-cell proliferation (MacKenzie et al. 2001). Furthermore, addition of antigen to eosinophil and T-cell cocultures promotes heightened T-cell proliferative responses (Shi et al. 2000). The capacity of eosinophils to present antigen has been debated in some publications. It is interesting to note that the failure of eosinophils to present antigen may be related to the methods used for isolating eosinophils. For example, lysis of erythrocytes with ammonium chloride, an inhibitor of lysosome acidification (needed for antigen presentation), negatively correlates with eosinophil antigen presentation activity (Shi et al. 2000; van Rijt et al. 2003). Eosinophils secrete an array of cytokines capable of promoting T-cell proliferation, and activation of Th1 or Th2 polarization (IL-2, IL-4, IL-6, IL-10 and IL-12) (Kita 1996; Lacy & Moqbel 2000; Shi et al. 2000; MacKenzie et al. 2001) (Fig. 12.6 and Table 12.2). Recent attention has been drawn to the ability of murine eosinophils to produce IL-4. Employing mice with enhanced green fluorescent protein (GFP) in the

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Inflammatory Cells and Mediators Cytokines IL-2, IL-3, IL-4, IL-5 IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, TGF-a/b GM-CSF, TNF-a/b, IFN-g and IL-12

Stimuli Tissue injury Viral and bacterial infections Allergens Allografts Tumors

Growth Factors Heparin-binding epidermal growth factor-like binding protein (HB-EGF-LBP) NGF PDGF VEGF SCF

TLR FcR/Ig

Lipid Mediators Leukotrienes-LTD4, LTE4 'Prostaglandins-PGE1, PGE2 15-HETE Platelet-activating factor

IDO KYN IL-4

Neuromediators Substance P NGF VIP Chemokines CCL3/MIP-1a, CCL5/RANTES CCL7/MCP-3, CCL8/MCP-2 CCL11/eotaxin-1, CCL13/MCP-4 IL-8

Tc

ell

MBP Activation

Antigen Presentation MHC-II CD80/CD86

IL-5

Cytotoxic secretory products EPO MBP ECP Ribonucleases EDN Others MMP-9

Mast cell

Fig. 12.6 Schematic diagram of an eosinophil and its multifunctional effects. Eosinophils are bilobed granulocytes with eosinophilic staining secondary granules. The secondary granules contain four primary cationic proteins designated eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil derived neurotoxin (EDN). All four proteins are cytotoxic molecules; in addition, ECP and EDN are ribonucleases. Eosinophils respond to diverse stimuli including nonspecific tissue injury, viral infections, allografts, allergens, and tumors. In addition to releasing their preformed cationic proteins, eosinophils can also release a variety of cytokines, chemokines, lipid mediators, and neuromodulators. Eosinophils directly communicate with T cells and mast cells in a bidirectional manner. Eosinophils activate T cells by serving as

antigen-presenting cells and eosinophil-derived MBP is a mast cell secretagogue. Eosinophils can also regulate T-cell polarization through synthesis of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in oxidative metabolism of tryptophan, catalyzing the conversion of tryptophan to kynurenines (KYN), a regulator of Th1/Th2 balance. 15-HETE, 15-hydroxyeicosatetraenoic acid; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NGF, nerve growth factor; PDGF, platelet-derived growth factor; RANTES, regulated on activation normal T-cell expressed and secreted; SCF, stem cell factor; VEGF, vascular endothelial growth factor; VIP, vasoactive intestinal polypeptide. See text for definition of other abbreviations. (Adapted from Rothenberg & Hogan 2006, with permission.) (See CD-ROM for color version.)

IL-4 gene locus (4get mice), eosinophils appear to be a primary source of IL-4 following parasitic infection or anti-IgD treatment (a strong Th2 stimulator). Notably, while the IL-4 gene locus is transcriptionally active in eosinophils, the amount of IL-4 protein production appears to be lower than in T cells and basophils (Shinkai et al. 2002; Khodoun et al. 2004; Voehringer et al. 2004). Furthermore, murine eosinophils promote IL-4, IL-5, and IL-13 secretion by CD4+ T cells (MacKenzie et al. 2001). Eosinophils can also regulate T-cell polarization through their synthesis of indoleamine 2,3-dioxygenase, an enzyme involved in oxidative metabolism of tryptophan, converting tryptophan to kynurenines. Kynurenines regulate Th1 and Th2 imbalance by promoting Th1 cell apoptosis (Odemuyiwa

et al. 2004). The eosinophil-mediated T-cell proliferative and cytokine secretion responses are dependent on costimulation. Indeed, blockade of CD80, CD86, and CTLA-4 by neutralizing antibodies inhibits eosinophil-elicited T-cell proliferation and cytokine secretion (Shi 2004). Fluorescent labeling studies have revealed that eosinophils instilled into the trachea of mice traffic into the draining peritracheal lymph nodes and localize to the T-cell-rich paracortical regions (B-cell zones) within 24 hours (Shi et al. 2000). Employing models of allergic airway disease and gastrointestinal allergy, investigators have demonstrated that inhalation of antigen promotes eosinophil homing to the draining endotracheal lymph nodes and Peyer’s patches (Korsgren

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et al. 1997; Mishra et al. 2000; Hogan et al. 2001; MacKenzie et al. 2001). Interestingly, a recent investigation suggests that eosinophils can only promote proliferation of effector T cells but not naive T cells (van Rijt et al. 2003). Moreover, eosinophils pulsed with ovalbumin peptide and cocultured with ovalbuminspecific T-cell receptor transgenic T cells (D011.10 T cells) induced effector T-cell proliferation; however, when cocultured with naive CD4+ T cells, no T-cell proliferation was observed. It is tempting to speculate that eosinophils traffic to draining lymph nodes in order to recruit activated effector T cells and promote proliferation of effector T cells.

with shared properties of basophils and eosinophils, and then into a separate eosinophil lineage (Boyce et al. 1995). Initial studies examining the the eos 47 gene (encoding EOS47, the avian ortholog of the mammalian melanotransferrin gene), a gene specifically expressed by bone marrow eosinophils, revealed that a 309-bp promoter region consisting of binding sites for Myb-, Ets-, c/EBP-, and GATA-type transcription factors were responsible for governing lineage-specific expression (McNagny et al. 1998). More recent investigations have supported these initial observations, demonstrating that eosinophil lineage specification is dictated by the interplay of at least three classes of transcription factors including GATA-1 (a zinc finger family member), PU.1 (an Ets family member), and c/EBP members (CCAAT/enhancer-binding protein family) (Nerlov & Graf 1998; Nerlov et al. 1998; McNagny & Graf 2002). Interestingly, these three transcription factors are expressed in a variety of hematopoietic lineages, although their synergistic mechanism of action in eosinophils promotes lineage specificity. The expression level of PU.1 specifies distinct cell lineage fates, with low levels specifying lymphocytic and high levels myeloid differentiation (De Koter & Singh 2000; Du et al. 2002). In most cell types, GATA-1 and PU.1 antagonize each other, but have synergistic activity in regulating eosinophil lineage specification (and eosinophil granule protein transcription) (Du et al. 2002). The specificity of these factors for eosinophils is conserved across species, for example c/EBP factors and GATA-1 drive differentiation of chicken progenitor cells into eosinophils (McNagny & Graf 2002). Of these transcription factors, GATA-1 is clearly the most important for eosinophil lineage specification as revealed by loss of the eosinophil lineage in mice harboring a targeted deletion of the high-affinity GATA-binding site in the GATA-1 promoter (Yu et al. 2002), and based on eosinophil differentiation experiments in vitro (Hirasawa et al. 2002). In particular, the specific activity of GATA-1 in eosinophils but not other GATA-1-positive lineages (mast cells, megakaryocytes, erythroid cells) appears to be mediated by a high-affinity palindromic (or “double”) GATA site (Du et al. 2002). This double GATA site is present in the downstream GATA-1 promoter and also in the regulatory regions of eosinophil-specific genes, including the eotaxin receptor CCR3, MBP, and the IL-5Rα gene, and accounts for eosinophil-specific gene expression (Zimmermann et al. 2000a; Du et al. 2002; Yu et al. 2002). For example, the tandem double GATA site in the human MBP-P2 promoter is required for both promoter activity in human eosinophil cell lines and for synergistic transactivation by GATA-1 and PU.1 (Du et al. 2002). Previous studies have identified cis-acting sequences (cis elements) as important regulators of GATA-1 expression, particularly a 3-cis-acting sequence known as upstream enhancer HS1/G1HE (HS1) as a major enhancer of GATA-1 expression (McDevitt et al. 1997; Onodera et al. 1997). However, studies have demonstrated that HS1 deletion in mice does not affect eosinophil

Mast cell regulation A substantial body of literature has emerged demonstrating that eosinophils have the capacity to regulate mast cell function (see Fig. 12.6). Notably, human umbilical cord bloodderived mast cells can be activated by MBP to release histamine, PGD2, GM-CSF, TNF-α, and IL-8 (Piliponsky et al. 2002). The activation of mast cells by MBP elicits not only exocytosis but also eicosanoid generation and cytokine production, both of which are prominent responses following FcεRI-dependent activation of mast cells (Piliponsky et al. 2002). Incubation of rat peritoneal mast cells with native MBP, EPO, and ECP (but not EDN) results in concentrationdependent histamine release (Zheutlin et al. 1984). Several studies have shown that MBP induces mast cell activation via a similar pathway to that observed with other polybasic compounds such as substance P, compound 48/80, and bradykinin (Piliponsky et al. 2001). Freshly isolated human lung mast cells are resistant to IgE-independent activation; however, highly purified lung mast cells cocultured with human lung fibroblasts are sensitive to IgE-independent activation by MBP (Piliponsky et al. 2002). Interestingly, activation of eosinophils with the mast cell protease chymase promotes production of eosinophil-derived stem cell factor, a critical mast cell growth factor. Eosinophils also produce nerve growth factor (NGF) (Solomon et al. 1998), a cytokine not only involved in survival and functional maintenance of sympathetic neurons but also in immune regulation. For example, NGF promotes mast cell survival and activation (Horigome et al. 1994; Bullock & Johnson 1996). NGF is preformed in eosinophils and acts in an autocrine fashion by activating release of EPO (Solomon et al. 1998). EPO activates rat peritoneal muscles to release histamine, suggesting a role of eosinophil-derived NGF in mast cell-eosinophil interactions. Thus, eosinophils and mast cells communicate in a bidirectional fashion.

Eosinophil development Eosinophils are produced in the bone marrow from pluripotent stem cells, which first differentiate into a hybrid precursor

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GATA-1 mRNA expression and eosinophil differentiation (Guyot et al. 2004). A number of the c/EBP members have also been implicated in the regulation of eosinophil lineage commitment (Nerlov et al. 1998). Phenotypic characterization of c/EBPα-deficient mice revealed that these mice are devoid of eosinophils (Zhang et al. 1997). This is consistent with studies demonstrating eosinophil formation from cord blood progenitors by enforced expression of c/EBPα (Iwama et al. 2002). Collectively these studies suggest that coexpression of GATA-1 and c/EBPα are required for efficient eosinophil formation (Hirasawa et al. 2002; Iwama et al. 2002; McNagny & Graf 2002). More recent investigations have demontrated that expression of eosinophil granule proteins is also regulated by c/EBPε and PU.1. Eosinophils from c/EBPε–/– mice have an abnormal phenotype (Yamanaka et al. 1997). Notably, in these mice neutrophil secondary granule gene expression is severely impaired, suggesting that c/EBPε may be involved in granule gene expression (Gombart et al. 2003). c/EBPε and GATA-1 proteins have been shown to weakly induce MBP expression; however, addition of PU.1 dramatically upregulates endogenous MBP expression, suggesting that PU.1 may regulate eosinophil-specific gene expression (Gombart et al. 2003). Consistent with these observations, MBP and EPX gene expression was attenuated in myeloid cell lines derived from PU.1–/– mice. Furthermore, PU.1 has been shown to be involved in the expression of other eosinophil-specific genes including EDN (van Dijk et al. 1998). Granulocytes are generated from a small number of hematopoietic stem cells and this process is highly regulated and maintained at a constant level under steady-state conditions (Metcalf 1991; Tenen et al. 1997; Zhu & Emerson 2002). However, under conditions of inflammation or cytokine stimulation, termed “emergency granulopoiesis,” the hematopoietic system greatly amplifies granulocyte formation. This process has been shown to be regulated by many cytokines (G-CSF, GM-CSF and IL-3). A recent study suggests that c/EBP factors (c/EBPα and c/EBPβ) also play an important role in the regulation of different aspects of steady-state and inflammatory stimuli-induced (emergency) granulopoiesis (Kincade 2006). Employing c/EBPβ-deficient mice, investigators have demonstrated normal steady-state granulopoiesis in the absence of c/EBPβ, suggesting that c/EBPα is sufficient for steady-state granulopoiesis. This has been confirmed by the demonstration of complete loss of granulocytes in c/EBPα-deficient mice (Zhang et al. 1997). Notably, emergency granulopoiesis induced by cytokine stimulation was ablated in c/EBPβ-deficient mice (Hirai et al. 2006), suggesting that c/EBPβ selectively regulates emergency granulopoiesis. In support of this hypothesis, cytokine treatment induced c/EBPβ but not c/EBPα or c/EBPε transcripts in granulocyte progenitors. Furthermore, granulocytes can be generated from c/EBPα–/– progenitors following cytokine stimulation in vivo (Hirai et al. 2006). These studies suggest

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that c/EBPα regulates steady-state and c/EBPβ emergency granulopoiesis (Hirai et al. 2006; Kincade 2006). Three cytokines, IL-3, IL-5, and GM-CSF, are particularly important in regulating eosinophil development (Lopez et al. 1986, 1988; Rothenberg et al. 1988; Takatsu et al. 1994). These eosinophilopoietins likely provide permissive proliferative and differentiation signals following the instructive signals specified by the transcription factors GATA-1, PU.1, and c/EBPs. These cytokines are encoded by closely linked genes on chromosome 5q31. They bind to receptors that share a common β chain and have unique α chains (Vadas et al. 1994). Of these three cytokines, IL-5 is the most specific to the eosinophil lineage and is responsible for selective differentiation of eosinophils (Sanderson 1992). IL-5 also stimulates the release of eosinophils from the bone marrow into the peripheral circulation (Collins et al. 1995). The critical role of IL-5 in the production of eosinophils is best demonstrated by genetic manipulation of mice. Overproduction of IL-5 in transgenic mice results in profound eosinophilia (Dent et al. 1990; Tominaga et al. 1991; Lee et al. 1997; Mishra et al. 2002) and deletion of the IL-5 gene causes a marked reduction of eosinophils in the blood and lungs after allergen challenge (Foster et al. 1996; Kopf et al. 1996). The overproduction of one or a combination of these three cytokines occurs in humans with eosinophilia, and diseases with selective eosinophilia are often accompanied by overproduction of IL-5 (Owen et al. 1989). The critical role of IL-5 in regulating eosinophils in humans has been demonstrated by several clinical trials with humanized anti-IL-5 antibody; this currently unapproved drug dramatically lowers eosinophil levels in the blood and, to a lesser extent, in the inflamed lung (Leckie et al. 2000; Flood-Page et al. 2003a; Kips et al. 2003).

Eosinophil trafficking Chemokine regulation of eosinophil and CD4+ T-cell trafficking Under baseline conditions, most eosinophils traffic into the gastrointestinal tract where they normally reside within the lamina propria of all segments except the esophagus (Mishra et al. 1999). The gastrointestinal eosinophil is the predominant population of eosinophils. Under baseline conditions, eosinophil levels in the gastrointestinal tract occur independent of lymphocytes and enteric flora, indicating unique regulation compared with other leukocytes (Mishra et al. 1999). Indeed, the recruitment of gastrointestinal eosinophils is regulated by the constitutive expression of eotaxin-1, as demonstrated by the marked decrease of this population of eosinophils in eotaxin-1-deficient mice. The importance of eotaxin-1 in regulating the baseline level of eosinophils is reinforced by the observation that mice with targeted deletion of CCR3 (but not eotaxin-2-deficient mice) also have a deficiency in gastrointestinal eosinophils (Humbles et al.

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2002; Pope et al. 2005a). In addition eosinophils homing into the gastrointestinal tract, thymus, mammary gland and uterus, under homeostatic conditions has been shown to be dependent on eotaxin-1 (Gouon-Evans et al. 2000; Rothenberg et al. 2001a). Notably, trafficking into the uterus is regulated by estrogen, as eosinophil and eotaxin-1 levels cycle with estrus (Gouon-Evans & Pollard 2001). The trafficking of eosinophils into inflammatory sites has been shown to involve a number of cytokines (most notably the Th2 cell products IL-4, IL-5, and IL-13) (Sher et al. 1990a; Moser et al. 1992; Horie et al. 1997), adhesion molecules (e.g., β1, β2, and β7 integrins) (Bochner & Schleimer 1994), chemokines such as RANTES and the eotaxins (Zimmermann et al. 2003), and other recently identified molecules (e.g., acidic mammalian chitinase) (Zhu et al. 2004) (Fig. 12.7). Of the cytokines implicated in modulating leukocyte recruitment, only IL-5 and the eotaxins selectively regulate eosinophil trafficking (Rankin et al. 2000). IL-5 regulates growth, differentiation, activation, and survival of eosinophils and has been shown to provide an essential signal for the expansion and mobilization of eosinophils from the bone marrow into the lung following allergen exposure (Collins et al. 1995). However, antigen-induced tissue eosinophilia can occur independent of IL-5, as demonstrated by residual tissue eosinophils in trials using anti-IL-5 in patients with asthma (Flood-Page et al.

2003a), and in IL-5-deficient mice (Foster et al. 1996; Hogan et al. 1997). Recent studies have demonstrated an important role for the eotaxin subfamily of chemokines in eosinophil recruitment to the lung (Zimmermann et al. 2003). Eotaxin was initially discovered using a biological assay in guinea pigs designed to identify the molecules responsible for allergen-induced eosinophil accumulation in the lungs (Jose et al. 1994; Rothenberg et al. 1995; Rankin et al. 2000). Subsequently, utilizing genomic analyses, two additional chemokine genes have been identified in the human genome that encode CC chemokines with eosinophil-selective chemoattractant activity, and have hence been designated eotaxin-2 and eotaxin-3 (Zimmermann et al. 2003). Eotaxin2 and eotaxin-3 are only distantly related to eotaxin-1 since they are only about 30% identical in sequence and are located in a different chromosomal position (Shinkai et al. 1999; Zimmermann et al. 2000b). The specific activity of all eotaxins is mediated by selective expression of the seventransmembrane spanning, G-protein-coupled receptor CCR3, primarily expressed on eosinophils (Murphy 1994; Daugherty et al. 1996; Ponath et al. 1996). Notably, the eotaxin chemokines cooperate with IL-5 in the induction of tissue eosinophilia. IL-5 increases the pool of eotaxin-responsive cells and primes eosinophils to respond to CCR3 ligands (Zimmermann et al. 2003). Furthermore, when given exogenously, eotaxins

Eosinophil differentiation Transcription factors: GATA-1, PU.1 and c/EBP members (a, b and e) Cytokines: IL-5, IL-3 and GM-CSF Bone marrow

IL-5

Blood Selectins L-selectin PSGL-1 Sialyl-Lewis X Selectins P-selectin E-selectin

Eosinophil adhesion Integrins b1-a4, a6 b2-aL, aM, aD, aX b7-a4 Adhesion receptors ICAM-1 VCAM-1 MAdCAM-1

Eosinophil chemotaxis Chemokine receptors CCR1, CCR3, CCR5, CCR6, CCR8, CXCR3 and CXCR4

Chemokines CCL3, CCL5, CCL8, CCL7, CCL-11, CCL13, CCL24, CCL26 and CCL28

Tissue

Fig. 12.7 Schematic representation of eosinophil trafficking. Eosinophils develop in the bone marrow where they differentiate from hematopoietic progenitor cells into mature eosinophils under the control of the critical transcription factors GATA-1, PU.1 and c/EBP members. The eosinophilopoietins IL-3, IL-5, and GM-CSF regulate eosinophil expansion, especially in conditions of hypereosinophilia. Eosinophil migration out of the bone marrow into the circulation is primarily regulated by IL-5.

Circulating eosinophils subsequently interact with the endothelium by processes involving rolling, adhesion, and diapedesis. Depending on the target organ, eosinophils cross the endothelium into tissues by a regulated process involving coordinated interaction between networks involving the chemokines, eosinophil adhesion molecules, and adhesion receptors on the endothelium. (Adapted from Rothenberg & Hogan 2006, with permission.) (See CD-ROM for color version.)

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cooperate with IL-5 to induce substantial production of IL-13 in the lung (Zimmermann et al. 2003). The finding that IL-4 and IL-13 are potent inducers of the eotaxin chemokines by a STAT6-dependent pathway provides an integrated mechanism to explain the eosinophilia associated with Th2 responses (Zimmermann et al. 2003). Recent studies have identified that eosinophil recruitment to the lung is dependent on STAT6 and a bone marrow-derived lung tissue resident nonT or B cell (Voehringer et al. 2004); in particular, eotaxin-2 production by airway macrophages likely accounts for this (Pope et al. 2005a,b). Of further interest, recently CCR3 has been shown to also deliver a powerful negative signal in eosinophils, depending on the ligand engaged. For example, pretreatment with the chemokine Mig inhibits eosinophil responses by a CCR3- and Rac2-dependent mechanism (Fulkerson et al. 2005). Utilizing eotaxin-1 and eotaxin-2 single and double genedeficient mice or neutralizing antibodies, both chemokines have been shown to have nonoverlapping roles in regulating the temporal and regional distribution of eosinophils in an allergic inflammatory site (Rothenberg et al. 1997; Gonzalo et al. 1998; Pope et al. 2005a). Utilizing a standard experimental asthma model induced by systemic sensitization with ovalbumin/alum followed by respiratory ovalbumin challenge, only a modest reduction in lung eosinophils was found in CCR3-deficient mice (Humbles et al. 2002). However, when the same CCR3-deficient mouse line was subjected to experimental asthma induction by epicutaneous ovalbumin sensitization, there was a marked deficiency of lung and BAL eosinophils (Ma et al. 2002). It was proposed that these apparently conflicting results may be related to the sensitization protocol (Ma et al. 2002), but the reason for this apparent discrepancy remains unclear. Notably, another CCR3-deficient mouse strain has recently been shown to have a profound reduction in eosinophil recruitment to the lung in the standard ovalbumin/alum systemic sensitization model (Pope et al. 2005b). There is now substantial preclinical evidence supporting a role for the eotaxin chemokines in human allergic disease (Zimmermann et al. 2003). Experimental induction of cutaneous and pulmonary late-phase responses in humans has revealed that the eotaxin chemokines are produced by tissue-resident cells (e.g., respiratory epithelial cells and skin fibroblasts) and allergen-induced infiltrative cells (e.g., macrophages and eosinophils). Following allergen challenge in the human lung, eotaxin-1 is induced early (6 hours) and correlates with early eosinophil recruitment; in contrast, eotaxin-2 correlates with eosinophil accumulation at 24 hours (Zimmermann et al. 2003). In another study, eotaxin-1 and eotaxin-2 mRNA were increased in patients with asthma compared with normal controls; however, there was no further increase following allergen challenge (Zimmermann et al. 2003). In contrast, eotaxin-3 mRNA was dramatically enhanced 24 hours after allergen challenge (Zimmermann et al.

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2003). The chemoattractant activity of BAL fluid from patients with asthma is inhibited by antibodies against RANTES, MCP-3, MCP-4, and eotaxin-1 (Zimmermann et al. 2003). Further support for an important role of eotaxin-1 in human asthma is derived from analysis of a single nucleotide polymorphism (SNP) in the eotaxin-1 gene. A naturally occurring mutation encoding a change in the last amino acid in the signal peptide (alanine→threonine) results in less effective cellular secretion of eotaxin-1 in vitro and in vivo (Nakamura et al. 2001). Notably, this SNP is associated with reduced levels of circulating eotaxin-1 and eosinophils and improved lung function (e.g., forced expiratory volume in 1 s or FEV1) (Nakamura et al. 2001). Furthermore, an SNP in the eotaxin-3 gene is associated with atopy in a Korean population and eosinophilic esophagitis in a white population (Chae et al. 2005; Blanchard et al. 2006). Recently, the activity of eotaxin-1 and eotaxin-2 in humans has been investigated by injection of these chemokines into the skin; both eotaxin-1 and eotaxin-2 induce an immediate wheal and flare response associated with mast cell degranulation and subsequent infiltrations by eosinophils, basophils, and neutrophils (MenziesGow et al. 2002). The infiltration by neutrophils is likely to be mediated indirectly by mast cell degranulation. These results provide substantial evidence that the biological activities attributed to eotaxins in animals are conserved in humans.

Role of eosinophils in disease Eosinophils are closely associated with infection by parasitic helminths, as production of Th2 cytokines, specifically IL-5, within cells in infected tissue promotes expansion of progenitor populations in the bone marrow, leading to blood and tissue eosinophilia (Pearce et al. 2004; Wynn et al. 2004; Jankovic et al. 2006). Although in vitro studies suggest that eosinophils can destroy these organisms via secretion of cytotoxic proteins and reactive oxygen species, the results from infection studies carried out in vivo remain unclear and controversial. Eosinophils are also closely associated with the pathogenesis of allergy, specifically in the respiratory tract, with the development of allergic asthma (Bochner & Busse 2005). Symptomatic wheezing and bronchoconstriction associated with asthma exacerbations can result from a superimposed respiratory virus infection, with common inciting agents including the paramyxovirus pathogen respiratory syncytial virus (hRSV) (Singh et al. 2007). At the same time, primary hRSV infection has intriguing associations with the development of childhood asthma (Everard 2006; Martin et al. 2006; Schaller et al. 2006). In connection with these observations, eosinophil recruitment and degranulation has also been observed in response to primary infection with hRSV, an observation that has been explored in human tissues, in culture systems, and in mouse models.

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Infection Eosinophils and parasitic helminth infection Profound blood and tissue eosinophilia are among the hallmark features of parasitic helminth infection, observed in response to activation of CD4+ Th2 lymphocytes at specific stages of the parasite life cycle. As noted earlier, although it seems logical to conclude that eosinophils serve as a means of host defense, there are no “errors of nature” (i.e., human conditions or syndromes characterized by a unique eosinophil deficiency) that might provide direct insight into eosinophil function in helminth-related disease. The only eosinophil-specific condition is hereditary EPO deficiency (Romano et al. 1994), which is detected by laboratory analysis and determined to be unrelated to an increased susceptibility to helminth infection in human studies. Interestingly, most of the human data available suggest a role for eosinophils in preventing reinfection (Hagan et al. 1985; Sturrock et al. 1996), a subject that might be addressed further in rodent infection models (Knopf et al. 1977). At the same time, controlled mouse model studies of primary infection have yielded results that are equivocal and, as such, no specific conclusions regarding the role of eosinophils in promoting host defense can be reached. There are several recent reviews that discuss these data in great depth, and provide significant insight into the ongoing controversy (Behm & Ovington 2000; Meeusen & Balic 2000; Klion & Nutman 2004). The primary points are summarized here. The initial paradigm, that eosinophils might provide host defense against parasitic helminths, came from studies in which activated human eosinophils in the presence of antibody and/or complement, as well as specific eosinophil secretory components alone (e.g., MBP, ECP, EPO), reduced the viability of various helminths in vitro (Butterworth & Franks 1975; Glauert & Butterworth 1977; Hamann et al. 1987). The availability of a monoclonal antibody directed against the eosinophilopoietic cytokine IL-5 permitted studies of the role of eosinophils in host defense against helminth infection to be performed in vivo. Although this reagent resulted in a large-scale reduction in circulating and tissue eosinophilia, there was no evidence for any change in the nature or extent of helminth infection in mouse model studies (Sher et al. 1990a; Herndon & Kayes 1992). Similar conclusions were reached in several studies performed in genetically altered IL-5 transgenic hypereosinophilic mice and in IL-5 and IL-5Rα gene-deleted, eosinophil-deficient mice (Hokibara et al. 1997; Takamoto et al. 1997; Le Goff et al. 2000), as well as more recently in studies performed in the Δdbl-GATA and TgPHIL eosinophil-ablated strains of mice (Swartz et al. 2006). However, among the notable exceptions, eosinophils did appear to play a role in reducing the parasite burden in several studies performed with nematode Strongyloides and Angiostrongylus species. Among these experiments, Korenaga et al. (1991) demonstrated increased recovery of S. venezuelensis worms from lungs of eosinophil-depleted mice treated with anti-IL-5


Table 12.3 Eosinophils and host defense against helminth parasites: results from mouse model studies. (Adapted from Klion & Nutman 2004, with permission.) Organism

Results

Cestodes Mesocestoides corti

−

Trematode Fasciola sp. Schistosoma sp.

− −

Nematodes Angiostrongylus sp. Heligmosomoides polygyrus Brugia sp. Nippostrongylus sp. Onchocerca sp. Strongyloides sp. Toxocara sp. Trichinella sp. Trichuris sp.

+ − + ± + + − ± −

−, Eosinophils shown to play no role; +, eosinophils shown to provide host defense; ±, conflicting information in the literature.

monoclonal antibody, and Sasaki et al. (1993) and Yoshida et al. (1996) demonstrated prolonged survival and increased recovery of A. cantonensis worms from anti-IL-5-treated and IL-5Rα-deficient mice, respectively. Given the unique tissuemigratory phase of Strongyloides and related nematode species, a role for eosinophils in host defense against specific tissueinvading helminths has been suggested (Klion & Nutman 2004). A compilation of the reports documenting the results of mouse model studies that have addressed the role of eosinophils in host defense against helminth pathogens is shown in Table 12.3. There are a number of publications that have focused on the role of eosinophils in promoting tissue pathology. A specific role for eosinophils in promoting pathology has been defined in mouse models of corneal inflammation characteristic onchocercal keratitis (Pearlman et al. 1998). However, in other models, such as those exploring the pathogenesis of Schistosoma mansoni and Nippostrongylus brasiliensis infection in eosinophil-deficient mice, the characteristic liver and lung lesions, respectively, are eosinophil depleted, but pathology remains otherwise unchanged (Coffman et al. 1989; Sher et al. 1990b; Swartz et al. 2006) (Fig. 12.8). There are many reasons why it may be difficult to discern a role for eosinophils in vivo in the mouse experimental system. Among the possibilities, many of these experiments are performed with human pathogens that do not naturally infect rodent species, and thus there is no assurance that one is

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

engaging innate host defense in an evolutionarily meaningful fashion. Related to this point, mouse eosinophils and human eosinophils are not necessarily functionally equivalent (Lee & Lee 2005). While EPO is highly conserved between mouse and human, the eosinophil ribonucleases and MBP are highly divergent. Likewise, mouse and human eosinophils display significant differences in morphology, surface protein expression, and propensity to degranulate in response to physiologic stimuli (Denzler et al. 2001; Clark et al. 2004; Lee & Lee 2005). The inability to observe eosinophils degranulating in response to infectious stimuli in vivo would appear to be a major factor hindering the identification of the role of this cell in host immunity against helminth infection. Different responses observed among various inbred strains of mice likewise add to the overall complexity (Dehlawi & Goyal 2003). Furthermore, it may be simply that we are asking the wrong questions. It remains possible that eosinophils do contribute to host defense, not necessarily by reducing the number of pathogens or eliminating immediate pathologic responses, but instead in a more subtle fashion by contributing to tissue remodeling (De Jesus et al. 2004; Reiman et al. 2006), propensity for reinfection (Knopf et al. 1977; Sturrock et al. 1983), and/or other long-term immunomodulatory sequelae.

Eosinophils and respiratory syncytial virus infection hRSV is a single-stranded negative-sense RNA virus pathogen of the family Paramyxoviridae, subfamily Pneumovirinae that causes respiratory tract infection, primarily among infants and toddlers. The severity of infection can extend from mild upper respiratory symptoms to full-blown bronchiolitis and pneumonia, and may progress to acute respiratory distress syndrome and death, particularly among highly susceptible populations (reviewed in Tripp 2004; DeVincenzo 2005).

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Fig. 12.8 Microscopic pathology of hepatic granulomas of Schistosoma mansoni-infected wild-type and eosinophil-ablated Ddbl-GATA and TgPHIL mice. Giemsa-stained liver tissue sections featuring granulomas from S. mansoni-infected BALB/c (a, b), eosinophilablated Ddbl-GATA (c, d), C57BL/6 (e, f), and eosinophil-ablated TgPHIL (g, h) mice, all at 12 weeks of infection. Arrows indicate examples of eosinophils. Original magnifications: ×10 (a, b, e, f); ×40 (c, d, g, h). (From Swartz et al. 2006, with permission.) (See CD-ROM for color version.)

There is no specific treatment for this infection other than primary support, as neither the antiviral agent ribavirin nor antiinflammatory glucocorticoids have proven impact on the course of disease (Randolph & Wang 1996). A safe and effective human vaccine is not available, although humanized monoclonal antibody directed against the virus fusion (F) protein is approved for treatment of high-risk infants (Cardenas et al. 2005). Several independent groups have detected eosinophils and/or their degranulation products in BAL washings taken from infants undergoing mechanical ventilation secondary to severe hRSV disease (Garofalo et al. 1992; Harrison et al. 1999; Dimova-Yaneva et al. 2004; Kim et al. 2006). The signals promoting eosinophil recruitment and degranulation have not been defined, although several potential eosinophil chemoattractants, including RANTES and MIP-1α have been detected in BAL from hRSV-infected infants (Harrison et al. 1999; Garofalo et al. 2001) (Fig. 12.9). Likewise uncertain is the role of eosinophils in primary hRSV infection, as there is no clear evidence from any human studies as to whether they promote host defense or serve to enhance immunopathology. Among these immunopathologies, there is a clear association between severe hRSV infection, particularly among the youngest infants, and the development of postinfection wheezing and asthma (Everard 2006; Martin et al. 2006; Schaller et al. 2006; Singh et al. 2007), the latter related, among other things, to age-dependent Th2 cytokine-mediated recruitment of proinflammatory eosinophils (Zhao et al. 2002; Kristjansson et al. 2005). Specific observations made in vitro provide potential insight into the role of eosinophils in primary hRSV disease. In correlation with their presence in human BAL washings, hRSVinfected respiratory epithelial cells in culture synthesize both RANTES (CCL5) and MIP-1α (CCL3) (Saito et al. 1997;

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+C 1

2 3

4 5

6 7

8

Eosinophils: Biological Properties and Role in Health and Disease 9 10 11 12 13 14 15 16 17 18 19 20

EDN

ECP

MIP-1a (CCL3; pg/mL/mg protein)

RNase activity (pg/mL/mg protein)

(a)

30 25 20 15 10 5 0

1

3

5

7

9

11

13

15

17

19

(b)

1200 1000 800 600 400 200 0

1

3

5

7

9

11

13

15

17

19

(c)

Fig. 12.9 Immunoreactive eosinophil granule proteins detected in lower airway secretions. (a) Western blots from lower airway secretions obtained from patients with RSV bronchiolitis (lanes 1–10) or unrelated diagnoses (lanes 11–20) were probed with polyclonal anti-EDN or anti-ECP antisera; +C, control: human eosinophil lysate (∼ 1 mg loaded). Detection of (b) ribonuclease activity (units/mL per mg protein) and (c) the proinflammatory chemokine MIP-1a (pg/mL per mg protein) in the same samples. (Adapted from Harrison et al. 1999, with permission.)

Harrison et al. 1999; Miller et al. 2004). In support of a role in host defense, Domachowske et al. (1998) have shown that human eosinophils mediate a dose-dependent reduction in hRSV infectivity, an effect directly dependent on degranulation of its unique secretory ribonucleases. Likewise, Adamko et al. (2001) demonstrated that EPO inhibited replication of the related rodent RNA virus pathogen parainfluenza type I in a similar in vitro assay system. The role of eosinophils in primary infection has also been addressed in the hRSV challenge model in mice. There are some differences between the human and mouse responses to the hRSV pathogen. As hRSV is a human pathogen, it undergoes little or no overt replication in mouse lung tissue and disease does not progress beyond a limited inflammatory state even in response to relatively large viral inocula. It is also important to recognize that most of the information relating to eosinophil recruitment in mice is based on studies of “enhanced disease,” which is an IL-5-mediated allergic response to formalin-fixed virus and virion components (Piedra 2003; Openshaw & Tregoning 2005) and not directly related to primary hRSV infection per se. However, in a recent study, Phipps et al. (2007) demonstrated accelerated clearance of hRSV after primary challenge in IL-5 transgenic hypereosinophilic mice, an effect that is directly dependent on signaling through TLR7. Interestingly, eosinophil recruitment in the primary hRSV challenge model appears to be a function

of age, with increasing numbers of eosinophils observed on infection of younger (neonatal) mice (Culley et al. 2002); neonatal challenge is also accompanied by enhanced recruitment of Th2 cells and progression to an allergen-responsive phenotype (You et al. 2006; Barends et al. 2004), similar to what has been observed in hRSV-infected human neonates. The role of eosinophils in promoting host defense against natural rodent paramyxovirus pathogens has been explored in some detail. In a study directed toward understanding airway hyperresponsiveness, Adamko et al. (1999) identified an eosinophil-dependent reduction in titers of rodent parainfluenza I virus in lungs of mice subjected to ovalbumin sensitization and challenge. Likewise, eosinophil recruitment has been observed as an early response to infection with pneumonia virus of mice (PVM), the cognate rodent pathogen most closely related to hRSV (Domachowske et al. 2000a; Easton et al. 2007). Eosinophil recruitment in response to PVM is not dependent on IL-5, but is blunted (along with recruitment of neutrophils) in mice devoid of the chemokine MIP-1α or its receptor CCR1 (Domachowske et al. 2000b). Preliminary studies suggest that, similar to studies perfomed with hRSV (Phipps et al. 2007), higher titers of virus are detected in lung tissue of the eosinophil-ablated Δdbl-GATA mice (Foster et al. 2007), although protection from the characteristic severe disease state is known to be related to factors other than absolute virus titer (Rosenberg et al. 2005).

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In summary, eosinophils are recruited and degranulate in lung tissue in response to hRSV infection. Experiments performed in vitro and in several different mouse models suggest mechanisms underlying eosinophil recruitment, and provide evidence consistent with a role for these cells in promoting virus clearance.

Asthma Asthma phenotypes Asthma is a heterogeneous disease with several clinical subtypes and a wide spectrum, ranging from mild, episodic, wheezy breathlessness to chronic, intractable, corticosteroiddependent chronic airway narrowing (Bel 2004). The classical IgE-associated allergic asthma phenotype starting in childhood is the most widely studied, not least because this form of the disease can be provoked in the clinical laboratory under controlled conditions by inhalation of allergen or allergenderived T-cell peptides. In these patients, airway cells have a predominant Th2 cytokine profile (i.e., IL-4+, IL-5+, IL-9+ and IL-13+ mucosal cells). Some asthmatics have late-onset nonallergic (so-called “intrinsic”) asthma in which sensitivity to allergens cannot be identified but in which airway eosinophilia and Th2 cells are also prominent (Humbert et al. 1999). The characteristic features of most asthma phenotypes, including allergic asthma, are airway inflammation, airway hyperresponsiveness (AHR), excessive airway mucus production due to goblet cell hyperplasia, and thickness of the airway wall. This airway thickness, often referred to as remodeling, is consequent to excessive repair processes following repeated airway injury and involves an increase in airway smooth muscle mass, deposition of collagen and other matrix proteins, and new blood vessel formation (Bousquet et al. 1990).

Association with eosinophils Increases in eosinophils in the tissues, blood, and bone marrow are a hallmark of most asthma phenotypes and, in general, elevated numbers correlate with disease severity (although “noneosinophilic/nonneutrophilic” asthma is characteristic of bacterial, viral, and pollutant triggers (Douwes et al. 2002)). This has led to the hypothesis that the eosinophil is the central effector cell responsible for ongoing airway inflammation. Thus, the cell has the potential to cause damage to the airway mucosa and associated nerves through the release of granule-associated basic proteins (which damage nerves and epithelial cells), lipid mediators (which cause bronchoconstriction and mucus hypersecretion), and reactive oxygen species (which generally injure mucosal cells). The inflammatory milieu promotes the survival of eosinophils by the elaboration of agents that delay apoptosis. These include epithelial-derived GM-CSF and neurotrophins (e.g., NGF and brain-derived neurotrophic factor) (Hahn et al. 2006). Eosinophils are also highly sensitive to Fas-mediated apoptosis. In a mouse model of asthma, Fas-positive T cells

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were found to regulate the resolution of airway inflammation since Fas deficiency on T cells produced long-term allergic airways disease (Tong et al. 2006). Blood eosinophils from patients with asthma have a number of phenotypic alterations, particularly in relation to their adhesive properties. Thus airway eosinophils recovered after antigen challenge have enhanced adhesion to VCAM-1 (CD106) and other ligands including albumin, ICAM-1 (CD54), fibrinogen, and vitronectin. These hyperadhesive properties seem to be mediated by upregulated and activated αMβ2 (CD11b/18) (Barthel et al. 2006). Asthmatic eosinophils also have increased expression of collagen receptors α1β1 and α2β1 integrins (Bazan-Socha et al. 2006). More attention is now given to a possible role for the eosinophil in repair and remodeling processes since there is a well-documented association of tissue eosinophilia and eosinophil degranulation with certain fibrotic syndromes and the cell is the source of several fibrogenic and growth factors, including TGF-α, TGF-β, fibroblast growth factor (FGF)-2, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-9, IL-1β, IL-13, and IL-17 (see Fig. 12.6).

Eosinophils and animal models of asthma Over the years animal models of asthma have often given conflicting results, especially those involving short-term sensitization (models of repeat allergen inhalation appear to be more reproducible). For example, two groups used experimental models of airway inflammation in mice genetargeted for complete and selective ablation of the eosinophil lineage. In one model eosinophils were targeted through transgenic expression of the diphtheria toxin A chain under control of the eosinophil peroxidase promoter (PHIL) (Lee et al. 2004). The authors of the study concluded that eosinophils were required for both AHR and mucus accumulation. Other workers have ablated the eosinophil lineage by deleting the high-affinity GATA-1 binding site on the palindromic GATA-1 promoter (Δdbl) but found that these same features of experimental asthma were unaffected by eosinophil depletion, although the cell did appear to play a critical role in airway remodeling (Humbles et al. 2004). Such anomalies have previously been explained by differences in the strains of animals used (Shinagawa & Kojima 2003) and different experimental protocols. Animal studies have demonstrated a role for eosinophils in airway remodeling. The Δdbl-GATA animals were clearly protected from peribronchiolar collagen deposition and increases in airway smooth muscle (Humbles et al. 2004). Furthermore, in a chronic repetitive allergen challenge model, Cho et al. (2004) found that IL-5 gene deletion suppressed lung eosinophilia, peribronchial fibrosis, collagen III, collagen V and total lung collagen content in parallel. These changes were associated with decreased TGF-β1 content of lung tissue, with evidence that eosinophils were the major source. Interestingly, epithelial cell expression of αvβ6, an integrin that

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Early Asthmatic Reaction

Late Asthmatic Reaction

• • • •

• • • • •

Mast cells IgE Histamine Cysteinyl leukotrienes

Airway hyperresponsiveness • • • •


Wheezy breathlessness

Neural pathways ASM T cells Mast cells

T cells Th2 cytokines (e.g., IL-13) DCs/IgE T cell/ASM interactions Mast cells (cysteinyl leukotrienes) Airway remodeling • Fibrogenic factors (e.g. TGF-b) • Growth factors (e.g. VEGF) • EOSINOPHILS

Natural Exacerbations • Viral/nonviral • Chronic inflammation • EOSINOPHILS Fig. 12.10 Pathways to wheezy breathlessness in asthma. Airway narrowing, the cause of wheezy breathlessness, can result from several mechanisms, many of which overlap. Early and late reactions are clinical models. The early asthmatic reaction can be largely blocked by antihistamines and anti-cysteinyl leukotrienes and is largely mast cell/IgE-mediated. The late asthmatic reaction is partially dependent on the early response and is “blunted” by leukotriene receptor antagonists. The late-phase reaction also has a T-cell component and may involve antigen trapping and focusing by IgE-bound dendritic cells prior to presentation

to, and activation of, Th2 cells. In airway hyperresponsiveness (AHR), nonspecific triggers such as smoke, dust, and fumes induce wheezing on a background of airway inflammation. The mechanisms underlying AHR include enhanced neural pathways, alterations in airway smooth muscle (ASM), and T cell- and mast cell-dependent pathways. Airway remodeling probably has a major eosinophil component as shown in both animals and human. Eosinophils are also prominent in natural exacerbations of the disease triggered by viral and nonviral agents. DC, dendritic cell; Th, T helper cell. (See CD-ROM for color version.)

activates latent TGF-β1, was also suppressed. Peribronchial smooth muscle thickness and epithelial mucus expression were also reduced. Others have observed reduced airway eosinophilia, TGF-β production, and remodeling in IL-5 knockout animals, and increased airway fibrosis in IL-5 transgenic animals after repeated allergen inhalation (Tanaka et al. 2004). It was surprising therefore that the reduced subepithelial cell fibrosis observed in the Δdbl-GATA line were apparently independent of TGF-β expression. However there are other potential fibrogenic pathway activators as shown diagrammatically in Fig. 12.10.

sites of inflammation. It stimulates the expansion and differentiation of eosinophil precursors and upregulates expression of its own specific receptor α chain during human eosinophil development. Anti-IL-5 has a good safety profile and anxieties regarding increased susceptibility to helminths and tumors appear, so far, unfounded. When asthmatics were given three infusions of anti-IL-5 (mepolizumab) this produced about a 90% reduction in blood and bronchial lavage eosinophils but only 55% reduction in bronchial mucosal eosinophils (Flood-Page et al. 2003b). However, even this modest effect produced significant reduction in tenascin, lumican, and procollagen III compared with placebo (Flood-Page et al. 2003c). There was also a significant reduction in the numbers and percentage of tissue eosinophils expressing mRNA for TGF-β1 as well as the concentration of TGF-β1 in BAL fluid. Although there were no appreciable improvements in clinical outcomes, the study was not powered to detect changes in lung function or AHR. Nevertheless, the results do provide strong evidence that there is a causal relation between eosinophils and matrix deposition in the extracellular matrix. The clinical significance of these findings is unclear, especially as fibroblast accumulation and airway smooth muscle cell hypertrophy in proximal airways seem to be more selective determinants of severe persistent asthma than matrix deposition beneath the basement membrane (Benayoun et al. 2003). A further complicating factor is the role of atopy. For example, airway eosinophilia and angiogenesis were observed in bronchial biopsies from atopic children without asthma (Barbato et al. 2006) and eosinophilic inflammation and

Remodeling in clinical asthma The precise clinical significance of airway remodeling is debated. One view is that the thickness of the wall, the overall consequence of remodeling, leads to a decrease in baseline caliber (i.e., the radius of the airway lumen), resulting in a disproportionate increase in airway resistance, which in turn enhances AHR. Others point out that corticosteroids, the mainstay antiinflammatory treatment in the disease, reverse some but not all the features of remodeling. Nevertheless there is a hard core of asthmatics who are steroid resistant and airway remodeling is often quite marked in asthma deaths. Studies in humans using anti-IL-5 antibodies also support a role for eosinophils in events surrounding deposition of certain matrix proteins within the reticular basement membrane (Kay et al. 2004). IL-5 is a key cytokine in eosinophil differentiation, maturation, recruitment, and activation at

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increased AHR have been observed in adult patients with allergic rhinitis (Tatar et al. 2005). On the other hand, intense bronchial mucosal eosinophilia is a feature of nonatopic (“intrinsic”) asthma. One interpretation of these findings is that eosinophil-mediated damage precedes the development of overt asthma irrespective of the atopic status. In order to provide definitive evidence that eosinophils are key cells in airway remodeling, more effective strategies are required to deplete tissue eosinophils. Even in animal models of asthma there was residual tissue eosinophilia in the airways after anti-IL-5 administration (Foster et al. 2001). In fact depletion of both IL-5 and eotaxin are required to abolish tissue eosinophils and AHR in mice, suggesting that IL-5 blockade alone is insufficient. Combination therapy with, for example, anti-IL-5 and a CCR3 antagonist may be more useful than IL-5 blockade alone, since this would have the theoretical advantage of inhibiting both bone marrow maturation (mainly an IL-5 effect) and tissue accumulation (predominantly a CCR3-dependent effect). In a mouse model, ablation of eotaxin chemokines prevented antigeninduced pulmonary eosinophilia (Pope et al. 2005b) and antagonism of CCR3 reduced eosinophil numbers and this was accompanied by a diminution in asthma pathology (Weigmann et al. 2007). Eosinophils are also known to localize to cholinergic nerves in a variety of inflammatory conditions including asthma. This effect appears to be the result of enhanced eotaxin production by neurons, possibly as a result of IL-4 and IL-13 upregulation (Fryer et al. 2006). These events result in damage of inhibitory M2 receptors by eosinophil MBP (Evans et al. 1997) and reduced catabolism of acetylcholine (Durcan et al. 2006).

Eosinophils and the late asthmatic reaction When clinical asthma is provoked experimentally by inhalation of allergen, there are two general patterns of airway narrowing, termed the early asthmatic reaction (EAR) and late asthmatic reaction (LAR). The EAR (as measured by changes in FEV1 as a test of airway narrowing) peaks within 15–30 min after allergen challenge and returns nearly to baseline by 1 hour. In this sense it is a “bronchospastic” reaction and involves the IgE-dependent release from mast cells of histamine and other mediators, including leukotrienes, prostaglandins, and tryptase. The LAR, on the other hand, is characterized by a delayed course and slow decrease in FEV1 (which peaks between 3 and 9 hours), and tends to resolve by 24 hours. The mechanism(s) of the LAR is controversial, although there is good evidence to suggest that there is a significant T-cell component because isolated LAR (i.e., without the EAR component) can be provoked by inhalation of allergen-derived T-cell peptides (which do not cross-link IgE and cause mast cell activation) (Haselden et al. 1999). Furthermore, the immunosuppressant cyclosporin A blocks the late, but not the early, asthmatic reaction (Sihra et al.

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1997). Thus the LAR may involve direct interaction between activated T cells (presumed Th2 cells) and airway smooth muscle subsequent to IgE-dependent trapping and focusing by airway dendritic cells. The role of the eosinophil in the LAR and in AHR remains uncertain. The original observation of Cockcroft et al. (1977) was that allergen inhalation increased AHR in dual asthmatic responders which was sustained for at least 7 days. The temporal association between increased inflammatory cell infiltration and increased AHR at 24 hours after allergen challenge in dual responders (Brusasco et al. 1990; Flood-Page et al. 2003b; Dorman et al. 2004a) has led to the suggestion of a causal relationship between the two. However, Kariyawasam et al. (2007) showed that allergen-induced increases in eosinophilic airway inflammation, while marked at 24 hours in dual asthmatic responders, were virtually resolved by 7 days. On the other hand, increases in AHR and expression of collagen markers of airway remodeling persisted. Other less direct evidence also supports the view that cellular inflammation, particularly eosinophil infiltration (Dorman et al. 2004b), does not necessarily directly relate to AHR (Djukanovic et al. 1990; Ollerenshaw & Woolcock 1992; Iredale et al. 1994; Crimi et al. 1998; Dorman et al. 2004b). In an allergen-induced study by Gauvreau et al. (1999) on the cellular kinetics of cells in induced sputum, eosinophilia remained elevated 7 days after allergen inhalation challenge, albeit at levels considerably less than at 24 hours. This is in contrast to the finding of others who observed resolution of mucosal eosinophils to baseline levels by this time point (Kariyawasam et al. 2007). The reason for this difference between measures of airway luminal eosinophils and tissue cells is not clear but may reflect eosinophils that have been cleared from the submucosa but can still be detected in the sputum at a time point when tissue infiltration has resolved. Attempts to deplete eosinophils selectively in humans have been largely unsuccessful and as such we have been unable to fully resolve the role of this cell in the asthma process. The first study in humans showed that a single infusion of anti-IL-5 produced a significant reduction in both blood and induced sputum eosinophils, but no appreciable changes in either the LAR or AHR (Leckie et al. 2000). However, a subsequent study (Flood-Page et al. 2003c) involving three infusions indicated that mepolizumab was unable to deplete tissue eosinophils, i.e., from the bronchial mucosa, making interpretation of the single infusion study problematic (O’Byrne et al. 2001; Flood-Page et al. 2002, 2003b; Leckie 2003). Nevertheless, further evidence against a role for eosinophils as causative of the late reaction comes from studies in human skin in which it has been shown that the kinetics of eosinophil accumulation can be dissociated from the time course of the late-phase allergic reaction and that profound reduction of eosinophils (again by anti-IL-5) does not affect the magnitude of the allergen-induced cutaneous swelling and edema (Phipps et al. 2004a).

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Eosinophils and airway hyperresponsiveness There is no firm evidence that eosinophils or their products are directly causative in AHR in clinical asthma. The correlation between blood and tissue eosinophils and the degree of AHR is generally weak or nonexistent. Following a single infusion of anti-IL-5 there was no change in AHR, even when patients were followed up for several weeks, although this study may have been flawed for the reasons already stated above (Leckie et al. 2000). Further evidence casting doubt on a role for eosinophils in AHR comes from studies of eosinophilic bronchitis (Brightling et al. 2002, 2003). In this condition there is a similar distribution of eosinophils in the airways to that found in asthma, although there is no wheezy breathlessness or AHR (Brightling et al. 2003). The only histopathologic feature distinguishing asthma from eosinophilic bronchitis was mast cells associated with airway smooth muscle cells.

Eosinophils and natural exacerbations of asthma Severe persistent asthma is characterized by viral- and nonviral-induced natural exacerbations on a background of chronic inflammation. An increase in blood or sputum eosinophils often predates deterioration in symptoms and lung function. In fact a management strategy directed at normalizing the sputum eosinophil count was more effective than traditional management strategy (based on lung function, assessment of symptoms, and use of rescue β2 agonists) in reducing the number of asthma exacerbations (Green et al. 2002). It is interesting to speculate that eosinophil-derived fibrogenic/growth factors amplify airway remodeling and associated mucus production relatively rapidly and that this in turn leads to deterioration in symptoms. In a recent study it was shown that even a single allergen inhalation could induce acute airway remodeling in mild atopic asthmatics (Phipps et al. 2004b). Endobronchial mucosal biopsies obtained 24 hours after challenge showed significant increases in Hsp47, a chaperone of collagen synthesis as well as STAT6 and phospho-Smad2 as evidence of IL-4/IL-13 and TGF-β activated cells respectively. There were also increases in the thickness of tenascin within the reticular basement membrane. Therefore (“eosinophilic”) airway remodeling in asthma may partly result from repeated acute activation of the epithelial mesenchymal trophic unit by allergen exposure.

Conclusions There are many areas of remodeling that require further investigation. The precise relationship between chronic inflammation and remodeling is still unclear. Some studies suggest that remodeling might even predate the first signs of inflammation and be an independent event that is amplified rather than caused by Th2 inflammation. The genetic predisposition to airway remodeling, including gene expression by resident cells from normal and diseased airways, is an important area for future research as are the precise effects of the various com-

Eosinophils: Biological Properties and Role in Health and Disease ponents of remodeling on airway function. For example, the consequence of subepithial fibrosis to chronic airway obstruction is still unknown and the importance of angiogenesis, one of the few components of remodeling reversed by corticosteroids, in inflammation and edema is poorly understood. Animal and human studies point to an important function of eosinophils in airway remodeling in asthma. The cell probably also plays a critical role in natural exacerbations of the disease. The significance of eosinophils in the LAR and in AHR, at least in the clinical situation, is far less certain. Studies on the role of the cell in AHR and mucus production, using eosinophil-lineage depletion in mice, has given diametrically opposite results, emphasizing the difficulties of animal models in mimicking the natural disease in humans.

Eosinophil-associated gastrointestinal diseases Eosinophil accumulation in the gastrointestinal tract is a common feature of numerous gastrointestinal disorders, including classic IgE-mediated food allergy (Saavedra-Delgado & Metcalfe 1985; Moon & Kleinman 1995), eosinophilic gastroenteritis (Keshavarzian et al. 1985; Torpier et al. 1988), allergic colitis (Sherman & Cox 1982; Hill & Milla 1990; Odze et al. 1995), eosinophilic esophagitis (Furuta 1998; Rothenberg et al. 2001b; Fox et al. 2002), inflammatory bowel disease (IBD) (Sarin et al. 1978; Dvorak 1980; Walsh & Gaginella 1991), and gastroesophageal reflux disease (Winter et al. 1982; Brown et al. 1984; Liacouras et al. 1998). In IBD, eosinophils usually represent only a small percentage of the infiltrating leukocytes (Walsh & Gaginella 1991; Desreumaux et al. 1999), but their level has been proposed to be a negative prognostic indicator (Nishitani et al. 1998; Desreumaux et al. 1999). Primary eosinophil-associated gastrointestinal diseases (EGIDs) such as eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, and eosinophilic colitis are defined as disorders that primarily affect the gastrointestinal tract with eosinophil-rich inflammation in the absence of known causes of eosinophilia (e.g., drug reactions, parasitic infections, malignancy). Patients with EGIDs suffer a variety of problems, including failure to thrive, abdominal pain, irritability, gastric dysmotility, vomiting, diarrhea, and dysphagia (Guajardo et al. 2002; Khan & Orenstein 2002). Evidence is accumulating to support the concept that EGIDs arise secondary to the interplay of genetic and environmental factors. Notably, a large percentage (∼ 10%) of patients suffering from EGID have an immediate family member with EGID (Guajardo et al. 2002). Additionally, several lines of evidence support an allergic etiology: (i) about 75% of patients with EGID are atopic (Caldwell et al. 1975; Cello 1979; Scudamore et al. 1982; Furuta et al. 1995; Iacono et al. 1996; Sampson 1997; Walsh et al. 1999; Spergel et al. 2002); (ii) the severity of disease can sometimes be reversed by institution of an allergen-free diet (Kelly et al. 1995; Walsh et al. 1999; Spergel et al. 2002); and (iii) the common finding of mast cell degranulation in tissue specimens (Oyaizu et al.

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1985; Bischoff 1996). Importantly, our recent models of EGID support a potential allergic etiology for these disorders (Rothenberg et al. 2001c). Interestingly, despite the common finding of food-specific IgE in patients with EGID, foodinduced anaphylactic responses only occur in a minority of patients (Sampson 1999). Thus, EGIDs have properties that fall between pure IgE-mediated food allergy and cell-mediated hypersensitivity disorders (e.g., celiac disease) (Sampson 1999).

Hypereosinophilic syndromes The term “hypereosinophilic syndrome” (HES) was introduced by Anderson and Hardy (1968) to designate patients with marked eosinophilia. They reported three patients, all males, between the ages of 34 and 47 who suffered from cardiopulmonary symptoms, fever, sweats, weight loss, and marked eosinophilia. Two of the patients died, and at autopsy their hearts were enlarged and showed mural thrombi. The treatment for HES is similar to that used for patients with chronic myelogenous leukemia, including tyrosine kinase inhibitors (e.g., Imatinib) prednisone, hydroxyurea (hydroxycarbamide), and interferon (IFN)-α and humanized anti-IL-5 antibodies. Chusid et al. (1975) formulated the diagnostic criteria for HES to include (i) persistent eosinophilia, with an eosinophil cell count of at least 1.5 × 109/L for a minimum of 6 months; (ii) lack of known causes for eosinophilia (e.g., parasitic or allergic triggers); and (iii) symptoms and signs of organ system involvement. Based on these diagnostic criteria, patients with EGID and blood eosinophil counts in excess of 1.5 × 109/L meet the diagnostic criteria. However, patients with EGID generally do not have the high risk of lifethreatening complications associated with classic HES (i.e., cardiomyopathy or central nervous system involvement). Notably, considerable heterogeneity among patients with HES has been recognized. For example, T-cell clones producing the characteristic Th2 cytokines IL-4 and IL-5 have been found in patients satisfying the diagnostic criteria for HES (Simon et al. 1999; Roufosse et al. 2003). However, perhaps the most striking advance in our understanding of HES has come about following treatment of HES patients with the tyrosine kinase inhibitor imatinib mesylate (Schaller & Burkland 2001; Ault et al. 2002; Gleich et al. 2002; Cools et al. 2003; Cortes et al. 2003). Imatinib was introduced for the treatment of chronic myelogenous leukemia and has had a remarkable effect in that disease. Treatment of many HES patients with imatinib mesylate causes a dramatic reduction of peripheral blood and bone marrow eosinophils, suggesting that certain HES patients express a novel kinase sensitive to imatinib mesylate. Further investigation of the ability of imatinib mesylate to treat HES patients revealed the existence of an 800-kb deletion in chromosome 4 bringing together an upstream DNA sequence homologous to a yeast protein, referred to as FIP1, and designated as like FIP1 or FIP1-L1, and the gene for the cytoplasmic domain of the platelet-derived growth factor α (PDGFRA) receptor (Cools

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et al. 2003; Griffin et al. 2003). This fusion gene is transcribed and translated to yield a novel kinase referred to as FIP-L1PDGFRA; FIP-L1-PDGFRA is exquisitely sensitive to imatinib in vitro, thus explaining the remarkable sensitivity of HES patients to this drug. The FIP-L1-PDGFRA fusion gene cooperates with IL-5 overexpression in a murine model of HES, suggesting that both pathogenic events cooperate in disease etiology (Yamada et al. 2006). The patients responsive to imatinib are those most characteristic of “classic” HES, namely males between the ages of 20 and 50 who present clinically with marked peripheral blood eosinophilia. Recently, these patients have been shown to meet minor criteria for systemic mastocytosis, having elevated levels of serum mast cell tryptase and high numbers of dysplastic mast cells in the bone marrow (Klion et al. 2003, 2004a). These patients go on to develop eosinophilic endomyocardial disease with embolization to peripheral organs including the extremities and the brain, and they strikingly resemble the patients originally designated by Hardy and Anderson. However, it appears that any disease that results in prolonged and marked eosinophilia can be associated with endomyocardial disease. For example, endomyocardial disease has occurred during the course of helminth infections and also in various malignancies associated with marked eosinophilia (Hussain et al. 1994; Yoshida et al. 1995; Andy et al. 1998). Thus patients with marked eosinophilia are at risk of developing cardiac disease regardless of the underlying etiology of the eosinophilia. Accordingly, routine surveillance of the cardiorespiratory system (e.g., echocardiography and plethysmography) in patients with EGID and peripheral blood eosinophilia is warranted. Based on these concerns, the diagnosis of HES in patients with EGID should always be considered, especially in those who develop extragastrointestinal manifestations (e.g., splenomegaly, or cutaneous, cardiac or respiratory). As such, additional diagnostic testing for HES should be considered including bone marrow analysis (searching for evidence of myelodysplasia), serum mast cell tryptase and vitamin B12 levels (both moderately elevated in classic HES), and genetic analysis for the presence of the FIP1-L1PDGFRA fusion event (Klion et al. 2003).

Anti-eosinophil therapeutics Numerous drugs inhibit eosinophil production or eosinophilderived products, including glucocorticoids, myelosuppressive drugs, leukotriene synthesis or receptor antagonists, tyrosine kinase inhibitors, IFN-α, and humanized anti-IL-5 antibodies. The etiology of the primary disease often specifies the best therapeutic strategy. For example, a subset of patients with HES have an 800-kb interstitial deletion on chromosome 4 (4q12) that results in the fusion of an unknown gene FIP1-L1 with PDGFRA (Cools et al. 2003, 2004a,b). This fusion gene produces a constitutively active tyrosine kinase

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(PDGFRA) that is exquisitively sensitive to the inhibitor imatinib mesylate, which is now approved for the treatment of HES (Gleevec). Although PDGFRA is not normally active in hematopoietic cells, the activated kinase renders cells growth factor independent, perhaps by activating STAT5 signal transduction. Thus, eosinophilic patients with FIP1L1-PDGFRA-positive disease are now treated with Gleevec as first-line therapy (Gleich et al. 2002). In addition, a variety of other activated tyrosine kinases have just been associated with HES, including PDGFR-β, Janus kinase-2, and FGF receptor 1. In most other individuals, glucocorticoids are the most effective agents for reducing eosinophilia (Rothenberg 1998). They suppress the transcription of a number of genes for inflammatory mediators, including the genes for IL-3, IL-4, IL-5, GM-CSF, and various chemokines including the eotaxins. Recently, the main action of glucocorticoids on eosinophil active cytokines has been shown to involve mRNA destabilization, thus reducing the half-life of cytokines such as eotaxins (Stellato et al. 1999). In addition, glucocorticoids inhibit the cytokine-dependent survival of eosinophils (Schleimer & Bochner 1994). Systemic or topical (inhaled or intranasal) glucocorticoid treatment typically causes a rapid reduction in eosinophils, but a few patients are glucocorticoid resistant and maintain eosinophilia despite high doses (Barnes & Adcock 1995). The mechanism of glucocorticoid resistance is unclear, but a reduced level of glucocorticoid receptors and alterations in transcription factor AP-1 (activator protein 1) appear to be at least partially responsible in some of them (Barnes & Adcock 1995). Glucocorticoid-resistant patients sometimes require other therapy such as myelosuppressive drugs (hydroxyurea, vincristine) or IFN-α (Rothenberg 1998). IFN-α can be especially helpful because it inhibits eosinophil degranulation and effector function (Aldebert et al. 1996). Notably, patients with myeloproliferative variants of HES can often go into remission with IFN-α therapy. Cyclophilins (e.g., cyclosporin A) have also been used because they block the transcription of numerous eosinophil-active cytokines (e.g., IL-5, GM-CSF) (Rothenberg 1998). Recently, lidocaine has been shown to shorten eosinophil survival, and its effects mimic those of glucocorticoids and are noncytotoxic (Bankers-Fulbright et al. 1998). Indeed, an early clinical trial has shown that nebulized lidocaine is safe and effective in subjects with asthma (Hunt et al. 2004). Drugs that interfere with eosinophil chemotactic signals include recently approved leukotriene antagonists and inhibitors. Inhibition of 5-lipoxygenase (e.g., zileuton) blocks the rate-limiting step in leukotriene synthesis and inhibits the generation of the eosinophil chemoattractant LTB4 and the cysteinyl leukotrienes (Kane et al. 1996). Cysteinyl leukotriene receptor antagonists block the muscle contraction and increased vascular permeability mediated by leukocyte-derived leukotrienes (Gaddy et al. 1992). Some of

the third-generation antihistamines inhibit the vacuolization (Snyman et al. 1992) and accumulation (Redier et al. 1992) of eosinophils after allergen challenge and directly inhibit eosinophils in vitro (Rand et al. 1988; Snyman et al. 1992). Cromoglycate and nedocromil inhibit the effector function of eosinophils such as antibody-dependent cellular cytotoxicity (Rand et al. 1988). The identification of molecules that specifically regulate eosinophil function and/or production offers new therapeutic strategies in the pipeline. Agents that interrupt eosinophil adhesion to the endothelium through the interaction of CD18/ICAM-1 (Wegner et al. 1990) or VLA-4/VCAM-1 may be useful (Kuijpers et al. 1993; Weg et al. 1993). Indeed, antibodies that block these pathways have recently been approved for other indications, but their anti-eosinophil activity has yet to be determined (von Andrian & Engelhardt 2003). Antibodies against IL-5, now humanized by two different pharmaceutical companies, are under active clinical investigation (Egan et al. 1995; Mauser et al. 1995) and look particularly promising for the treatment of HES (Garrett et al. 2004; Klion et al. 2004b, 2006) and eosinophilic esophagitis (Stein et al. 2006). While its utility for asthma may be limited due to redundant pathways, anti-IL-5 is particularly promising for HES. Numerous inhibitors of the eotaxin/CCR3 pathway, including small-molecule inhibitors of CCR3 and a human anti-eotaxin-1 antibody, are being developed (Zimmermann et al. 2003). Early results with a phase I trial of human antieotaxin-1 antibody in patients with allergic rhinitis have demonstrated the ability of this apparently safe drug to lower levels of eosinophils in nasal washes and nasal biopsies, and to improve nasal patency (Zimmermann et al. 2003). Antihuman IL-13 antibody is now in early clinical trials (Blanchard et al. 2005) and looks promising for lowering tissue eosinophil levels and improving features of asthma. Finally, a recently identified eosinophil surface molecule, Siglec-8, may offer a therapeutic opportunity (Nutku et al. 2003). Siglec-8 is a member of the sialic acid-binding lectin family and contains ITIMs (immunoreceptor tyrosine-based inhibitory motifs) that can induce efficient eosinophil apoptosis when engaged by anti-Siglec-8 cross-linking antibodies. It is interesting to note that Siglec-8, as well as CCR-3 and CRTH2, are coexpressed by other cells involved in Th2 responses including Th2 cells, mast cells, and basophils. Thus, agents that block these receptors may be particularly useful for allergic disorders.

Conclusions Historically, eosinophils have been considered end-stage cells involved in host protection against parasites. However, numerous lines of evidence have now changed this perspective by showing that eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of

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adaptive immunity by directly activating T cells. As normal constituents of the mucosal immune system, particularly in the gastrointestinal tract, eosinophils are likely to have a physiologic function. Indeed, eosinophils have been implicated in innate immunity by being an early and possibly instrumental source of cytokines (e.g., IL-4) and have been shown to have a role in developmental processes such as mammary gland development. Analysis of recently generated genetically engineered eosinophil-deficient mice will soon answer critical questions concerning the true involvement of this cell type in a variety of processes. Breakthroughs in identifying key eosinophil regulatory cytokines such as IL-5 and the eotaxin subfamily of chemokines have uncovered mechanisms that selectively regulate eosinophil production and localization at baseline and during inflammatory responses. In particular, an integrated mechanism involving Th2 cell-derived IL-5 regulating eosinophil expansion in the bone marrow and blood and Th2 cell-derived IL-13 regulating eotaxin production now explains the means by which T cells regulate eosinophils. Based on these findings, it is predicted that targeted therapy against key eosinophil regulators (e.g., humanized anti-IL-5 and CCR3 antagonists) will likely transform medical management of eosinophilic patients.

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Neutrophils: Biological Properties and Role in Health and Allergic Diseases Alison M. Condliffe, Andrew S. Cowburn and Edwin R. Chilvers

Summary The polymorphonuclear leukocyte or neutrophil is the most abundant yet shortest lived of all myeloid cells. It develops and matures in the bone marrow under the influence of interleukin (IL)-3, granulocyte colony-stimulating factor (GCSF), and granulocyte–macrophage colony-stimulating factor (GM-CSF) and is released into the circulating pool to fulfil its normal and vital functions of bacterial and fungal surveillance and phagocytic removal. In recent years, molecular steps underlying bone marrow release, neutrophil migration, pathogen recognition, phagocytosis, degranulation, activation of NADPH oxidase, and the precise mechanisms underlying pathogen destruction have been elucidated. Neutrophils are remarkably well adapted to survive and function in highly acidic and hypoxic environments such as an abscess cavity or infected joint space, in part through their capacity for anaerobic glycolytic metabolism. The numerical dominance, migratory potential, and highly destructive capacity of these cells results in their frequent and pathogenic role in many inflammatory diseases, the most notable being acute respiratory distress syndrome (ARDS), inflammatory bowel disease, and rheumatoid arthritis. In addition, a more prominent role has recently been attributed to the neutrophil in allergic diseases, in particular in the development of asthma exacerbations and in steroid-resistant asthma. New studies have also highlighted additional functions for the neutrophil, including the production of neutrophil extracellular traps comprising granule proteins and chromatin designed to trap and neutralize extracellular bacteria, a role in early antigen processing and transfer to dendritic cells, and a role in facilitating the migration of other inflammatory cells such as the eosinophil. In vivo trafficking studies using 111In- and 99mTclabeled autologous neutrophils have now defined the major role of the spleen, liver, and bone marrow in human neutrophil margination, and complementary studies in mice have shown the potential for a minor fraction of neutrophils to migrate from an inflamed site back into the circulatory pool. The capacity for neutrophils to injure tissues demands tight regulatory control


via the processes of priming (which dictates the speed and magnitude of NADPH oxidase or respiratory burst response) and apoptosis (which regulates the functional longevity and clearance route for these cells). This latter pathway is now established as a major mechanism underlying the removal of neutrophils from the inflamed site, a process that involves recognition and uptake by inflammatory and tissue resident macrophages. The monitoring of neutrophilic inflammation and the therapeutic targeting of these cells, both to inhibit activation and to facilitate apoptotic clearance, remain important research targets.

Introduction Neutrophil granulocytes are the most abundant white blood cells and originate from the myeloid series in the bone marrow. They have an average volume of 330 fL and a mean diameter of 10 μm (Schmid-Schonbein et al. 1980). Together with the eosinophil and basophil they form the class of polymorphonuclear cells (PMN), so called because their nuclei have a characteristic multilobulated shape (Fig. 13.1) in contrast to the mononuclear white blood cells (monocytes and lymphocytes) whose nuclei are unsegmented. In health the normal peripheral blood neutrophil count is 2.5–7.5 × 109/L. Circulating neutrophils are short-lived cells with a half-life of just 6–8 hours. As a consequence, neutrophils must be continually replenished from hematopoietic stem cells within the bone marrow by the process of granulopoiesis. The bone marrow manufactures approximately 1011 neutrophils per day, with the capacity to increase production still further when required. The initial stages of neutrophil development involve the commitment and differentiation of stem cells through pluripotent common myeloid progenitors and bipotent granulocyte–macrophage progenitors toward unipotent progenitors committed to granulocyte lineage (Fig. 13.2). The early stages of neutrophil development are governed by a transcriptional program that progressively downregulates the genes associated with multiple lineages (Akashi et al. 2003); terminal differentiation into bone marrow neutrophils is directed by a transcriptional program that promotes cell

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Inflammatory Cells and Mediators cycle exit and acquisition of the neutrophil-specific proteins that enable these cells to fulfil their role within the framework of the overall immune system (Theilgaard-Monch et al. 2005).

Neutrophil structure

Fig. 13.1 Electron micrograph of an activated human neutrophil granulocyte showing the multilobed nucleus and cytoplasmic granules.

Antimicrobial and proteolytical granule proteins

Bone marrow Promyelocytes Cell cycle Metamyelocytes PM Exit MY

Human neutrophils are defined by the segmentation of their nucleus into three to five lobes connected by heterochromatin filaments. The physiologic purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation, although a role for the centrosome and associated microtubules has been suggested in the latter process (Olins & Olins 2005). Nuclear segmentation occurs during granulocyte development and maturation. In myeloid leukemias, the normal differentiation process is arrested and hence immature cells with large rounded nuclei may be seen in the circulation. During severe sepsis, neutrophils may be released from the bone marrow before completing nuclear maturation; such ‘band’ cells have indented rather than segmented nuclei. When maturation is delayed, as in megaloblastic anemia, hypersegmentation may occur. Finally, during apoptosis, dramatic chromatin condensation occurs to produce the characteristic dense and pyknotic nucleus of programmed cell death.

Blood PMN

az–GP3 Azurophil-granule proteins SPe GPs Specific gel GPs

Cytokines and chemokine

Receptors

Apoptosis Cell cycle

Gelatinase CDK2, 4, 6 E2F target genes

Death Rec. pathway TLR-2 apoptosis pathway

P53 pathway

Rec. for multiple lineage HGF

(Hematopoietic growth factors)

Rec. for proinflammatory cytokines/chemokines

Proinflammatory cytokines and chemokines

Fig. 13.2 Neutrophil origins and ontogeny. GP, granule protein. (Adapted from Theilgaard-Monch et al. 2006, with permission.)

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Neutrophils: Biological Properties and Role in Health and Allergic Diseases

Neutrophils have been called the secretory organs of the innate immune response and contain heterogeneous and numerous populations of secretory granules. The manufacture of these granules and their contents is regulated by cytokines and transcription factors, many of which are shared with the neutrophil differentiation program; this ensures that granules develop at the correct stage of cell maturation (Gullberg et al. 1999). Neutrophil differentiation is characterized by the sequential formation of azurophil (primary), specific (secondary), and gelatinase (tertiary) granules, and this is synchronized with the synthesis and packaging of their specific granule proteins (Fig. 13.2). This has given rise to the “targeting-by-timing” hypothesis, which states that the packaging of proteins into distinct granule subtypes is determined principally by the time of their biosynthesis. Therefore granule proteins are characterized both by gene expression during neutrophil development and by subcellular localization (Faurschou & Borregaard 2003). Azurophil granules contain proteins that are released into the phagocytic vacuole at the end of the engulfment process or are secreted onto opsonized surfaces, including proteinases, hydrolases, and microbicidal proteins such as defensins and bactericidal/ permeability-inducing protein. Specific granules incorporate enzymes such as lysozyme and collagenase together with lactoferrin and vitamin B12-binding protein, and also provide an important intracellular reserve of membrane components. There appear to be several subtypes of tertiary granules, containing enzymes such as gelatinase and lysozyme, plus membrane components including constituents of the NADPH oxidase, in a form that can be rapidly mobilized to the cell surface (Faurschou & Borregaard 2003). It is likely that most granule proteins have evolved to aid the neutrophil’s rapid migration through tissues and to promote effective and targeted bacterial killing; it can be readily appreciated, however, that many of these agents can be highly toxic to normal tissue if released in an uncontrolled manner. The plasma membrane is of great importance to this highly motile and responsive cell, since it contains the receptors used to detect inflammatory events – such as adhesion molecules and ligands enabling interactions with the activated endothelial cell surface, pathogen-recognition receptors including Toll-like receptors (TLRs), and inflammatory cytokine receptors – and the molecules involved in the transduction of external signals into cellular responses, including locomotion, phagocytosis, secretion, and the oxidative burst. Therefore intimate communication arrangements must exist between the membrane and the granule populations that replenish these membrane components. The neutrophil also possesses a highly developed cytoskeletal framework responsible for coordinating its complex motility functions (see below). It was previously believed that neutrophils possess few or even no mitochondria: neutrophils utilize glycolysis rather than oxidative phosporylation to generate energy, mitochondrial poisons such as cyanide have little effect on neutrophil

function, and few intact mitochondria are evident on electron microscopy of neutrophil specimens. However, the use of mitochondrial-specific dyes has confirmed that mitochondria do exist in neutrophils, organized as a tubular network (Fossati et al. 2003). Functionally their role seems to be largely restricted to the regulation of apoptosis (Maianski et al. 2004). The endoplasmic reticulum (ER) is the largest endomembrane system within eukaryotic cells, and subserves a variety of functions, including calcium uptake and release, protein and lipid synthesis, posttranslational protein modification and export to the Golgi apparatus. Terminal differentiation of myeloid cells is associated with a decrease in ER content (Clark et al. 2002), perhaps reflecting decreased protein synthetic activity. Despite this reduction, mature neutrophils retain a plentiful ER network, participating in the calcium signaling pathways that are of fundamental importance to these rapidly responsive cells; it is also sufficient to induce the ER stress or unfolded protein response in patients with mutations in neutrophil elastase, with the resultant activation of pro-apoptotic signaling pathways producing congenital and cyclical neutropenia syndromes (Kollner et al. 2006). Likewise, while the Golgi apparatus is more prominent in immature neutrophils consistent with its importance in granule packaging and transport, it still has a role in maintaining homeostasis in the terminally differentiated cell; for example, monomeric lipopolysaccharide (LPS) is delivered to the neutrophil Golgi apparatus for processing, and defective function of the glycosylation/fucosylation enzymes located within the Golgi membrane results in leukocyte adhesion deficiency (LAD) type II, in which abnormal processing of selectin ligands leads to a profound defect in leukocyte rolling and hence an immunodeficient state (Etzioni & Tonetti 2000). Thus neutrophil development is tailored to produce a terminally differentiated cell, uniquely adapted to its role. Its highly developed and rapidly responsive cytoskeletal network allows rapid recruitment to sites of infection and the efficient engulfment of bacteria, the lack of dependence on mitochondrial respiration permits optimal function even in profoundly hypoxic environments, and the rapid mobilization of preformed granules and assembly of the NADPH oxidase complex allows rapid and efficient bacterial killing. The neutrophil is truly “fit for purpose.”

Neutrophil origin, maturation, and bone marrow release The production of neutrophils accounts for more than half of the cellular output of the bone marrow. The development of mature neutrophils from CD34+ stem cells typically takes 14 days and is dependent on growth factors such as IL-3, GM-CSF, and G-CSF, the latter being available for use as a therapeutic agent to enhance neutrophil production in vivo (Bai et al. 2005). Stromal cells in the marrow appear to be a

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particularly important source of these factors (Takagi 2005). In addition to this constitutive role, stromal cells are of great importance in the control of inflammation, as their release of growth factors is exquisitely sensitive to cytokines such as IL-1 and tumor necrosis factor (TNF)-α. Thus granulopoiesis can be increased several fold as part of the systemic response to infection and inflammation (Burman et al. 2005). Granulopoiesis is also subject to negative regulatory feedback. Neutrophils that have migrated into tissue compartments undergo apoptosis and are phagocytosed by resident macrophages and dendritic cells, with resultant suppression of IL-23, IL-17 and hence G-CSF production; neutrophils from mice lacking β2 integrins exhibit reduced tissue migration, hence this regulatory loop is lost, granulopoiesis is enhanced, and the mice have a persistent neutrophilia unrelated to infection (Stark et al. 2005). The events underlying neutrophil release from the bone marrow are also becoming clearer, with opposing roles for CD18 and CD49d (VLA4) in mediating retention and release respectively demonstrated in a rat model (Burdon et al. 2005). Neutrophil responses are regulated in large part by cytokines, chemokines, and microorganisms signaling through specific cell-surface receptors. The ontogeny of receptor expression during the different stages of neutrophil maturation has recently been analyzed by cDNA microarray (Theilgaard-Monch et al. 2006). Receptor expression for many inflammatory stimuli is very low in neutrophil progenitors but increases progressively as the cell matures, with terminally differentiated bone marrow neutrophils expressing a wide repertoire of cytokine, chemokine, growth factor, and pathogen-recognition receptors. Among these are the interferon receptors (αR-1 and -2, γR-1 and -2), interleukin receptors (IL-1, -4, -6, -10, -13, -17, -18), TNF receptors (TNF-R1 and -R2), and CXC and CC chemokine receptors (CXCR-1, -2, -4 and CCR-1, -2 , -3 respectively). In addition, the expression of a number of proinflammatory cytokines and chemokines is also increased in mature neutrophils, including IL-1, TNF-α, IL-8, and GRO-α. Thus overall the expression pattern for receptors and inflammatory cytokines suggests a marked increase in responsiveness toward inflammatory stimuli during terminal differentiation (Theilgaard-Monch et al. 2005).

Neutrophil kinetics The proportion of neutrophils circulating freely comprises only a small fraction of the total neutrophil cell pool. The bone marrow contains a large number of mature and nearmature neutrophils available for mobilization, and neutrophils within the vascular compartment also reside in the so-called “marginated” pools of the spleen, liver and bone marrow as well as in the active circulation. Margination represents prolonged physiologic transit of neutrophils through these organs with, for example, mean transit times in the

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spleen of 10 min. The mechanisms regulating the migration, capture, and release of neutrophils from these organs are poorly understood. Likewise, the function of the marginated neutrophil pool is unclear, although the rapid release of cells back into the active circulation from these sites through the shedding of CD62L may provide a mechanism for elevating neutrophil numbers acutely during stress or injury. Splenic transit may also afford a mechanism for sensing and removing damaged cells and/or marking them for subsequent removal. The presence and true size of a marginated pool within the lung is debated; early experiments showing significant neutrophil retention in the pulmonary circulation may have been influenced by inadvertent neutrophil priming during cell isolation (Peters 1998). Radiolabeled neutrophils leave the blood in an exponential and apparently random manner with a half-life of approximately 7 hours. This corresponds to a mean intravascular lifespan of 10 hours and a physiologic disposal rate of approximately 108 cells/min. In health the sites of neutrophil disposal are restricted to the reticuloendothelial system (RES), in particular the liver and bone marrow. Hematologic analysis of healthy subjects has failed to identify any evidence of circulating apoptotic cells, which suggests either a very efficient clearance mechanism for such cells or, more likely, that this program of cell death is not activated within the circulation. Once neutrophils undergo apoptosis in the RES they are recognized and removed by tissue resident macrophagelike cells. A study by Shi et al. (2001) has indicated that P-selectin expression on hepatic endothelial cells is functionally important in the clearance of neutrophils by Kupffer cells, which have been shown to contain apoptotic bodies. Neutrophil expression of CXCR4 and the chemotactic response to its cognate ligand SDF-1α have been shown to influence the release of mature neutrophils from the bone marrow and their subsequent return. Newly released neutrophils are low expressers of CXCR4, and in mice it has been shown that as neutrophils age they upregulate this receptor and acquire the ability to migrate toward SDF-1α, which is constitutively expressed by bone marrow stromal cells. Senescent CXCR4high neutrophils therefore appear to migrate back to the bone marrow and allow efficient clearance of effete cells from the circulation (Martin et al. 2003). Whether this mechanism exists in humans has not yet been determined.

Physiologic role of the neutrophil Enormous numbers of short-lived neutrophils are produced by the bone marrow, and are only rarely called upon to protect against significant microbial invasion. This high-turnover system allows a very rapid increase in neutrophil numbers following insult or injury to the host. Neutrophils are highly chemotactic and hence are usually the first immune cells to

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reach an inflamed site where, using their oxidative responses and array of granule proteins, they are rapidly effective in ingesting and killing fungal and bacterial pathogens. The ability of the neutrophil to employ glycolytic metabolism and thus to migrate into, and function and survive within, the highly hypoxic and acidic environment characteristic of an inflamed site is likewise an important aspect of their adaptable nature. Until quite recently the neutrophil was widely perceived as being an immutable end-stage cell with the sole capacity of detecting and removing bacteria, before finally disintegrating. However, it is now clear that the neutrophil is a key player in several aspects of the evolving inflammatory response. Moreover, each stage of its behavior at the inflamed site, including its final removal, has the potential to be finely controlled in order to limit incidental injury to normal tissues and to facilitate the resolution of inflammation and tissue repair, so that tissues can be restored to their normal homeostatic function once the bacterial invasion has been wiped out. For example, (i) the neutrophil can be involved in the initial generation of inflammatory edema (see below), (ii) the migration of eosinophils and monocytes appears to depend on the initial migration of neutrophils (Kikuchi et al. 2006), (iii) neutrophils play an important role in antigen digestion and processing (reviewed by Appelberg 2007), and (iv) it is now recognized that the neutrophil can generate a variety of important cytokines and chemokines that may amplify inflammation and attract additional cells to the inflamed site. It is also clear that different aspects of neutrophil behavior during the inflammatory response, including sequestration, endothelial adhesion, capillary transmigration, bacterial phagocytosis, oxidative burst, degranulation and removal, are interlinked. Exposure of the neutrophil to physiologic concentrations of inflammatory cytokines such as LPS, GM-CSF, or TNF-α engenders a “primed” state in which the functional responses [phagocytosis, degranulation, and generation of reactive oxygen species (ROS)] to subsequent stimuli are greatly upregulated (reviewed in Downey et al. 1995 and Condliffe et al. 1998) and also profoundly influences neutrophil longevity (Fig. 13.3). This dual effect may augment the ability of the innate immune response to clear a bacterial challenge, but may likewise underlie the tissue-damaging potential of the neutrophil in inflammatory disease.

Neutrophil recruitment to inflamed sites Neutrophil recruitment to an inflammatory site is a complex multistep process involving coordinated and sequential interactions of neutrophil adhesion molecules and ligands with those of vascular endothelium. Failure of recruitment, as exemplified by LAD [due to deletion of the gene coding for the β2 integrins (LAD type I) or failure to process the glycoprotein ligands recognized by the selectin adhesion

Priming/activation

Apoptosis

Injury

Resolution

Fig. 13.3 Regulation of neutrophil function and longevity. Mature neutrophils are poorly responsive to secretagogue agonists unless first exposed to a priming agent, such as lipopolysaccharide, granulocyte–macrophage colony-stimulating factor or platelet-activating factor; these agents on their own cause minimal activation but serve to induce cell polarization and substantially upregulate the cell’s secretory response to a second agent. Apoptosis, in contrast, is a constitutive process designed to limit the functional longevity of cells and aid macrophage clearance and the resolution of neutrophilic inflammation. (See CD-ROM for color version.)

molecules (LAD type II)], leads to recurrent bacterial infections, impaired wound healing, and early death. In response to an inflammatory (allergic or infective) stimulus, resident cells such as alveolar macrophages and mast cells release cytokines and chemokines including TNF-α and IL-8, which modify the local microcirculatory environment and lead to upregulation of vascular endothelial adhesion molecules. In addition, neutrophils exposed to circulating inflammatory mediators or such molecules presented on the endothelial surface themselves become “stickier” and less deformable. These two processes result in neutrophil sequestration and transmigration through the endothelial barrier. Neutrophils subsequently migrate toward the inflammatory focus by detecting and moving along concentration gradients of chemoattractant molecules (chemokines, complement components and bacterial products), a process known as chemotaxis. When the migrating neutrophil is exposed to higher concentrations of chemoattractants it becomes immobilized and an array of “activation” responses are progressively engaged, preparing the cell to phagocytose and kill invading bacteria.

Neutrophil sequestration and adhesion Egress of neutrophils from the circulation depends on the sequential interactions of cellular adhesion molecules (CAMs)

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(Fig. 13.4) and, in some circumstances, on factors influencing neutrophil deformability. The relative contribution of individual components of the recruitment cascade may vary according to the stimulus and site involved. In the systemic circulation following insults such as ischemia–reperfusion (Zahler et al. 2002), ventilator-induced lung injury (Miyao et al. 2006), and mast cell degranulation (Kubes & Kanwar 1994), neutrophils roll along the wall of the postcapillary venule. Rolling is principally mediated by the interaction of the selectin family of CAMs with their glycoprotein ligands (Fig. 13.4b); stored endothelial P-selectin is mobilized to the endothelial cell surface by inflammatory stimuli (McEver et al. 1989), including thrombin, histamine and complement, and binds to P-selectin glycoprotein ligand (PSGL)-1, which

is constitutively expressed on the cell surface of neutrophils, eosinophils, monocytes, and lymphocytes. The interaction of endothelial E-selectin and neutrophil L-selectin with their respective ligands also contributes to rolling (Jung & Ley 1999) (Fig. 13.4b), as may certain integrins such as the α4 integrins (Barringhaus et al. 2004). During severe sepsis, rolling of neutrophils may be compromised (Swartz et al. 2000), perhaps due to shedding of L-selectin from the neutrophil surface induced by inflammatory cytokines (Condliffe et al. 1996). Rolling neutrophils “taste” the endothelial surface and integrate a range of signals to determine whether they will detach or whether their rolling velocity will decrease, enabling initiation of firm adhesion and transendothelial migration (reviewed by Ley 2002). Factors that promote adhesion

G protein-coupled receptor Neutrophil L-selectin PSGL-1 (P-selectin ligand) VE-cadherin (a)

Endothelial L-selectin ligand P-selectin b2 integrin (inactive conformation) b2 integrin (active conformation) Chemoattractant Cytokine (b)

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Fig. 13.4 Neutrophil adhesion molecules and recruitment (rolling, firm adhesion, and transmigration). (a) Circulating neutrophils do not interact with quiescent endothelial cells. (b) Cytokines such as TNF-a (here shown derived from inflammatory macrophages) activate the vascular endothelium to express P-selectin, which interacts with neutrophil P-selectin glycoprotein ligand (PSGL)-1 to induce rolling. Interaction of other selectins with their ligands may contribute to rolling. Chemokines (such as IL-8) may be bound by the endothelium and with circulating inflammatory mediators lead to activation of b2 integrins. (c) The activated endothelium expresses intercellular adhesion molecule (ICAM)-1, which binds activated b2 integrins on the neutrophil surface, leading to arrest and firm adhesion. (d) Endothelial cells adjacent to adherent neutrophils may experience a rise in intracellular calcium, leading to endothelial retraction. The interaction of neutrophils with endothelial junctional adhesion molecules (JAMs), of neutrophils with endothelial PECAM-1, and of integrins with their ligands may all encourage transmigration, which preferentially occurs at sites in the basement membrane with low protein expression. (See CD-ROM for color version.)

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include circulating inflammatory cytokines, interaction of selectins with their corresponding glycoprotein ligands, and interaction of neutrophil receptors with so-called “arrest chemokines” such as IL-8 and platelet-activating factor (PAF) tethered to the endothelial surface (Lorant et al. 1991; DiVietro et al. 2001; Laudanna & Alon 2006) (Fig. 13.4b). These varied inputs summate to induce a conformational change in the neutrophil β2 integrins (principally LFA-1 and Mac-1). In their active confirmation, these integrins bind to endothelial intercellular adhesion molecule (ICAM)-1, whose expression is substantially upregulated by inflammatory cytokines such as TNF-α and IL-1. Binding of neutrophil β2 integrins to ICAM-1 leads to firm adhesion to the endothelial surface (Lawrence & Springer 1991; Phillipson et al. 2006) (Fig. 13.4c), and induces further intracellular signaling events to prepare the neutrophil for transmigration. In the lung, diseases such as asthma and bronchiectasis affect the proximal airways, which are fed by the bronchial circulation, while the distal bronchi, bronchioles and alveoli (relevant to processes such as acute lung injury and bacterial pneumonia) derive their blood supply from the pulmonary circulation (Doerschuk 2000). Recruitment of neutrophils from the bronchial circulation is likely to follow the rolling– tethering–transmigration paradigm outlined above, and increased expression of relevant CAMs (including selectins and ICAM-1) has been demonstrated in the bronchial microvasculature in disease processes, for example in asthmatic versus nonasthmatic lungs (Feuerhake et al. 1998; Hirata et al. 1998). In contrast, in the pulmonary circulation, neutrophils emigrate from the pulmonary capillary bed, where spatial constraints preclude rolling, and even under normal flow conditions neutrophils must deform to squeeze through the narrow pulmonary capillaries (Doerschuk et al. 1993). This leads to a modest prolongation in neutrophil transit time in the lung under physiologic conditions (see above); however, inflammatory challenge has a major effect on the biomechanical properties of neutrophils and results in a significant increase in the sequestration of these cells in the pulmonary microvasculature. In response to inflammatory cytokines, and in a model of bacterial pneumonia (Yoshida et al. 2006), neutrophils become stiffer and less deformable due to changes in the actin cytoskeleton and such stiffened cells are preferentially sequestered in pulmonary capillaries. Once the neutrophils have arrested they may subsequently exit the capillary, and the dependence of this process on CAMs is highly variable according to the causative inflammatory insult (Doerschuk 2000; Reutershan & Ley 2004). Many studies on neutrophil recruitment have been performed in vitro using isolated cell populations. More recently, the role of other cellular components of the immune system in this process has been investigated in vivo, revealing a new layer of complexity. Neutrophils may be recruited to the lungs by the products of alveolar macrophages or mast cells, but they are themselves a potent source of cytokines and chemo-

kines, thus initiating a possible feed-forward inflammatory loop. Neutrophil–platelet aggregates have been detected in many inflammatory conditions and such aggregates may be more prone to sequestration due to their biomechanical properties; additionally, platelets adherent to the endothelium may interact with circulating leukocytes to promote their recruitment. Additionally, platelets and neutrophils may activate each other and there are intriguing suggestions that such interactions may enhance neutrophil-mediated tissue injury (Singbartl et al. 2001). Finally, as noted above, neutrophils may also promote the recruitment of eosinophils under certain conditions, thereby aggravating allergic inflammatory states (Kikuchi et al. 2006).

Neutrophil transmigration To exit the circulation, adherent neutrophils squeeze between endothelial cells and through the basement membrane and pericyte layer (diapedesis), usually with minimal disruption of vascular integrity. Engagement of leukocyte–endothelial cell adhesion molecule interactions leads to “loosening” of the interendothelial cell junctions with loss of VE-cadherin (Shaw et al. 2001) to allow the leukocyte to transmigrate (Fig. 13.4d). Endothelial cell-selective adhesion molecule (ESAM) has likewise been implicated in triggering the opening of endothelial cell contacts (Wegmann et al. 2006). Sequential homophilic interactions between PECAM-1 and CD99 expressed on both leukocyte and endothelial cells, plus interactions of endothelial cell junctional adhesion molecules (JAMs) with neutrophil JAMs and additionally neutrophil integrins, also seem to be integral to this process (reviewed by Muller 2003) (Fig. 13.4d). Endothelial cells adjacent to transmigrating neutrophils experience a rise in intracellular free calcium, which leads to endothelial retraction and is essential for diapedesis to occur. The second key barrier of the venular wall that the neutrophil must traverse is the perivascular basement membrane (BM). Transmigrated neutrophils express the protease elastase on their surface (Wang et al. 2005) and proteolytic cleavage of BM constituents such as laminin is a possible (though not universally accepted) mechanism by which leukocytes are thought to penetrate this membrane. In addition, neutrophils seem to utilize areas of the BM with reduced expression of matrix proteins as specific exit points (Wang et al. 2006).

Neutrophil chemotactic factors Interaction of chemoattractant molecules with specific neutrophil cell-surface receptors engages signaling mechanisms that result in the development of polarity and directional movement. The principal groups of neutrophil chemoattractants are (i) products of the complement (C5a) and eicosanoid (LTB4 and PAF) cascades, (ii) bacterial-derived peptides (classically N-formyl-methionyl-leucyl-phenylalanine or fMLP) and (iii) members of the chemokine family.

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Allergen–antibody complexes stimulate mast cells and macrophages to release LTB4, a potent chemoattractant for both neutrophils and eosinophils. Neutrophils also release LTB4 on stimulation, conferring the potential for positive feedback. Studies in mice lacking the LTB4 receptor BLT1 (Medoff et al. 2006) demonstrate that it is required for the early recruitment of both eosinophils and neutrophils to the airways following allergen challenge, but that the later phases of granulocyte recruitment and the development of airway hyperresponsiveness were not affected. Mechanisms leading to airway inflammation in asthma are likely to be complex and redundant, and it is possible that the LTB4 pathway may play a more important role in those asthmatics with neutrophil-predominant inflammation. fMLP and several other bacterial-derived and synthetic peptides bind the high-affinity formyl peptide receptor (FPR) (Selvatici et al. 2006) and mice lacking this receptor exhibit impaired antibacterial host defense (Gao et al. 1999). More recently, annexin 1 (lipocortin 1) has been identified as an endogenous FPR agonist (Walther et al. 2000). The loweraffinity FPR homolog FPR1 has also now been found to interact with other host-derived chemotactic agonists, including serum amyloid A (Su et al. 1999), peptides derived from prion protein (Le et al. 2001), and the neutrophil-granule protein cathelicidin (Yang et al. 2000). The significance of these endogenous agonists for FPRs is as yet uncertain, but they suggest a much broader role in inflammatory processes than has been hitherto appreciated. Chemokines are an extensive family of small heparinbinding proteins that direct the movement of leukocytes in both physiologic and pathologic situations (Baggiolini & Loetscher 2000; Charo & Ransohoff 2006). There are approximately 50 chemokines organized into four families: the CC chemokines (where the first two of four cysteine residues are adjacent to each other) act on T cells, monocytes, eosinophils, basophils, mast cells, and dendritic cells, while some CXC chemokines (where a single amino acid is interposed between the first two cysteine residues) are powerful neutrophil chemoattractants. Neutrophils express the chemokine receptors CXCR1 and CXCR2, enabling them to bind CXCL8 (IL-8, the prototypical neutrophil chemokine), CXCL1 (Gro-α), CXCL2 (Gro-β), CXCL5 (ENA-78), CXCL3, and CXCL6. Classically, IL-8 has been implicated in the pathogenesis of ARDS (Donnelly et al. 1993), but elevated IL-8 in sputum or bronchoalveolar lavage (BAL) fluid has also been detected in adults suffering acute severe asthma, where it correlates with the presence of a predominantly neutrophilic inflammatory response (Fahy et al. 1995; Lamblin et al. 1998; Tonnel et al. 2001). Elevated IL-8 in sputum is also associated with markers of epithelial damage and with neutrophil elastase in children with asthma exacerbations (Yoshihara et al. 2006). Finally, IL-8 has been found to be increased in allergic bronchopulmonary aspergillosis and to be correlated with airway neutrophilia and airway obstruction (Gibson et al. 2003). Whether these signs

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of neutrophil mobilization in the airway lumen reflect an increased number of neutrophils in the surrounding airway tissue has not yet been determined. Nonetheless it seems that chemokines, and in particular IL-8, have an important role in mediating the recruitment of neutrophils in several inflammatory and allergic disease states, and have hence been identified as potential therapeutic targets (see below).

Neutrophil chemotaxis Neutrophils exiting the circulation may be exposed to gradients of the chemoattractant molecules described above, in which case they develop polarity (i.e., form distinct front and back ends) and migrate in amoeboid fashion by extension of pseudopods at the leading edge, enabling directed movement toward the source of the stimulus (chemotaxis). The development of polarity is fundamental to chemotaxis and is associated with a dramatic redistribution of cytoskeletal proteins, with enrichment of F-actin at the leading edge and assembly of myosin posteriorly (reviewed by Parent 2004 and Niggli 2003). Chemoattractants generate intracellular signals by binding transmembrane receptors coupled to heterotrimeric G proteins. This binding induces dissociation of the G protein into α and βγ subunits, which activate an array of downstream effectors. While both receptors and G proteins are uniformly or near-uniformly distributed over the cellular surface (Servant et al. 1999; Jin et al. 2000), the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), the product of class 1 phosphatidylinositol 3-kinase (PI3K), accumulates at the leading edge (Fig. 13.5), and this seems to underpin subsequent responses (Servant et al. 2000). Studies using knockout mice have suggested a critical role for the PI3K-γ isoform in this process (Hirsch et al. 2000; Li et al. 2000; Sasaki et al. 2000) and PI3K-δ has also been implicated by studies employing isoform-specific PI3K inhibitors (Sadhu et al. 2003); both PI3K-γ and PI3K-δ are highly expressed in neutrophils. Precisely how the internal PIP3 gradient is generated remains unclear, but it may involve selective recruitment of PI3Ks to the leading edge together with a concentration of phosphatases that metabolize PIP3 (PTEN in Dictyostelium, SHIP-1 in mammalian neutrophils) at the trailing edge (Li et al. 2003; Nishio et al. 2007). PIP3 activates the small GTPases Rac and CDC42, which relay signals to the actin cytoskeleton resulting in actin polymerization and pseudopod protrusion (Srinivasan et al. 2003) (Fig. 13.5); activated Rac may additionally promote PIP3 formation in a feed-forward loop, reinforcing cellular polarity (Weiner et al. 2002). At the rear of the cell, in the absence of predominant PIP3-directed signaling, activation of the small GTPase RhoA seems to be important in organizing myosin-based contraction and retraction of the cell posterior (Xu et al. 2003) (Fig. 13.5). The net result of this spatially and temporally coordinated signaling process is the highly purposeful movement of the neutrophil to the source of the inflammatory cascade at speeds of up to 20 μm/min.

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Formation of protrusions at leading edge; directional locomotion

PIP3

PIP2

Ga/Gbg Ga/Gbg

PI3Kg/d Gbg

F-actin polymerization

GTP-Rac GTP-Cdc42

Direction of movement

Myosin activation

SHIP-1 PtdIns (3,4)P2

GTP-RhoA

PIP3

Tail retraction Rear release Allows directional locomotion

Fig. 13.5 Chemotaxis signaling in neutrophils. Stimulation of G protein-coupled receptors by chemoattractants leads to stimulation of phosphatidylinositol 3-kinase (PI3K)-g or PI3K-d, with the generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), activation of small GTPases (Rac and CDC42), F-actin polymerization, and hence the development of a leading edge. At the rear of the cell, PIP3 is metabolized by phosphatases such as SH2-containing inositol phosphatase (SHIP)-1, and activation of myosin leads to the development of a rear end or uropod. The net result of these diverse signaling events is the development of polarity and directional migration toward the concentration gradient. PIP2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate. (See CD-ROM for color version.)

Neutrophil activation Neutrophil phagocytosis Neutrophils and macrophages are professional phagocytes, highly evolved to recognize, engulf, and eliminate invading pathogens (Lee et al. 2003). Pathogen recognition occurs via TLRs, which initiate proinflammatory signaling pathways to trigger the innate immune response (reviewed in Wagner & Bauer 2006 and Pinheiro & Ellar 2006). TLRs identify molecular motifs of infectious agents (pathogen-associated molecular patterns); so far, expression of 10 TLRs is known in humans. The best characterized is TLR4, which recognizes LPS. Bacterial lipopeptides are recognized by TLR-1, TLR-2, and TLR-6; foreign (viral and bacterial) DNA is sensed by

TLR-3, TLR-7, TLR-8, and TLR-9; and various bacterial, protozoal, and yeast proteins are sensed by TLR2 and TLR5. The adapter proteins, myeloid differentiation factor 88 and Toll IL-1 receptor (TIR) domain-containing adapter inducing interferon (IFN)-β, are the key players in the TLR signaling cascade leading to the activation of NF-κB and interferon regulatory factor-3. Ingestion by the phagocyte is usually greatly facilitated if the target is coated (opsonized) by specific immunoglobulins (Fig. 13.6a) or by complement components (C3b), with immunoglobulins binding to neutrophil Fc receptors, principally FcγRIIa (CD32) and FcγRIIIb (CD16), and complement components binding to the β2 integrin CD11b/CD18. Receptor clustering initiates signaling events (unique to the receptor engaged), resulting in particle engulfment. In the case of Fcγ receptors, receptor phosphorylation by src-family kinases leads to recruitment and activation of the tyrosine kinase Syk (Kiefer et al. 1998), with subsequent stimulation of PI3K and local production of PIP3 at the evolving phagocytic cup. As during chemotaxis, PIP3-directed activation of Rac1 and CDC42 are thought to be required for actin assembly and hence pseudopodial extension; PIP3 additionally leads to local activation of phospholipase C (PLC) and protein kinase C (PKC), which have also been implicated in the uptake process. Since professional phagocytes can engulf multiple particles, large volumes of surface membrane may be internalized by a process that is compensated for by the delivery of cellular endomembranes to the vicinity of the nascent phagosome (Bajno et al. 2003). The newly formed and sealed phagosome is not initially antimicrobial, but must undergo maturation to acquire microbicidal enzymes and components of the NADPH oxidase (see below) that enable killing and disposal of the ingested microorganisms. In addition to their “classical” role in phagocytosis and pathogen destruction, neutrophils have also been shown to express molecules associated with antigen presentation, including major histocompatibility complex class II (DR) antigen (Sandilands et al. 2003); moreover, these molecules are translocated to the plasma membrane on treatment of neutrophils by a variety of stimuli. In the case of some pathogens, including Listeria monocytogenes, neutrophils have been shown to provide nonsecreted bacterial antigens for crosspresentation to the adaptive immune system (Tvinnereim et al. 2004) in addition to their role in innate defense against infection.

Neutrophil degranulation Neutrophil granules and secretory vesicles constitute an important reservoir of antimicrobial proteins, proteases, components of the NADPH oxidase, and a wide range of receptors for adhesion molecules, matrix proteins and bacterial products (Faurschou & Borregaard 2003). Regulated granule exocytosis is intimately involved in all aspects of neutrophil function, including recruitment, phagocytosis and

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SG

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p91 p47 P p40 p22 p67 P GTP-Rac2

ROS release P p91 p40 p67 P p47 p22 P GTP-Rac2

(b)

killing, while dysregulated extracellular release of granule contents contributes to host cell damage and organ dysfunction in disease processes. All granule subsets are defined by a phospholipid bilayer that encompasses an intragranular matrix, but the proteins within the matrix differ between the granule subsets (Table 13.1). As introduced earlier, primary (azurophil) granules bud off from the Golgi apparatus in promyelocytes, are characterized by the presence of myeloperoxidase (MPO), and contain the structurally related serine proteases neutrophil elastase, cathepsin G, and proteinase-3. Secondary (specific) granules and tertiary (gelatinase) granules are formed as a continuum in myelocytes, metamyelocytes, band cells, and segmented neutrophils, but differ in protein content and secretory properties. Specific granules are rich in antibacterial proteins

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O2–

Fig. 13.6 Phagocytosis, degranulation, and assembly of the neutrophil’s NADPH oxidase. (a) In a resting neutrophil, the components of the NADPH oxidase reside in the cytoplasm (p47phox, p67phox, p40phox, and GDP-Rac2) or on secretory vesicle (SV) membranes (gp91phox, p22phox). Granule populations (azurophil granules, AG; gelatinase granules, GG; specific granules, SG) are distributed throughout the cytoplasm. (b) On phagocytosis of a bacterium (B), components of the oxidase become activated (phosphorylation of p47phox and p67phox, conversion of GDP-Rac2 to GTP-Rac2) and both the cytoplasmic and membraneassociated proteins are assembled at the phagosomal membrane and reactive oxygen species (ROS) are generated. Granules, particularly protease-containing AGs, are discharged into the phagosome, and ROS promote their liberation from proteoglycan matrix to digest the engulfed bacterium. If the neutrophil is instead stimulated by soluble agonists, these events may occur at the plasma membrane, leading to degranulation and external ROS release. (See CD-ROM for color version.)

such as lactoferrin, transcobalamin, and lysozyme, plus the matrix metalloproteinases (MMPs) gelatinase, collagenase and leukolysin and also a number of membrane receptors and proteins including CD11b/CD18 and cytochrome b558 (Gallin 1985). Tertiary granules contain abundant gelatinase (MMP-9) but no lactoferrin. Secretory vesicles are endocytic vesicles that form a reservoir to replenish plasma membrane lost during phagocytosis, and are also an important source of membrane receptors, which can be mobilized to allow the neutrophil to respond more readily to environmental signals. Such is the functional diversity of these granules that neutrophils can attack bacteria in a number of ways. For example, constituents such as lysozyme exert antimicrobial activity directly by disrupting bacterial membranes, lactoferrin binds and sequesters iron and copper to interfere with the

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Table 13.1 Neutrophil granule constituents. Azurophil

Specific

Gelatinase

Secretory

Membrane proteins

CD63, CD66

CD11b/CD18 (Mac1), CD18, CD66, CD67, Rap, receptors (TNF, fMLP, laminin, fibronectin, vitronectin), cytochrome b558

CD11b/CD18, cytochrome b558, LAMP-2, NRAMP-1, SNAPs, receptors for fMLP IgE, laminin

CD10, CD11b/CD18, CD13, CD14, CD16, CD35, CD45, alkaline phosphatase, cytochrome b558, decay accelerating factor, fMLP receptor, CR1

Proteinases

Elastase, cathepsin G, proteinase-3,

Collagenase, gelatinase, leukolysin

Gelatinase

Enzymes

MPO, lysozyme, azurocidain, neuraminidase

Lysozyme, neuraminidase

MPO, lysozyme

Hydrolases

Cathepsins B and D, bgalactosidase, b-glucuronidase, a-mannosidase, N-acetyl-bglucosaminidase

Lactoferrin, apolactoferrin, vitamin

Inhibitors

a1-antitrypsin, heparin-binding protein

B12-binding protein, transcobalamin-1, histaminase, heparinase

Others

Defensins, BPI, ubiquitin

Lipocalin, b2-microglobulin

Acetyltransferase

Plasma proteins

BPI, bactericidal/permeability-inducing protein; LAMP, lysosome-associated membrane protein; NRAMP, Natural resistance-associated protein; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein. See text for definition of other abbreviations.

metabolic pathways in bacteria, and MPO is both directly toxic and participates in the formation of oxygen radicals (see below). Several of the constituents, for example defensins and azurocidin, are also chemotactic toward CD4 and CD8 T cells, thus amplifying and diversifying the inflammatory response. The intracellular granule populations also differ in their availability for exocytosis and are thus mobilized in a hierarchical fashion (Sengelov et al. 1995). Secretory vesicles have the highest propensity for extracellular release, and are recruited to the cell surface by low concentrations of soluble agonists such as TNF-α, LPS, fMLP, and C5a, enabling the rapid deployment of additional membrane receptors to enhance cellular responses. Tertiary, secondary, and primary granules are progressively less prone to mobilize to the cell surface (a control mechanism reducing the likelihood of extracellular release of the potentially destructive serine proteases). The hierarchical mobilization of neutrophil granule populations can be reproduced in vitro by progressive elevations of intracellular calcium (Sengelov et al. 1993), suggesting that activation of PLC may be critical in controlling this response. Primary granule exocytosis is preferentially targeted to the emerging phagosome, a process that seems dependent on the microtubule network (Tapper et al. 2002) and on the GTPase Rac2 (Abdel-Latif et al. 2004). The precise molecular mechanisms providing this fine control of granule deployment are not

fully understood, but are likely to involve differential granule expression of SNARE (soluble N-ethylmaleimide-sensitive attachment protein receptor) proteins and differences in SNARE complex formation (Mollinedo et al. 2006). In addition to the classical degranulation response, neutrophils have also been shown to release a combination of primary and secondary granule proteins together with chromatin, forming an extracellular fiber network (so-called neutrophil extracellular traps). These neutrophil extracellular traps have been shown to be formed both in vitro and in vivo, to trap bacteria, and to kill both bacteria and fungi (Brinkmann et al. 2004; Urban et al. 2006). While the importance of this recently described phenomenon is as yet unclear, it provides another weapon in the neutrophil armamentarium, and likewise may contribute to the injurious potential of these cells. The essential role of neutrophil proteases in bacterial killing has been confirmed by gene targeting. Mice deficient in neutrophil elastase exhibit profound susceptibility to Gramnegative organisms and to some fungi, but not Gram-positive bacteria (Belaaouaj et al. 1998). While the deletion of cathepsin G alone had little effect on bacterial killing, mice lacking both cathepsin G and elastase are susceptible to both Grampositive and Gram-negative organisms (Tkalcevic et al. 2000), conferring a phenotype similar to that seen in chronic granulomatous disease (CGD), in which there is defective NADPH oxidase function (see below). Thus a combination of a

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functional respiratory burst and an intact degranulation response is required for optimal bacterial killing (see below).

Neutrophil NADPH oxidase The neutrophil NADPH oxidase is a multicomponent enzyme with a redox core that allows electron transfer from cytoplasmic NADPH to intraphagosomal or extracellular molecular oxygen, generating superoxide anions (O2–). The oxidase is composed of membrane-associated (p22phox, gp91phox) and cytosolic (p67phox, p47phox, p40phox, and Rac2) components (see Fig. 13.6a) and is assembled when the neutrophil becomes activated, by translocation of the cytosolic elements to dock with the membrane proteins. In resting cells, the membraneassociated cytochrome b558 (a heterodimer comprising one molecule of p22phox and one molecule of gp91phox) is predominantly located within the intracellular granule membranes (Fig. 13.6a), with only 15% present at the plasma membrane (Calafat et al. 1993); much of the initial oxidase assembly occurs on the intracellular granules, with subsequent movement to the phagosomes or to the plasma membrane (Kobayashi et al. 1998). The molecular interactions between the oxidase components and the mechanisms by which the multimeric oxidase is assembled have been extensively reviewed elsewhere (Roos et al. 2003; Sheppard et al. 2005). Neutrophil priming and/or activation results in phosphorylation of p47phox at several serine residues. The precise residues and the kinase responsible may vary according to the stimulus, but isoforms of PKC activated by PI3K have been heavily implicated. p47phox phosphorylation, and to a lesser extent phosphorylation of p67phox, induces conformational changes in the cytosolic complex of p47phox/p67phox/p40phox, thereby exposing new binding domains (SH3, proline-rich and PX domains). This results in translocation of the cytosolic phox proteins to the membrane and their association with cytochrome b558 (see Fig. 13.6b). Membrane recruitment may be further enhanced by the local activation of PI3Ks with consequent generation of 3-phosphorylated lipids (particularly phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4bisphosphate), which interact with the PX domains of p40phox and p47phox (Ellson et al. 2001; Kanai et al. 2001). Neutrophil activation also results in conversion of inactive GDP-Rac2 to active GTP-Rac2, which migrates to the membrane and binds p67phox. The assembled NADPH oxidase enables electron transfer that generates O2−, a short-lived radical that can be converted to other cytotoxic products (ROS) such as hydrogen peroxide and hypochlorous acid, or which can react with nitric oxide to form reactive nitrogen species. The importance of the NADPH oxidase in bacterial killing is exemplified by the rare genetic condition CGD. In this disease, mutations in genes coding for the oxidase components (gp91phox, p22phox, p47phox, or p67phox) lead to a failure of ROS generation and a profound immunodeficiency state characterized by infections with Staphylococcus aureus, Aspergillus, enteric

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Gram-negative bacteria, and Burkholderia cepacia. While no cases of CGD have been found to be due to mutations in p40phox, recent evidence from knockout mice (Ellson et al. 2006) and studies employing cellular reconstitution (Suh et al. 2006) have supported the critical role this protein plays in the generation of ROS at the phagosome. The mechanism by which such phagosomal ROS kill bacteria was assumed to be direct toxicity, but elegant experiments (Reeves et al. 2002) have suggested that an indirect mechanism is important. The influx of electrons consequent on NADPH oxidase activity results in influx of potassium ions (charge compensation), which are instrumental in liberating the serine proteases elastase, cathepsin, and proteinase-3 from the proteoglycan matrix within the granules, and it is suggested that these liberated proteases are the predominant microbicidal factor (Segal 2005). Generation of ROS in response to phagocytosed particles is tightly restricted to the phagosomal membrane, and soluble stimuli such as C5a and formylated peptides liberate minimal extracellular ROS release from quiescent neutrophils. However, cells that have been primed by prior engagement of adhesion receptors, by cytokines such as TNF-α, or by bacterial LPS exhibit massive upregulation (10–20 fold) of NADPH oxidase activity at the plasma membrane (see Fig. 13.6b), potentially contributing to tissue injury in a range of disease states (Condliffe et al. 1998). Degranulation responses are similarly augmented. The molecular basis for priming is complex and poorly understood, but enhanced activation of PI3-kinase, phosphorylation of p47phox and recruitment of tyrosine kinases may all contribute to the establishment of the primed state.

Neutrophil deactivation and removal The intense histotoxic potential of the neutrophil dictates that powerful control mechanisms need to be in place to regulate the activation status of these cells. As indicated, the normal short half-life of these cells and the presence of a marginated pool and significant bone marrow reserve of mature cells support a system that can respond rapidly to any demand on neutrophil numbers. In the same manner, healthy circulating cells fail to generate a large respiratory burst or extracellular degranulation response when challenged unless first primed (see above). Moreover, in contrast to unprimed cells, fully primed/activated neutrophils tend to be poorly motile, which may trap these cells within the immediate inflamed site. Priming of circulating neutrophils is a feature of several systemic inflammatory conditions, including vasculitis, graft-versus-host disease, and inflammatory bowel disease, and these cells can be identified by their polarized (“shape-change”) morphology, the upregulation of their cell-surface β2 integrins, and shedding of CD62L (Condliffe et al. 1996). As a consequence these cells are held up within the pulmonary capillary bed, but do not cause lung injury unless there is a secondary trigger, for example local

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infection, causing tissue migration and activation. It is also apparent that, at least in vitro, neutrophils can recover from this primed state and return to their previous quiescent form (Kitchen et al. 1996). Unfortunately, very little is known about the capacity for neutrophils to de-prime in vivo or the role of the lung and the spleen in this process. As described below, there is also a major interaction between neutrophil priming and survival, with the majority of priming agents having the further function of delaying apoptosis, the second vital control mechanism regulating the function and fate of these cells.

Neutrophil apoptosis Despite the importance of tissue residence time and the rate of removal of neutrophils in the control of neutrophil tissue “load,” there has been little formal study of the mechanisms controlling these events. It had been widely assumed that neutrophils inevitably undergo necrosis and disintegrate in situ before removal of their fragments by local macrophages (Hurley 1983). However, if this was the case, healthy tissues would be continuously exposed to injurious neutrophil contents. Although a number of pathologic descriptions have favored neutrophil necrosis as a major mechanism operating in inflammation, most of these examples have been derived from histologic observation of diseased tissues rather than “beneficial” inflammatory responses, such as the response to infection. Furthermore, there has been evidence from the classical observations of Metchnikoff (1886) more than a century ago of an alternative fate for neutrophils, whereby the intact cell is removed by local macrophages. More recently, it has been shown that a major mechanism available for the removal of intact neutrophils and other granulocytes at the inflamed site is the process of apoptosis or programmed cell death, which has major implications for the control of inflammation (Haslett 1992, 1999). There is now clear evidence of a role for apoptosis in the clearance of neutrophils from tissues in a variety of acute inflammatory disorders (Grigg et al. 1991; Cox et al. 1995; Brazil et al. 2005; Rossi et al. 2006). Moreover, it appears that apoptosis represents a pivotal control point that determines the functional longevity of granulocytes and which can be modulated by a variety of inflammatory mediators. In mice, a distinct subpopulation of circulating neutrophils has recently been identified that are CD54high and CXCR1low and appear to represent cells that have undergone reverse endothelial cell transmigration, from an inflamed site back into the circulation (Buckley et al. 2006). These cells appear to have a delayed rate of apoptosis and can account for up to 2% of the circulating neutrophil population. Thus neutrophil exit strategies include apoptosis, transepithelial migration into the airways and bowel, transendothelial migration back into the circulation, and necrosis; clearly the balance is dictated by the nature, site, and stage of the inflammatory response. Unlike necrosis, apoptosis is a process that occurs where death is predictable or physiologic, such as thymic involution,

or where cell turnover is physiologically rapid, for example crypt cells in the gut epithelium (Wyllie et al. 1980). During apoptosis, ultrastructural studies show that cells shrink yet the plasma membrane remains intact, the ability to exclude vital dyes is preserved, and the cell retains organelles including granules (Fig. 13.7). Apoptotic cells are ingested very rapidly by phagocytes (particularly macrophages) in vivo; for example, in tissue sections of the remodeling embryo, apoptotic cells are rarely seen outside phagocytes (Koenig & Yoder 2004). Furthermore, in these and other examples of programmed cell death there is no evidence of local tissue injury or the induction of an inflammatory response, suggesting that apoptosis may represent a tissue injury-limiting mechanism for the removal of neutrophils. Human neutrophils undergo spontaneous apoptosis both in vitro and in vivo and this process has been proposed as a key mechanism underlying the resolution of granulocytic inflammation. Following the emigration of neutrophils into tissues, a switch from prostaglandin and leukotriene production to lipoxin formation occurs and this, together with the local production of resolvins and protectins from docosahexaenoic acid, serves to limit further neutrophil recruitment and initiate apoptosis (Serhan & Savill 2005). The process of apoptosis involves the promotion of controlled cell death while maintaining cellular integrity; with the possible exception that apoptotic neutrophils may still be able to release macrophage migration inhibitory factor (MIF) (Daryadel et al. 2006), this process prevents the release of hazardous proinflammatory mediators and promotes the recognition and

Fig. 13.7 Electron micrograph of an apoptotic human neutrophil. Note the intact cell membrane, the retention of granules, and the nuclear chromatin condensation and cytoplasmic vacuolation that characterize apoptosis.

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Regulation of neutrophil apoptosis: LPS, IL-1b, TNF-a, IL-6, IFN-g G-CSF, GM-CSF, C5a, IL-8, NGF calcium, cAMP, corticosteroids Normal cell

INHIBIT

Apoptotic cell

ACCELERATE E. coli ingestion Oil red ingestion Peritoneal dialysate UV irradiation HIV infection

TNF-a/Fas-L CD11b/CD18 cross-linking Protein synthesis inhibitors Sodium arsenite, ROS, NO IL-10, hyperosmolarity

Fig. 13.8 Factors that influence rates of neutrophil apoptosis in vitro. cAMP, cyclic AMP; IFN, interferon; NGF, nerve growth factor; NO, nitric oxide; UV, ultraviolet. See text for definition of other abbreviations. (See CD-ROM for color version.)

phagocytic elimination of effete cells from the site of inflammation. Thus neutrophil apoptosis modulates the duration and intensity of the inflammatory granulocytic response and hence the extent of neutrophil-mediated tissue damage. A wide range of proinflammatory cytokines and mediators that serve as neutrophil priming or activating agonists also inhibit the progression of apoptosis in vitro and these two processes are thought to be important in the establishment of chronic inflammatory diseases. GM-CSF, IL-8, LPS, C5a, LTB4, insulinlike growth factor 1, respiratory syncytial virus infection, and hypoxia have all been documented to delay neutrophil apoptosis, whereas TNF-α, Fas-L, TRAIL, and ultraviolet radiation promote apoptosis (Ward et al. 1999a; Scheel-Toellner et al. 2004; Walmsley et al. 2005; Lindemans et al. 2006) (Fig. 13.8). In addition to physiologic and pathologic stimuli, several pharmacologic agents have been reported to modify rates of apoptosis. For example, corticosteroids have been documented to promote neutrophil survival, an effect in contrast to their major pro-apoptotic effect in eosinophils (Cox 1995; Meagher et al. 1996), whereas NF-κB and cyclin-dependent kinase inhibitors accelerate this process (Ward et al. 1999b; Rossi et al. 2006).

Signaling pathways regulating neutrophil apoptosis (Fig. 13.9) Caspases are essential proteases for the initiation and execution of apoptosis. The caspase family is subdivided into three groups, initiators (caspase-8, -9, and -10) and executioners (caspase-3, -6, and -7) of apoptosis, and cyokine-processing (caspase-1, -4, and -5). The initiators sense death signals and activate downstream executioner caspases, which cleave cellular substrates mediating the changes associated with apoptosis. Neutrophil apoptosis can be initiated by activation of either the extrinsic or the intrinsic cell death pathways (Akgul & Edwards 2003). The extrinsic pathway is initiated by death receptor–ligand interaction (e.g., Fas/FasL, TNF-α/

308

TNF-R1 and -R2), leading to the formation of the deathinducing signaling complex and the cleavage of caspase-8. The intrinsic (or stress) pathway, typically initiated by ultraviolet radiation, rapid temperature shifts, or oxidant damage, leads to increased mitochondrial membrane permeability and a reduction in membrane potential; as a result cytochrome c and apoptosis-inducing factor are liberated from the mitochondria, activating caspase-9. Activation of the initiator complexes (Apaf-1, caspase-8) by either of these routes leads to cleavage of the effector caspases caspase-3 and caspase-7, the main proteolytic executioners of cell death. The intrinsic and extrinsic pathways can also interact to amplify the death initiation signal since the pro-apoptotic Bcl-2 protein Bid is cleaved by caspase-8 and translocates to the mitochondria, resulting in activation of Bak/Bax (Thornberry & Lazebnik 1998). Caspase-10, in contrast, is activated in spontaneous but not death receptor-mediated neutrophil apoptosis (Thornberry, Lazebnik & Goepel et al. 1998). The differential expression and phosphorylation status of Bcl-2 and Bcl-2-like proteins, which serve as a common checkpoint for several distinct death signaling pathways, appear to be important in controlling the apoptotic threshold of neutrophils. Pro- and anti-apoptotic members of the Bcl-2 family regulate death signaling through their ability to form complex homodimers and heterodimers, which ultimately influence the insertion of Bax and Bax-like proteins into the outer mitochondrial membrane. This event plays a key role in triggering neutrophil apoptosis and results in the release of cytochrome c and activation of the caspase-9/Apaf-1 apoptosome (Dibbert et al. 1999; Pryde et al. 2000). High levels of pro-apoptotic Bcl-2 family members have been identified in normal neutrophils and the presence of these may account for the short half-life of the neutrophil. Both Bax and Bak have been identified at the mRNA and protein level in mature neutrophils (Weinmann et al. 1999; Santos-Beneit & Mollinedo 2000); while the selective deletion of either Bax or Bak in mice does not influence neutrophil longevity, in dual Bax–/–/Bak–/– animals there is a significant increase in neutrophil accumulation suggesting a degree of functional crossover between these proteins (Strasser 2005). Neutrophils also express “BH3-only” members of the Bcl-2 family, such as Bad and Bim; deletion and mutagenesis analysis of the BH3 domain have shown this to be the minimal death sequence required for heterodimerization and the promotion of apoptosis (Ottilie et al. 1997). These BH3-only proteins show no intrinsic or independent cell destructive properties; instead they appear to function as dominant inhibitors of the anti-apoptotic Bcl-2-like proteins. Bad has been reported to be phosphorylated at Ser 112 and Ser 136 by GM-CSF, which was associated with its translocation into the cytosol (Cowburn et al. 2002). Phosphorylated Bad is unable to bind to Bcl-XL, and consequently Bcl-XL can block Bax-like proteins and delay apoptosis. Genetic deletion of Bim in mice again promotes the accumulation of neutrophils

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Cytokine withdrawal

Death receptor CD9SL

TNF-a

CD9SL Plasma membrane

TRAFF

TRADD

PUMA

BIM

BAD

HRK

Bcl-XL A1 Mcl-1 Pro-survival BCl-2-family

NOXA PUMA BIM P53

NOXA PUMA

DNA damage

Bid

FADD Pre-caspase-8

Caspase-8

Effector caspase (caspase 3)

BAX/BAK-like protein

Mitochondria

Cell death

Cytochrome c

Effector caspase (caspase 3) APAF1

Caspase-9 Caspase-9

Fig.13.9 Signaling pathways in neutrophil apoptosis.

in vivo and prolongs neutrophil survival in vitro (Strasser et al. 2000; Strasser 2005). Neutrophils appear to express a rather limited and individual array of anti-apoptotic Bcl-2 family members. Hence, mature circulating neutrophils do not express Bcl-2 itself but rather Mcl-1 and A1 with Bcl-XL also reported at mRNA

level. The expression of Mcl-1 has been described as transient, and in the absence of survival stimuli the protein is rapidly degraded below a protective threshold (Moulding et al. 1998). Notably, GM-CSF increases or maintains Mcl-1 and A1 expression in neutrophils in vitro (Derouet et al. 2004).

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Several intracellular signaling pathways have been proposed as involved in cytokine-mediated inhibition of neutrophil apoptosis. For GM-CSF (the growth factor most clearly implicated in the aberrant survival of neutrophils in ARDS), tyrosine phosphorylation of Lyn and subsequent activation of the JAK/STAT pathway together with activation of the PI3K and p42/44 mitogen-activated protein kinase (MAPK) pathways appear to be the dominant survival routes. Vlahos et al. (1995) first identified the contribution of the PI3K/Akt pathway in GM-CSF-stimulated neutrophil survival, with possible downstream targets including phosphorylation of the pro-apoptotic BH3-only Bcl-2 family member protein Bad, suppression of BAX translocation to the mitochondria, and phosphorylation of the inhibitor of NF-κB, IκB (Ward et al. 1999a; Cowburn et al. 2005; Walmsley et al. 2005; see below). The role of MAPK in neutrophil survival is more controversial, with some groups reporting only a marginal effect of specific inhibitors (Cowburn et al. 2002), whereas others report significant attenuation of GM-CSF-induced survival (Klein et al. 2000). TNF-α is a complex pluripotent cytokine, first reported to inhibit neutrophil apoptosis through activation of NF-κB (Colotta et al. 1992), but subsequently demonstrated to also stimulate apoptosis at early times via a caspase-dependent mechanism requiring the coligation of both TNF-R1 and TNF-R2 (Murray et al. 1997). NF-κB is a key transcription activator of several anti-apoptotic proteins including Bcl-2, Bcl-X, Bcl-2A1, and IL-8 (Chen et al. 2000; Glasgow et al. 2001). The activation of of NF-κB is regulated by a complex family of proteins termed IκBα/β/ε that sequester the transcription factor in an inactivated state in the cytosol. Phosphorylation of the IκB complex leads to its polyubiquitination and subsequent degradation, enabling NF-κB to translocate to the nucleus. NF-κB may also be important in the regulation of constitutive rates of neutrophil apoptosis; hence, under in vitro culture conditions both IκB and NF-κB expression decreases in these cells in a time-dependent manner and this may represent an essential trigger for the onset of apoptosis (Walmsley et al. 2005). The central role of NF-κB in neutrophil survival is reinforced by studies demonstrating that the introduction of a NEMO (NF-κB essential modulator)-binding peptide into the neutrophil using an HIV-TAT transduction shuttle causes selective inhibition of IκKγ (IκB-kinase-γ; NEMO)/IκKβ interaction and NF-κB activation, and a major increase in neutrophil apoptosis (Choi et al. 2003).

Phagocytic removal of apoptotic neutrophils The presence of neutrophils in tissues is predominantly observed at injured or inflamed sites, from which their effective elimination is essential for resolution of the inflammatory response. Intact apoptotic neutrophils can be cleared from tissues by phagocytosis, principally by inflammatory and tissue resident macrophages. Neutrophil apoptosis triggers this recognition and clearance event, and the process

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of phagocytosis initiates the migration of engorged macrophages from the inflamed site to the draining lymphatic system. Neutrophil apoptosis is associated with downregulation of several cell-surface proteins including CD16 and CD62L, as well as exposure of phoshatidylserine (PS) and “enabled” FcγRII that supports the binding of immune complexes (Dransfield & Rossi 2004). Bridging molecules including thrombospondin-1 appear to enhance the binding of apoptotic cells to phagocytes either directly or indirectly. The interaction of oxidized PS and these other bridging molecules with phagocyte receptors, including αvβ3 integrin and CD36, engages apoptotic cells (Savill et al. 1991, 1992; Greenberg et al. 2006). The involvement of the macrophage PS receptor in the process of apoptotic cell uptake is currently uncertain (Fadok et al. 1992; Devitt et al. 2003). The uptake of apoptotic neutrophils by inflammatory macrophages stimulates the release and activation of transforming growth factor (TGF)β1 and other antiinflammatory factors as a further mechanism to facilitate the resolution of the inflammatory response (Fadok et al. 1998). Finally, semiprofessional phagocytes, such as fibroblasts and mesangial cells, have the capacity to recognize and ingest apoptotic neutrophils but not with the same facility or capacity as inflammatory macrophages. The in vivo significance of this latter observation is uncertain but it could represent a “back-up” mechanism for the macrophage system. Thus apoptosis not only determines the functional longevity of granulocytes at the inflamed site but can be modulated and controlled by external mediators of relevance to the control of inflammation. In contrast with necrosis, it provides a neutrophil removal mechanism that again is influenced by inflammatory mediators and which, by a variety of mechanisms, would tend to limit rather than promote bystander injury to healthy tissues.

Neutrophils in disease Role of neutrophils in inflammatory disease Depletion of neutrophils by cytotoxic drugs confers an increased risk of bacterial and fungal sepsis, proportional to the decrease in the neutrophil count; rare genetic diseases that compromise neutrophil function likewise lead to recurrent infections. To evade killing by the innate immune system, many bacteria have evolved virulence factors to circumvent specific neutrophil functions. For example, certain Salmonella species can interfere with the assembly of the NADPH oxidase (Vazquez-Torres et al. 2000; Gallois et al. 2001), certain pathogenic streptococci inhibit azurophil granule fusion with phagosomes (Staali et al. 2006), and Anaplasma phagocytophila (the causative agent of tick-borne ehrlichiosis) downregulates the expression of oxidase components (Carlyon et al. 2002; Thomas et al. 2005). Thus pathogens and the host immune response are in a perpetual arms race, and neutrophils

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comprise the frontline defense. Not surprisingly, given the potency of the neutrophil’s bactericidal armamentarium, host damage from “friendly fire” may occur, and in some cases have serious consequences. Neutrophil-mediated vascular endothelial injury, both direct and indirect, has been implicated in the genesis of inflammatory edema in several animal models and human disease states. Neutrophil numbers correlate spatially and temporally with edema formation in simple models of inflammation, for example carrageenan-induced paw edema in rodents (Houle et al. 2005; Rocha et al. 2006). Similar correlations can be seen in more complex animal models, e.g., adjuvant-mediated arthritis (Barsante et al. 2005) and corneal inflammation (Sonoda et al. 2005), and in human inflammatory conditions such as wound healing in pressure ulcers (Diegelmann 2003). Multiple effectors may summate to induce vascular leakage; for example, both natural killer cells and neutrophils are required for IL-2-induced pulmonary vascular leak syndrome (Assier et al. 2004). In models of cerebral ischemia, the presence of neutrophils and the expression of MMP-9 have been associated with blood–brain barrier leakage (Fujimura et al. 1999; Rosell et al. 2006), but neutrophil depletion did not influence several indicators of ischemic brain damage including cerebral edema and infarct size (Harris et al. 2005). Thus the importance of neutrophilic inflammation in edema formation varies according to the stimulus or disease state. Neutrophils can migrate to tissues, perform their phagocytic and microbicidal functions, and be cleared from the inflammatory site without causing tissue injury, as occurs in lobar pneumonia (Haslett 1999). The activation status of the cell and the precise anatomic location at the time of activation seem to be crucial in determining whether a beneficial or a harmful response is elicited. Priming of neutrophils has been shown to be a requirement for neutrophil-induced endothelial injury in vitro (Smedly et al. 1986; Jacobi et al. 2006) and primed neutrophils have been demonstrated either in the circulation or at the sites of tissue injury in a variety of disease states (El Benna et al. 2002; Bhatia et al. 2006; Chen et al. 2006). Close apposition of the neutrophil with a host cell is likewise thought to facilitate injury, enabling labile ROS to reach and react with target proteins and excluding tissue antiproteases from the interacting surfaces (Campbell et al. 1982; Rice & Weiss 1990). Catalytically active neutrophil elastase has been demonstrated at the neutrophil plasma membrane; indeed, in vitro studies have shown that up to 12% of stored human neutrophil elastase is present at the cell surface after priming with TNF-α or activation with IL-8 (Owen et al. 1997). The protease/antiprotease theory of emphysema postulates that an imbalance between neutrophilderived proteases (principally elastase) and locally produced antiproteases (such as α1-antitrypsin) leads to the destruction of matrix proteins such as elastin and hence to the airspace enlargement typical of this condition. Factors such as cigarette smoking and antiprotease deficiency (α1-antitrypsin deficiency)

are thought to tip the balance in favor of protease-mediated tissue injury. While these mechanisms are still thought to have an important role in some forms of tissue injury, neutrophils are no longer believed to be the only source of elastolytic enzymes within the lung, and the role of MMPs produced by many cell types including macrophages is increasingly felt to be of significance (Parks & Shapiro 2001). The deleterious role of the neutrophil in inflammatory disease has been studied in greatest depth in ARDS, a state characterized by high-permeability pulmonary edema and refractory hypoxemia occurring in the setting of the systemic inflammatory response syndrome. This condition serves as a paradigm for other disease states in which the neutrophil is a key player. Neutrophil depletion is protective in a range of animal models of acute lung injury (Flick et al. 1981; Heflin & Brigham 1981; Shasby et al. 1982; Looney et al. 2006), and in patients with ARDS the degree of neutrophilic inflammation correlates with the severity of lung injury (Fowler et al. 1987). BAL fluid concentrations of the neutrophil chemokine IL-8 predict the development of ARDS in an at-risk trauma group (Donnelly et al. 1993) and an excess of neutrophil proteases (Lee et al. 1981; Christner et al. 1985) have likewise been identified in BAL fluid from patients with ARDS. Zimmerman et al. (1983) found evidence of activated neutrophils in the pulmonary circulation of patients with ARDS, and the extent of NF-κB activity in peripheral blood neutrophils predicts the outcome of ARDS (Yang et al. 2003). Thus neutrophil recruitment and activation status appear to correlate with the development and clinical course of ARDS, and strategies that prevent neutrophil recruitment are protective in experimental models. Neutrophils are also implicated to varying degrees in the pathogenesis of a wide range of other inflammatory conditions with various targets (Table 13.2). Such neutrophilic injury may be localized, for example to an acutely inflamed joint in crystal-induced arthropathy, or occur in the setting of a multisystem disorder such as Wegener’s granulomatosis. A characteristic feature of Wegener’s granulomatosis is the presence of pathogenic autoantibodies directed against neutrophil proteins (MPO and proteinase-3); interaction of these antibodies with their target antigens results in intense perivascular and systemic activation of neutrophils, contributing to widespread vasculitic injury. Thus in the future, either local targeting of neutrophils at the disease site or inhibition of specific neutrophil activation pathways may provide treatments for inflammatory diseases where current treatments are ineffective or rely on generalized immunosuppression (see below). Finally, recent research has suggested that failure of the innate immune response to clear pathogens may induce chronic inflammatory disease states, not just chronic infections. In patients with Crohn disease, trauma to the gut mucosa or skin was shown to induce defective neutrophil accumulation, and the acute inflammatory reaction to subcutaneous

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Total tissue neutrophil load

Table 13.2 Noninfectious diseases in which neutrophils are implicated. Respiratory system Acute respiratory distress syndrome Bronchiectasis (including cystic fibrosis) Chronic obstructive pulmonary disease Asthma (especially corticosteroid-resistant asthma) Silicosis Hypersensitivity pneumonitis Idiopathic pulmonary fibrosis

Disposal Apoptosis

Lifespan

Disintegration

Activation/secretion

+

+ – Neutrophil products

+

Antiproteases, antioxidants +

Tissue injury

Tissue protection

Cardiovascular system Acute coronary syndromes Gastrointestinal system Crohn disease Ulcerative colitis Acute pancreatitis Musculoskeletal system Rheumatoid arthritis Gout Chondrocalcinosis Multisystem disorders Vasculitides (ANCA positive and negative) Ischemia–reperfusion (including post transplantation and post infarct) Multiorgan failure complicating SIRS Genetic disorders Familial Mediterranean fever ANCA, antineutrophil cytoplasmic antibody; SIRS, systemic inflammatory response syndrome.

Escherichia coli was greatly attenuated, responses that were shown to be at least partly due to reduced production of IL-8 by macrophages (Marks et al. 2006). Such delayed or incomplete removal of bacteria could initiate the granulomatous inflammation seen in Crohn disease, a hypothesis strengthened by the fact that granulomatous inflammation is the hallmark of CGD. A further mechanism of ineffective neutrophil pathogen clearance leading to chronic inflammatory disease was suggested by van Zandebergen et al. (2004), who demonstrated that under certain conditions neutrophils can serve as Trojan vectors by conveying Leishmania parasites into macrophages (which phagocytose infected and apoptotic neutrophils). Thus the innate immune response must be exquisitely fine-tuned, not only to avoid tissue injury secondary to overexuberant responses, but also to achieve effective pathogen clearance so that overwhelming infection or the establishment of chronic infection/inflammation does not occur (Fig. 13.10).

Role of neutrophils in allergic disease Eosinophilic inflammation is widely regarded as the hallmark of allergic disease, and until recently the neutrophil

312

Fig. 13.10 The “balance” of injurious and protective influences in the determination of inflammatory tissue injury.

has been dismissed as having no role in this spectrum of disorders. However, recent studies have suggested that in clinically relevant subsets of patients, the neutrophil may be a key effector of “allergic” responses. Neutrophils have been demonstrated in induced sputum and in bronchial biopsies in the allergen-induced late asthmatic response (Lopuhaä et al. 2002; Kariyawasam et al. 2007). Allergen provocation was likewise associated with a prominent neutrophil influx in both the early and late phase responses in allergic rhinitis (Fransson et al. 2004). Although the role of the eosinophil in mediating mild and moderate atopic asthma is well established, eosinophilic inflammation is present in the airway lumen of only 50% of asthmatic subjects (Douwes et al. 2002), intense eosiniphilic inflammation in the setting of eosinophilic bronchitis does not induce asthma (Brightling et al. 2003), and certain antieosinophilic strategies are poorly efficacious in asthma (Bryan et al. 2000; Leckie et al. 2000; Kips et al. 2003); thus the eosinophil appears to be neither necessary nor sufficient to induce asthma alone (reviewed in Kamath et al. 2005). However, a strong association has been established between airway neutrophilia and severe asthma (Wenzel et al. 1997; Jatakanon et al. 1999; Little et al. 2002), corticosteroid-resistant asthma (Wenzel et al. 1999; Green et al. 2002), asthma exacerbations (Fahy et al. 1995; Lamblin et al. 1998; Tonnel et al. 2001), and acute fatal asthma (Sur et al. 1993). Where measured, neutrophil numbers correlate with markers of neutrophil degranulation, implying that these cells are also activated. Airway neutrophilia may be triggered by physical agents such as bacteria and viruses (Stenfors et al. 2002; Wark et al. 2002), and IL-8 may be critically involved in the recruitment of these cells to the asthmatic airway (Ordonez et al. 2000). While in severe asthma eosinophils and neutrophils are often found together, neutrophils may gradually replace eosinophils in proportion to the severity and/or duration of the disease, perhaps reflecting the ability of corticosteroids to induce eosinophil apoptosis while inhibiting this process in neutrophils (Tanizaki et al. 1993; Meagher et al.

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1996; Hauber et al. 2003). In addition to the direct effects of proteases and ROS on airway epithelia, neutrophil elastase has been shown to stimulate TGF-β release from airway smooth muscle cells (Lee et al. 2006) and to convert latent TGF-β into the biologically active form (Taipale et al. 1995; Hyytiäinen et al. 1998), suggesting a potential role in the airway remodeling that occurs in chronic asthma. Bronchoscopic studies (Martin et al. 1991) have suggested that neutrophils are in a rapid state of flux in asthma, with a short half-life (< 8 hours) in the asthmatic airway. This plasticity of airway neutrophilia is supported by an equine model of asthma, where complete resolution of the neutrophilic airway response occurred over a few days following removal of allergen challenge (Brazil et al. 2005). Thus strategies aimed at blockade of neutrophil recruitment may be efficacious in this condition, which may be of particular importance given the disproportionate healthcare resources required to manage severe and corticosteroid-resistant asthma (Serra-Batlles et al. 1998).

Antineutrophil strategies IL-8 is a major neutrophil chemoattractant released by macrophages and epithelial cells and in many lung inflammatory situations appears to account for the majority of the neutrophil chemotactic signal (Kunkel et al. 1991; Beeh & Beier 2006). Fully human anti-IL-8 antibodies and CXC-chemokine receptor antagonists have now been developed (Yang et al. 1999) and early clinical trials in conditions such as chronic obstructive pulmonary disease are beginning to be published (Mahler et al. 2004). Targeting LTB4 is also possible, either through inhibition of its synthesis via the 5-lipoxygenase pathway or by using LTB4 receptor antagonists and these agents appear to show efficacy in reducing neutrophilic inflammation at least in antigen-challenged animal models (Silbaugh et al. 2000). The final current strategy to prevent neutrophil recruitment involves the use of selectin antagonists such as TBC1269, which targets P-, L- and E-selectin and following inhalation has been shown to cause potent inhibition of allergic airway responses and neutrophilic inflammation in a sheep model of allergy (Abraham et al. 1999). The most promising targets for reducing neutrophil activation and mediator release include adenosine A2A receptor agonists (which inhibit NADPH oxidase activation, degranulation, and adherence), elastase and metalloproteinase inhibitors (especially MMP-9), anti-TNF-α strategies, phosphodiesterase IV inhibitors, macrolide antibiotics, and various antioxidants. Of these, neutralizing monoclonal antibodies against TNF-α appear to be showing most promise (Howarth et al. 2005; Berry et al. 2006). These agents are reviewed extensively elsewhere (Beeh & Beier 2006; Yamagata & Ichinose 2006). Lastly, recent insights into the molecular mechanisms that regulate neutrophil apoptosis have offered valid new targets aimed at either blocking survival signaling or driving apop-

tosis itself. Such targets include inhibition of NF-κB, PI3K-γ, a Gβγ and p101-regulated PI3K isoform with restricted expression in the neutrophil, cyclin-dependent kinases, and Bad (Ward et al. 1999b; Rossi et al. 2006). Overcoming the redundancy in neutrophil signaling and testing of these agents in animal and human models and disease settings are the immediate challenges ahead.

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Basophils: Biological Properties and Role in Allergic Diseases Gianni Marone, Giuseppe Spadaro and Arturo Genovese

Summary Basophils and mast cells are the only cells expressing the tetrameric (αβγ2) structure of the high-affinity receptor for IgE (FcεRI) and synthesizing histamine in humans. These cells differ immunologically, biochemically and pharmacologically, which suggests that they play distinct roles in the appearance and fluctuation of the allergic phenotype. Monoclonal antibodies are now available to identify specific basophil epitopes (e.g., basogranulin), representing a breakthrough in clarifying the role(s) of these cells in allergic disorders. Recent data indicate the complexity of the involvement of human basophils in allergic diseases and have shed light on their control of recruitment and activation in different human tissues. Basophils may play a role in inflammatory angiogenesis through the expression of several forms of vascular endothelial growth factor (VEGF) and their receptors. Preliminary evidence suggests that these cells are not always harmful but, in some circumstances, they might exert a protective effect by influencing certain aspects of innate and acquired immunity and allergic inflammation.

Introduction Human basophils and mast cells, described by Paul Ehrlich in 1879, are unique in that they are the only cells that both express the tetrameric (αβγ2) high-affinity receptor for IgE (FcεRI) and synthesize histamine (Ehrlich 1879). Despite these similarities, however, they differ in various aspects. Mast cells are tissue-resident cells, while basophils are normally found only in peripheral blood. Both cell types are highly mobile and readily infiltrate tissues at sites of inflammation (Ying et al. 1999; de Paulis et al. 2004a). However, mast cells and basophils have different strategic microlocalizations in the human


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compartments and synthesize different sets of proinflammatory mediators, cytokines and chemokines. For instance, basophils, not found in normal lung tissue, infiltrate the sites of allergic airway inflammation (Ying et al. 1999; KleinJan et al. 2000; de Paulis et al. 2006). A wide range of newly identified chemotactic receptors (e.g., CC and CXC chemokine receptors, c-kit, CRTH2, FPR, FPRL1, FPRL2, C3aR, C5aR), selectively displayed on basophils and mast cells, are responsible for their recruitment to different compartments of skin and lung tissues. The mast cell’s role in the pathogenesis of allergic disorders has been extensively investigated, whereas the basophil’s role is less well defined, mainly because of limitations in the experimental models available at present. There are four canonical mechanisms whereby cross-linking IgE high-affinity receptors for the IgE (FcεRI) network can induce the release of mediators from human basophils in vitro and, presumably, in vivo (Fig. 14.1). In the classical model, a multivalent antigen cross-links at least two specific IgE bound to these cells and can serve as a stimulus for histamine and cytokine release from basophils. There is also evidence that lectins such as concanavalin A induce histamine release from basophils and mast cells through interaction with the carbohydrate moiety of IgE (Magro, 1974; Siraganian & Siraganian 1974). In addition, anti-human IgE antibodies cross-link two binding sites on the Fcε region of IgE. Similarly, antibodies directed against an epitope of the α chain of human FcεRI can trigger the release of mediators from FcεRI+ cells. Finally, immune complexes containing IgG against human IgE can activate human basophils (Marone et al. 2004). A fifth mechanism was based on the observation that IgE-mediated activation of human basophils and mast cells can also be induced by endogenous (e.g., protein Fv), bacterial (e.g., protein A and protein L) and viral superallergens (e.g., gp120) (Marone et al. 2004; Marone 2007) (Fig. 14.2). The binding of certain types of monomeric IgE to FcεRI can influence murine mast cell survival, apoptosis (Asai et al. 2001) and cytokine production (Kalesnikoff et al. 2001; Lam et al. 2003; Pandey et al. 2004). Exposure of mouse bone marrow-derived mast cells to monomeric IgE induces histidine decarboxylase expression and consequently affects levels of stored histamine (Tanaka et al. 2002). Donald MacGlashan reported that three human monoclonal IgE

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Anti-lgE Allergen

IgE

IgE

FceRI e

Fig. 14.1 The four canonical mechanisms by which cross-linking of the IgE–FceRI network can induce the release of mediators from human basophils and mast cells. (a) A multivalent antigen can cross-link at least two specific IgE molecules bound to FceRI+ cells to release mediators. (b) Anti-human IgE (anti-IgE) antibodies possess two binding sites for the Fce region of human IgE and activate mediator secretion from these cells. (c) Antibodies directed against an epitope of the a chain of FceRI (anti-FceRIa) can also trigger the release of mediators. (d) Immune complexes containing IgG anti-IgE and anti-IgG can activate human basophils in vitro. (From Marone et al. 2004, with permission.)

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Fig. 14.2 A fifth potential mechanism by which endogenous (protein Fv), bacterial (protein A and protein L) and viral proteins (gp120) can activate human FceRI+ cells, acting as superallergens. (a) Protein Fv, synthesized in small amounts in normal liver, is released in biological fluids of patients with acute and chronic viral hepatitis. Protein Fv has six binding sites for the VH3 region of human immunoglobulin and is a potent stimulator of histamine and cytokine release from FceRI+ cells through the interaction with VH3+ IgE. (b) Protein A, a cell-wall protein of Staphylococcus aureus, has a classical binding site for Fcg and an alternative site that binds the Fab portion of 15–50% of human polyclonal IgM, IgA, IgG and IgE. Protein A

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induces mediator release from human basophils and mast cells through interaction with the VH3 domain of IgE. (c) Peptostreptococcus magnus is a bacterium expressing a cell-wall protein L that binds human Ig regardless of the heavy-chain class, through high-affinity interaction with k light chains, and is thus an immunoglobulin superantigen. Protein L is a potent stimulator of the activation of human FceRI+ cells through interaction with the k light chain of membrane-bound IgE. (d) HIV-1 gp120 from divergent HIV-1 isolates from different viral clades of various geographical regions activate human FceRI+ cells through interaction with membrane-bound VH3+ IgE.

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antibodies did not induce signaling or mediator release from human basophils (MacGlashan 2003). Clarification of the underlying molecular mechanism(s) and their relevance in vivo and in human in vitro models will shed further light on these observations.

Origin of human basophils Basophils circulate in human peripheral blood where they amount to less than 1% of total leukocytes. Peripheral blood also contains basophil precursors (Denburg et al. 1983). Interleukin (IL)-3 is the principal cytokine responsible for human basophil growth and differentiation (Valent et al. 1989; Valent et al. 1990) from CD34+ pluripotent progenitor cells (Kirshenbaum et al. 1992). Other cytokines, such as granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-4, IL-5, and nerve growth factor (NGF), are also important for basophil growth, differentiation, and mediator production (Denburg 1995).

Morphologic and ultrastructural characteristics of human basophils Human basophils have polylobed nuclei with condensed chromatin (Fig. 14.3). They are commonly identified by metachromatic staining with basic dyes, such as toluidine

Fig. 14.4 A basophil in the ileum of a patient with Crohn disease shows piecemeal degranulation in vivo. Empty granules (arrows), full granules and partially depleted granules are present in the cytoplasm (× 10 700). (From Dvorak 1988, with permission.)

blue. On the surface there are irregular, broad processes. The cytoplasm contains membrane-bound secretory granules, mitochondria, and small Golgi structures. The secretory granules of basophils are of two types: the most numerous are large, round, and filled with electron-dense particles and/or finely granular material. A small, paranuclear granule with homogeneous content has also been observed, but much less frequently (Dvorak 1995, 2005). Human basophils extrude secretory granules to the external microenvironment when stimulated with a variety of triggering agents. Bridging the IgE–FcεRI network in vitro triggers a series of biochemical and ultrastructural changes, termed “anaphylactic degranulation,” which culminate in fusion of cytoplasmic granule membranes with the plasma membrane (Dvorak 2000). Ann Dvorak showed that in certain immunologic responses in man the basophils progressively lost their cytoplasmic granule contents, with no evidence of anaphylactic degranulation. This vesicle-mediated release of granule content is termed “piecemeal degranulation” (Fig. 14.4). Until a few years ago, technical limitations hampered the identification of basophils infiltrating the sites of inflammation. In fact, it is hard to identify partially degranulated basophils with conventional morphologic techniques. The availability of monoclonal antibodies (BB1 and 2D7) that identify specific basophil epitopes (e.g., basogranulin) (Irani et al. 1998; McEuen et al. 2001; de Paulis et al. 2006) led to a technological breakthrough in these studies.

Mediators of human basophils

Fig. 14.3 The human peripheral blood basophil has a polylobed nucleus, irregular, short broad cell surface processes, cytoplasmic glycogen and large secretory granules filled with particles (× 18 000). (From Dvorak 1992, with permission.)

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Human basophils contain about 1 pg/cell of histamine (Fig. 14.5). Basogranulin, a basophil-specific granule protein recognized by monoclonal antibody BB1, used to identify basophils in tissues (McEuen et al. 2001; de Paulis et al. 2006), is secreted together with histamine in response to IgE-dependent

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Fig. 14.5 Human peripheral blood basophils synthesize histamine, which is stored in secretory granules as a preformed mediator (∼1 pg/cell). Basophils contain basogranulin in their secretory granules, a specific granule protein recognized by the monoclonal antibody BB1, and mature and enzymatically active tryptase at levels of less than 1% of those of mast cells. Immunologic activation of basophils leads to the production of a restricted profile of Th2-like cytokines (IL-4 and IL-13). Activated basophils also produce the chemokines IL-8/CXCL8 and MIP-1a/CCL3 and several isoforms of VEGF-A. Immunologically activated basophils produce only LTC4 (∼30 ng/106 cells); no cyclooxygenase metabolite has been conclusively demonstrated in these cells. Activated basophils also synthesize PAF and AAGPC in a ratio of 1 : 2. (From Marone et al. 2005c, with permission.) (See CD-ROM for color version.)

activation (Mochizuki et al. 2003). Basophils express mature and enzymatically active tryptase at levels less than 1% of those of mast cells (Foster et al. 2002; Jogie-Brahim et al. 2004). Immunologically activated human basophils produce leukotriene (LT)C4 (∼30 ng/106 cells). Arachidonic acid for conversion to LTC4 in basophils is supplied almost exclusively by the cytosolic group IV phospholipase (PL)A2 (Triggiani et al. 2004), even though these cells contain one or more secretory PLA2 isoforms (Hundley et al. 1998). No cyclooxygenase metabolite has been conclusively demonstrated in these cells. However, PGD2, unlike other prostaglandins, enhances histamine release from immunologically activated basophils. It also reverses the inhibition of release by drugs and hormones that activate adenylate cyclase to increase cellular cyclic AMP (cAMP) (Peters et al. 1984). Immunologically activated basophils produce two acetylated phospholipids, AAGPC and plateletactivating factor (PAF), in a ratio of 2 : 1 (Triggiani et al. 1991, Triggiani et al. 1995) which, interestingly, have different effects on basophils primed with cytokines (Brunner et al. 1991; Columbo et al. 1993a). Immunologic activation of human basophils leads to an increase in IL-4 mRNA transcription and IL-4 secretion (MacGlashan et al. 1994; Mueller et al. 1994; Gibbs et al. 1996; Ochensberger et al. 1996; Redrup et al. 1998; Genovese et al. 2003). Basophils secrete an average of 30 pg/106 cells of IL-4 (range 10– 80 pg/106 cells); IL-4 secretion starts 1 hour after stimulation and peaks at 6 hours. The kinetics of production

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of IL-13 are different: there is a lag period of 4 hours and a plateau is reached 18–24 hours after immunologic stimulation (Fig. 14.6). Under these conditions there was no evidence of interferon (IFN)-γ mRNA. Therefore it appears that human basophils are unique because they express large amounts of IL-4 and IL-13 without any Th1-like cytokine (e.g., IFN-γ) and without most of the proinflammatory cytokines [tumor necrosis factor (TNF)-α, IL-1, IL-6] (Dahinden 2000). Thus, basophils are not simply inflammation effector cells, but also play a key immunoregulatory role by skewing immune responses towards the Th2 type. Differently from mast cells, basophils express a restricted cytokine profile. The different profiles of cytokines synthesized and released by basophils and mast cells provide indirect evidence that these cells have distinct roles in the appearance and fluctuation (exacerbations and remissions) of the allergic phenotype. Basophils also express the chemokines IL-8/CXCL8 and MIP-1α/CCL3 on IgE receptor cross-linking (Li et al. 1996). Vascular endothelial growth factor (VEGF)-A has also been recently identified in the supernatants of activated basophils (de Paulis et al. 2006; Marone et al. 2007a).

Surface markers of human basophils Basophils express an impressive array of cell–cell signalling molecules and this might explain why these cells can be attracted and/or activated by a very wide variety of inflammatory and immune stimuli (Fig. 14.7). In addition to FcεRI, human basophils express the inhibitory receptor FcγRIIb (Kepley et al. 2000). Coligation of FcγRIIb and FcεRI inhibits mediator release from basophils. Of particular importance for the response to agonists is the priming effect of certain cytokines that enhance effector functions, such as mediator release and cytokine expression. IL-3, IL-5, GM-CSF, and NGF prime basophils with different potencies (IL-3 > NGF ≥ IL-5 ≥ GM-CSF) (Bischoff et al. 1990; Bischoff & Dahinden

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Fig. 14.7 Selective display of membrane receptors on human basophils. Basophils express the tetrameric high-affinity receptor for IgE (FceRI). They also express FcgRIIb, whose cross-linking inhibits mediator release, and Tolllike receptor 2 (TLR2), TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9. Basophils express various receptors for chemokines (CCR1, CCR2, CCR3, CXCR1, and CXCR4), and cytokines (IL-3Ra, IL-5Ra, GM-CSFRa, and TRAK-A), whose activation induces chemotaxis and/or mediator release. These cells express the seven-transmembrane receptor CRTH2, whose activation by prostaglandin D2 (PGD2) induces chemotaxis, and a receptor for PAF. They also express at least two receptors for cysteinyl leukotrienes (CysLTR1 and CysLTR2) and three formyl peptide receptors (FPR, FPRL1 and FPRL2). They express receptors for anaphylatoxins (C3a and C5a), for VEGF (VEGFR-2/KDR) and for IGF (IGF-1R and IGF-2R) and the urokinase plasminogen (uPA) receptor (uPAR). Human basophils express the histamine H2 receptor, whose activation inhibits the release of mediators. Basophils express low levels of LIR3 and LIR7; cross-linking of LIR7 induces the secretion of mediators, while coligation of LIR3 and FceRI inhibits mediator release. (From Marone et al. 2005c, with permission.) (See CD-ROM for color version.)

1992; Brunner et al. 1993). The priming effect of IL-3, IL-5, and GM-CSF depends on the expression of the common β chain in their receptors (Ochensberger et al. 1999). The priming and modulatory effects of NGF are due to the expression of the high-affinity receptor TRK-A (Burgi et al. 1996), and highlights the link between the nervous system and allergic inflammation. An important function of basophils is their ability to migrate from peripheral blood to sites of allergic inflammation, thanks to a complex interplay of different chemotactic factors of various origins that act on a wide spectrum of surface receptors. Basophils express various receptors for chemokines (CCR1, CCR2, CCR3, CXCR1, and CXCR4), whose activation induces chemotaxis and/or mediator release. The eotaxin receptor CCR3 is expressed on the majority (∼ 80%) of human basophils (Uguccioni et al. 1997; de Paulis et al. 2000, 2001), and also on human lung mast cells (∼ 20%) (Romagnani et al. 1999, 2000; de Paulis et al. 2000). CCR3

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is activated by eotaxin/CCL11, eotaxin-2/CCL24, eotaxin-3/ CCL26 and RANTES/CCL5 and governs mostly chemotaxis to sites of allergic inflammation. Basophils also express high levels of CCR2, which can be activated by MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, and MCP-4/CCL13. Activation of CCR2 seems mainly to cause mediator release from basophils with only weak migratory responses (Bischoff et al. 1992; Uguccioni et al. 1997). PGD2, a major mast cell mediator released during the allergic response (Schleimer et al. 1985; Genovese et al. 2000), is chemotactic for basophils, eosinophils, mast cells, and Th2 cells through activation of the CRTH2 receptor (Hirai et al. 2001). The anaphylatoxins C3a and C5a are potent chemoattractants for basophils and induce mediator release, particularly when these cells are primed with certain cytokines (IL-3, IL-5, GM-CSF) (Bischoff et al. 1990; Ochensberger et al. 1995, 1996). The effects of anaphylatoxins are due to the specific seven-transmembrane (STM) G protein-coupled receptors C3aR and C5aR. In this family of STM, de Paulis and collaborators have identified at least three receptors in human basophils that bind several natural N-formyl peptides, including the prototype N-formyl-methionyl-leucyl-phenylalanine (FMLP) (de Paulis et al. 2004a,b). Basophils express the high-affinity receptor FPR and its homologs FPR-like-1 (FPRL1) and FPR-like-2 (FPRL2). Both receptors serve as chemotactic receptors for endogenous or viral products. For example, two HIV-1 gp41 peptides act as chemoattractants for basophils by interacting with FPRL1 (de Paulis et al. 2002). It has also been found that urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for FPRL1 and FPRL2 (de Paulis et al. 2004b). These results indicate that the uPA/ uPAR system is involved in allergic disorders. Activated FcεRI+ cells express the ligand for CD40 (CD40L), which can provide the cell contact signal required for IgE synthesis by human B cells (Gauchat et al. 1993). Basophils may therefore play a key role in allergic disorders not only by producing inflammatory and fibrogenic mediators but also by directly (CD40L) and indirectly (IL-4 and IL-13) regulating IgE synthesis independently of T cells. Recent evidence indicates that human basophils contribute to various aspects of angiogenesis. They synthesize and release such potent proangiogenic factors as (VEGF). Furthermore, VEGF and placental growth factor (PlGF) are both chemotactic for basophils through the activation of VEGF receptors (Marone et al. 2005a; de Paulis et al. 2006). The leukocyte immunoglobulin-like receptors (LIRs) are a family of cell-surface receptors that include both activating and inhibitory receptors (Arm 2004). Human basophils express low levels of LIR3 and LIR7 (Sloane et al. 2004). Cross-linking of LIR7 induces the secretion of proinflammatory mediators and IL-4, whereas coligation of LIR3 and FcεRI inhibits mediator release.

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CD203c (ecto-nucleotide pyrophosphatase/phosphodiesterase 3), a transmembrane protein, has been described as being selectively expressed on basophils, mast cells and their CD34+ progenitors. As CD203c is rapidly upregulated after allergen challenge in sensitized patients, it has been proposed as a new tool for allergy diagnosis (Boumiza et al. 2003).

Evidence of basophil involvement in bronchial asthma On the basis of some apparent similarities to mast cells, basophils have often been considered (and neglected) as minor and possibly redundant “circulating mast cells.” It is now evident, however, that they differ morphologically, ultrastructurally, and biochemically and produce a different set of preformed and de novo synthesized mediators. More importantly, basophils express a restricted profile of Th2-like cytokines (IL-4 and IL-13), whereas mast cells synthesize a broad array of proinflammatory and immunoregulatory molecules (Marone et al. 2005b). These marked differences suggest that the two cells play different roles in the pathogenesis of allergic disorders. Basophils, usually confined to the circulation, are not found in normal tissues. However, they infiltrate the sites of allergic inflammation (Irani et al. 1998; Ying et al. 1999; KleinJan et al. 2000). In addition, they have been found in the airways of asthmatics (Liu et al. 1991; Gauvreau et al. 2000; Macfarlane et al. 2000), in postmortem cases of fatal asthma (Koshino et al. 1993; Kepley et al. 2001), after antigen challenge of airway mucosa (KleinJan et al. 2000; Nouri-Aria et al. 2001), and in nasal polyps (de Paulis et al. 2006). From a functional viewpoint, IgE-mediated basophil releasability (i.e., the ability of a basophil/mast cell to release a certain percentage of histamine in response to a given immunologic stimulus) is increased in asthma and allergic rhinitis (Casolaro et al. 1990). More importantly, allergen-induced asthmatic responses are accompanied by infiltration of basophils that express IL-4 mRNA (Nouri-Aria et al. 2001). Thus, basophils might be an important source of Th2-like cytokines (IL-4 and IL-13) in the lung microenvironment. Techniques for the isolation and purification of basophils from peripheral blood, and monoclonal antibodies (BB1 and 2D7) that recognize specific epitopes in these cells (Irani et al. 1998; McEuen et al. 2001), should permit further studies of the function of basophils in allergic diseases (Falcone et al. 2000; Marone et al. 2005b).

Basophil recruitment to allergic airways Human basophils constitutively express several chemokine receptors (CCR1, CCR2, CCR3, CXCR1, CXCR3, CXCR4) and about 80% express CCR3, which can be activated by eotaxin/

Basophils: Biological Properties and Role in Allergic Diseases CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, MCP-3/CCL7 and MCP-4/CCL13 (Uguccioni et al. 1997; Romagnani et al. 1999). Eotaxin increases FcεRI-dependent IL-4 and IL-13 generation, respectively, by basophils (Devouassoux et al. 1999) and mast cells (Price et al. 2003). It is likely that the production of RANTES/CCL5, eotaxin/ CCL11, and eotaxin-2/CCL24 by human airway epithelial cells (Stellato et al. 1997) and smooth muscle (Hirst et al. 2002) partly accounts for the recruitment of basophils and mast cells to the lung during allergic inflammation. Two cytokines, IL-4 and TNF-α, produced by basophils and mast cells, respectively, increase eotaxin mRNA stability in airway epithelial cells, further amplifying the recruitment of inflammatory cells (Atasoy et al. 2003). Insulin-like growth factor (IGF)-1 and IGF-2 have been identified as selective basophil chemoattractants in human nasal polyps (Hartnell et al. 2004). These factors induce chemotaxis of basophils, but not eosinophils or neutrophils, and may therefore help explain the preferential infiltration of basophils in certain allergic disorders, although in vivo studies are required to confirm this. Nasal polyps involve hyperplasia of the mucosal epithelium and submucosal mucous glands with underlying areas of infiltrating inflammatory cells and proliferating blood vessels (Kakoi & Hiraide 1987). Vascular endothelial growth factor and its receptors have been implicated in nasal polyposis (Wittekindt et al. 2002). Nasal polyp tissues from patients undergoing polypectomy were examined by immunohistochemistry using the monoclonal antibody BB1, which specifically recognizes human basophils in tissue (McEuen et al. 1999; Ying et al. 1999), and a rabbit polyclonal anti-VEGF-A. Figure 14.8 shows BB1+ basophils in the perivascular area stained positive for VEGF-A as well. Figure 14.8 also shows the colocalization of BB1 and VEGF-A. Thus, VEGF-A appears to be present not only in peripheral blood but also more importantly at sites of chronic inflammation. This suggests that VEGF-A synthesized and released from basophils plays a dual role in inflammatory angiogenesis. First, VEGF-A released from circulating basophils might activate VEGF receptors on circulating endothelial cell precursors and immune cells; second, VEGF-A released from basophils infiltrating the sites of chronic inflammation might serve as a local source of an important angiogenic and chemotactic factor. A variety of other receptors are implicated in basophil chemotaxis. Human basophils express specific receptors for C3a and C5a (Füreder et al. 1995). A recent intriguing example is how the FMLP receptors FPRL1 and FPRL2 mediate the activation and chemotaxis of basophils in response to viral (gp41 of HIV-1) and bacterial [Hp (2–20)] peptides (de Paulis et al. 2002, 2004a). Consequent to these observations, it became evident that basophils express several receptors involved in the innate pattern recognition of microbes (FPR, FPRL1, FPRL2, and complement receptors) and of danger signals (VEGFR-2/KDR,

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uPAR, IGF-1/2/R). Thus the concept emerged that basophils receive danger signals in infection and injury and release mediators that initiate a protective leukocyte response that facilitates repair and healing. Amplification of these processes, however, could lead to persistent inflammation, angiogenesis, and tissue remodeling.

Fig. 14.8 Confocal micrographs of nasal polyps stained for basophils (green) and VEGFA (red). (a) A cluster of BB1-immunoreactive basophils. (b) VEGF-A-positive cells. (c) Colocalization of basogranulin and VEGF-A in basophils in nasal polyps. (From de Paulis et al. 2006, with permission.) (See CD-ROM for color version.)

classical mechanisms of FcεRI+ cell activation. We have demonstrated that immunoglobulin superantigens of various origins (endogenous, bacterial, and viral) can activate FcεRI+ cells to release proinflammatory mediators and cytokines (Marone et al. 2007b).

Endogenous and viral immunoglobulin superallergens

Superallergens in allergic disorders A mechanism by which human FcεRI+ cells can be activated by immunoglobulin superantigens has recently been identified (Marone et al. 2004, 2007b; Marone 2007). This might play a physiologic protective role in certain bacterial and viral infections (see Fig. 14.2). Amplification of this mechanism could lead to superantigen/superallergen activation of FcεRI+ cells, enabling certain viruses and bacteria to cause exacerbations in allergic disorders. A conventional antigen can usually stimulate less than 0.001% of the naive lymphocyte pool, whereas a superantigen can stimulate more than 5% (Silverman 1998). This immunologic property derives from the superantigen’s unique ability to interact with most lymphocytes that express antigen receptors from a particular variable (V) region gene family (Silverman 1997). Classical superantigens are T-cell superantigens (staphylococcal enterotoxins and toxic shock syndrome toxin-1). Some naturally occurring proteins are B-cell superantigens with unconventional immunoglobulin-binding capacities. The best-characterized immunoglobulin superantigen is Staphylococcus aureus protein A (Inganäs et al. 1980; Graille et al. 2000). Other B-cell superantigens are the gp120 envelope glycoprotein of HIV-1 (Townsley-Fuchs et al. 1997), a human gut-associated sialoprotein known as “protein Fv” (Guihard et al. 1997), and protein L from Peptostreptococcus magnus (Björck 1988). The concept of immunoglobulin superantigens applied to the pathophysiology of allergic disorders could be translated as “superallergens” to indicate proteins of various origin able to activate FcεRI+ cells by interacting with membranebound IgE. A significant proportion of allergic diseases (e.g., certain cases of intrinsic asthma) cannot be explained by the four

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About 5% of patients with viral hepatitis suffer urticarial reactions (Segool et al. 1975; Popp et al. 1981; Vaida et al. 1983). Protein Fv is a sialoprotein produced in the human liver and released in biological fluids during hepatitis A, B, C, and E (Bouvet et al. 1990). A single protein Fv molecule can bind six F(ab′)2 fragments (Bouvet et al. 1991a) of human IgM, IgG, and IgE (Bouvet et al. 1990, 1991a,b). Protein Fv binds to the VH3 region of immunoglobulins in a domain outside the conventional antigen-binding pocket (Bouvet et al. 1991a,b). We found that protein Fv is the most potent IgE-mediated stimulus for the activation of human basophils and lung mast cells (Patella et al. 1998) by interacting with IgE VH3+. This is the largest immunoglobulin family in the human repertoire (∼ 50%) (Karray & Zouali 1997; Silverman 1997; Karray et al. 1998). Therefore, protein Fv can function as an endogenous immunoglobulin superantigen frequently interacting with IgE VH3+ bound to FcεRI+ cells (Patella et al. 1993, 1998). Low concentrations of protein Fv induce IL-4 secretion from basophils through interaction with IgE VH3+ (Patella et al. 1998). IL-4 is a critical cytokine in the regulation of IgE synthesis by B lymphocytes and it is intriguing that some patients with viral hepatitis have high serum IgE levels (Van Epps et al. 1976). HIV-1-infected patients have higher than normal prevalence and/or severity of allergic reactions (Kaplan et al. 1987; Coopman et al. 1993). Serum IgE levels are high in HIV-1infected children (Viganò et al. 1995; Koutsonikolis et al. 1996; Onorato et al. 1999) and adults (Wright et al. 1990; Paganelli et al. 1995; Rancinan et al. 1998). Immunoglobulin VH3+ is a ligand for gp120 (Berberian et al. 1993; Karray & Zouali 1997). We found that four recombinant gp120 from different HIV-1 isolates from viral clades of varying geographic origin stimulated the release of cytokines (IL-4

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Bacterial immunoglobulin superallergens Staphylococcus aureus can exacerbate atopic dermatitis (Leung et al. 1998) and certain forms of asthma (Suh et al. 2004). Most clinical isolates of S. aureus synthesize protein A, a cell wall protein that has unique immunoglobulin-binding properties. Protein A has a classical site that binds the Fcγ of IgG, and an alternative site that binds the Fab portion of 15–50% of human polyclonal IgG, IgM, IgA, and IgE (Inganäs 1981). Staphylococcus aureus Cowan 1, which synthesizes protein A and soluble protein A, gradually increased histamine release from basophils (Marone et al. 1982) and lung mast cells (Genovese et al. 2000). Protein A activated basophils through interaction with the alternative binding site of IgE VH3+. These observations raise the possibility that exacerbations of atopic dermatitis (Leung et al. 1998) and certain forms of asthma (Suh et al. 2004) associated with S. aureus infection might be caused through this mechanism. Protein L, a cell-wall protein synthesized by the bacterium Peptostreptococcus magnus (Björck 1988), consists of up to five repeated immunoglobulin-binding domains (B1–B5) (Kastern et al. 1992) and is a determinant of virulence (Ricci et al. 2001). Each homologous domain binds with high affinity to the variable domain of the VKI, VKIII and VKIV subgroups (Nilson et al. 1992). Protein L binds human Ig regardless of the H chain class, is mitogenic for B cells (Axcrona et al. 1995), and is an immunoglobulin superantigen (Silverman 1997). We found that protein L induced the release of proinflammatory mediators and cytokines (IL-4 and IL-13) from basophils by interacting with the κ light chains of IgE (Patella et al. 1990; Genovese et al. 2000; Genovese et al. 2003). The relation between certain viral and bacterial infections and the induction and/or exacerbation of allergic reactions is well established (Johnston 1997; Leung et al. 1998; Gern & Busse 2000; Holtzman et al. 2002; Suh et al. 2004). Although there is evidence that certain viruses may exert an inhibitor effect on human basophils (Shiratori et al. 2005), our results point to a novel mechanism by which these infections might be involved (Marone 2007; Marone et al. 2007b). The in vivo implications of IgE-mediated activation of human FcεRI+ cells by these immunoglobulin superallergens remain to be defined.

Role of basophils in allergic angiogenesis The formation of new blood vessels (angiogenesis) is vital for numerous inflammatory and immune disorders including asthma, atopic dermatitis, polyposis, and rheumatic diseases (Carmeliet 2003). It is a prerequisite for airway remodeling. Several growth factors may be important in allergic inflammation and angiogenesis. VEGF and (PlGF) are among the most potent proangiogenic factors (De Falco et al. 2002; Nagy et al. 2003). FcεRI+ cells, which are closely associated with blood vessels and are increased at angiogenic sites, contribute to various aspects of angiogenesis (Ribatti et al. 2002; Hiromatsu & Toda 2003). Mast cells synthesize and release proangiogenic factors (histamine, tryptase, transforming growth factor β, IL-8, VEGF, I-309/CCL1) (Boesiger et al. 1998; Grützkau et al. 1998; Gilchrest et al. 2003). PGE2 and other cAMP-elevating agents increase the production of VEGF by mast cells (Abdel-Majid & Marshall 2004). We have explored the expression of VEGF and its receptors, and their functional interactions in human basophils (Marone et al. 2005a). Basophils constitutively express several isoforms of VEGF-A (VEGF-A121, VEGF-A165, and VEGF-A189) and their immunologic activation induces the release of VEGF-A (Fig. 14.9). This factor is chemotactic for basophils, presumably through interaction with the VEGFR-2/KDR, a receptor phenotypically expressed on the vast majority of these cells. Basophils also constitutively express mRNA for the soluble VEGFR-1 (sVEGFR-1), which is biologically active in blocking endogenously expressed VEGF activity (Eubank et al. 2004).

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and IL-13), parallel to histamine secretion from basophils (Florio et al. 2000; Patella et al. 2000). The viral superantigen gp120 activates basophils through an interaction with IgE VH3+ (Fig. 14.8). The mechanism of FcεRI+ cell activation by protein Fv and gp120 represents a new pathogenic cascade consisting of viral infection, immunoglobulin superantigen production, activation of FcεRI+ cells and tissue injury (see Fig. 14.2). This sequence of events raises the possibility that further immunoglobulin superantigens induced by viruses cause tissue injury in allergic inflammation through this mechanism involving FcεRI+ cell activation.


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Time (hours) Fig. 14.9 Kinetics of VEGF-A and histamine release from human basophils. Basophils were incubated with buffer (spontaneous release) or with anti-IgE (0.3 μg/mL). At each time point, supernatants were collected and centrifuged (1000 × g, 4°C, 5 min). VEGF-A release from basophils induced by anti-IgE is indicated by filled circles and histamine release by open circles. VEGF-A and histamine release in the supernatants was determined by ELISA and fluorometric techniques, respectively. The values are mean of duplicate determinations from a typical experiment.

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Most basophils express neuropilin 1 (NRP1) (de Paulis et al. 2006), a coreceptor for VEGF-A 165 (Soker et al. 1998; Mamluk et al. 2002), which enhances VEGFR-2/KDR-induced responses (Yamada et al. 2003). Basophils also express NRP2 mRNA and protein. Neuropilin 1 has no known enzymatic activity and therefore participates in signal transduction events by forming a complex with tyrosine kinase receptors. However, it appears to support the autocrine functions of VEGF in cells lacking VEGFR-2 expression (Wang et al. 2003). This raises the possibility that in certain cells NRP1 functions either alone or with other tyrosine kinase-linked receptors to transduce VEGF signaling. Neuropilin 1 on cells other than endothelial cells can induce angiogenesis too (Yamada et al. 2003). Therefore, NRP1 highly expressed on basophils might enhance angiogenesis even when VEGF is not abundantly expressed. The urokinase plasminogen activator (uPA) and its highaffinity receptor (uPAR) are involved in tissue remodeling and vessel sprouting (Carmeliet 2003). The uPAR is expressed by human basophils (de Paulis et al. 2004b) and mast cells (Sillaber et al. 1997). uPA is a potent chemoattractant for basophils through exposure of a chemotactic uPAR epitope, which is a ligand for FPRL1 and FPRL2 (de Paulis et al. 2004b). The expression and functions of angiopoietins and their receptors in human basophils remain to be elucidated. These findings illustrate the complex roles of basophils in fine regulation of the homeostatic control of angiogenesis during chronic inflammation. The VEGF/VEGFR system is emerging as an important regulator of angiogenesis and tissue remodeling in inflammatory disorders. We believe that this VEGF/VEGFR network warrants consideration as a target for future therapeutic intervention in allergic diseases.

Pharmacologic modulation of human basophils Basophils, mast cells and their mediators play a pivotal role in most allergic diseases in humans. This has prompted a race within the pharmaceutical industry to achieve optimal therapeutic targeting of FcεRI+ cells in these disorders. It is now clear that this ambitious target is more elusive than expected, on account of the complexity of the human FcεRI+–IgE system. First, mast cells differ immunologically, biochemically, and pharmacologically from human basophils. Second, basophils and mast cells synthesize different sets of proinflammatory mediators, cytokines, and chemokines. Moreover, different subpopulations of mast cells and basophils might have different roles – even, in some cases, protective – in the appearance and disappearance of the allergic phenotype. In addition, under some circumstances, FcεRI+ cells are important in homeostasis (Marone et al. 2005b,c). Finally, human FcεRI+ cells can be activated by a variety of immunologic and nonimmunologic stimuli, besides IgE-cross-linking.

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Blockade of activating receptors There are four main areas for therapeutic targeting of basophils and mast cells in allergic disorders. The first is interfering with activating receptors (FcεRI, C3aR, C5aR, cytokine and chemokine receptors, etc.) on the cells. Using a monoclonal antibody anti-IgE this strategy has been partially successful in certain forms of allergic asthma (Milgrom et al. 1999). The partial efficacy might be because other receptors besides FcεRI trigger the release of mediators from human FcεRI+ cells. Chemokine receptor antagonists are another area with pharmacologic potential for the prevention or treatment of allergic disorders. Several chemokine receptors are selectively displayed on human basophils and mast cells. Their antagonism can interfere with chemotaxis and/or secretion of FcεRI+ cells (Lukacs 2001). The redundancy of chemokines and their receptors is a major obstacle to achieving specific inhibitory effects. VEGF and its receptors are expressed in human basophils (Marone et al. 2005a; de Paulis et al. 2006). Small-molecularweight antagonists of VEGF receptors are candidates for the prevention/treatment of certain aspects of tissue remodeling in asthma (Wood et al. 2000). Alternative approaches to VEGF-induced angiogenesis include the use of humanized monoclonal antibodies against VEGF (Presta et al. 1997) or its receptor Flk-1/KDR (Prewett et al. 1999), or a decoy of this receptor (Wood et al. 2000).

Inhibition of signal transduction A second therapeutic strategy for allergic diseases is to inhibit FcεRI+ cell activation by interfering with one or more biochemical events essential for signal transduction. Excellent reviews have focused on the molecular consequences of basophil (MacGlashan et al. 2000a; Lusková & Dráber 2004) and mast cell (MacGlashan et al. 2000b; Bastan et al. 2001; Siraganian 2003; Wymann et al. 2003; Blank & Rivera 2004) activation and their pharmacologic manipulation. Several steps in the signal transduction process appear to be promising targets for the action on mediator release, cell growth, proliferation, and survival of FcεRI+ cells. In particular, tyrosine kinases (Syk, Btk, Lyn, Fyn, etc.), protein kinase C, phosphatidylinositol 3-kinase, and adapter molecules (Gab1, Gab2, Cbl, LAT, Grb2, Vav, etc.) might qualify as drug targets for the treatment of allergic disorders. The pharmacologic effects of most of these compounds have been so far evaluated in vitro using rodent and/or mast cell lines. Their pharmacologic properties must be confirmed using human mast cells and basophils before they can be considered for clinical trials.

Adenylate cyclase activators and phosphodiesterase inhibitors Catecholamines inhibit IgE-mediated histamine release from basophils and mast cells by binding β2-adrenergic receptors

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(Lichtenstein & Margolis 1968; Marone et al. 1984). Phosphodiesterases (PDEs) are responsible for cAMP hydrolysis. At least 11 different classes of cAMP PDE isoenzymes have been identified; cAMP PDE3 and PDE4 have both been found in basophils and mast cells (Essayan 2001). PDE4 has attracted particular attention and four variants have been identified (PDE4A, PDE4B, PDE4C, PDE4D) (Giembycz 2000). The four gene products of PDE4 show sensitivity to inhibition by rolipram, which inhibits PAF- and anti-IgE-induced mediator release from basophils too (Peachell et al. 1992; Columbo et al. 1993b). PDE4 inhibitors also regulate mast cell functions (Torphy et al. 1992). In contrast, PDE3 and PDE5 inhibitors do not inhibit mediator release. Thus, it appears that PDE4 is the isoenzyme mainly responsible for action on FcεRI+ cells.

Glucocorticoids Glucocorticoids are one of the therapeutic mainstays in allergic disorders. In humans, intravenous glucocorticoids cause rapid basopenia, while skin tissue histamine remains unchanged (Dunsky et al. 1979). In contrast, prolonged treatment with topical glucocorticoids reduces skin mast cell density and inhibits the allergen-induced wheal-and-flare response (Lavker & Schechter 1985). High doses of inhaled glucocorticoids in mild-to-moderate asthma reduce mast cell density and the number of bronchial vessels (Chetta et al. 2003). Prolonged (12–24 hours) incubation with glucocorticoids inhibits IgE-mediated histamine release from basophils (Schleimer et al. 1981). In contrast, up to 24-hour incubation of mast cells isolated from lung parenchyma with glucocorticoids does not alter their immunologic release of histamine, PGD2, or LTC4 (Schleimer et al. 1983). These examples highlight the pharmacologic differences between human basophils and mast cells. Short preincubation (∼1 hour) of human basophils with glucocorticoids inhibits IL-4 release (Schroeder et al. 1997), without any inhibitory effect on histamine. These results suggest that the mechanisms controlling the release of cytokines differ from those controlling the release of histamine from basophils.

Immunophilin ligands Cyclophilin (CyP) is a protein with high affinity for cyclosporin A (CsA) (Hait et al. 1986) that belongs to a family of intracellular proteins, the immunophilins, which includes the FK-binding proteins (FKBPs) (Standaert et al. 1990). FK-506 (tacrolimus) binds with high affinity to FKBPs (Standaert et al. 1990). CsA–CyP and FK-506–FKBP complexes bind to calcineurin (Cn) (Stellato et al. 1992), which has a catalytic A subunit (CnA) and a regulatory B subunit (CnB). The CnA subunit has a binding site for calmodulin (CaM) and for the CnB subunit. Complexes of CsA–CyP or FK-506–FKBP inhibit the CaM-dependent protein phosphatase 2B, which is essential in the signal transduction pathway for basophils

Basophils: Biological Properties and Role in Allergic Diseases (Cirillo et al. 1990; de Paulis et al. 1991) and mast cells (Cirillo et al. 1990; de Paulis et al. 1992; Stellato et al. 1992). Low concentrations of CsA prevent histamine and LTC4 release from basophils and mast cells challenged with IgEmediated stimuli (Cirillo et al. 1990; de Paulis et al. 1991, 1992; Stellato et al. 1992), by interacting with CyP. A single oral dose of CsA rapidly inhibited histamine release from basophils ex vivo, providing a rare example of how a drug administered in vivo can affect basophil releasability ex vivo (Casolaro et al. 1993). CsA and tacrolimus are also potent inhibitors of the de novo synthesis of IL-4 and IL-13 from immunologically activated basophils (Patella et al. 1998; Florio et al. 2000; Genovese et al. 2003). Tacrolimus ointment is rapidly effective in patients with atopic dermatitis (Ruzicka et al. 1997). Skin mast cells and infiltrating basophils are important in atopic dermatitis (Mitchell et al. 1982). Tacrolimus exerts its potent antiinflammatory effects by inhibiting the release of histamine and eicosanoids from skin mast cells (de Paulis et al. 1991, 1992). These findings, together with its efficacy in atopic dermatitis, suggest that the beneficial effects of this compound in vivo are largely due to its antiinflammatory properties (Marone 1998).

Receptor antagonists of Fc εRI+ cell-derived mediators Antagonists of receptors activated by mediators synthesized by human FcεRI+ cells are widely used in the treatment of allergic disorders. The main property of H1 receptor antihistamines is to antagonize the effects of this mediator at the H1 receptor in different organs (Marone 1997). Certain antihistamines prevent the release not only of histamine, but also of other proinflammatory mediators, such as LTC4, PAF, and PGD2 (Church & Gradidge 1980; Patella et al. 1996; Genovese et al. 1997; Marone et al. 1999). Loratadine and desloratadine inhibit histamine release and cytokine production from human basophils induced by IgE-dependent mechanisms (Genovese et al. 1997; Schroeder et al. 2001); in human lung mast cells, desloratadine inhibits preformed (histamine and tryptase) and de novo synthesized mediators (LTC4 and PGD2) (Genovese et al. 1997). Not all H1 antagonists have antiinflammatory activity in vitro. For instance, mizolastine inhibits the de novo synthesis of LTC4 from basophils, but potentiates the secretion of histamine (Triggiani et al. 2004). Thus, the inhibitory effect of certain H1 receptor antagonists on mediator release from human FcεRI+ cells is not class-specific and is not a general property of all antihistamines. Selective cysteinyl (Cys)LT1 receptor antagonists, such as montelukast, have been employed in certain allergic disorders (Barnes et al. 2005). CysLT1 and CysLT2 receptors are also expressed on mast cells (Mellor et al. 2001) and leukocytes (Figueroa et al. 2001). It is possible that cysteinyl leukotriene antagonists influence certain aspects of their activation.

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PGD2 activates two important receptors: the DP receptor, whose activation elicits bronchoconstriction in asthmatics (Hardy et al. 1984), and the CRTH2 receptor present on human basophils and mast cells (Hirai et al. 2001). Specific antagonists of these receptors are being evaluated for use in allergic diseases. β Tryptase and chymase, stored and released by human mast cells, exert several biochemical and proinflammatory effects (Caughey 2004; Stevens et al. 2004). Specific smallmolecular-weight inhibitors of β tryptase and chymase are under investigation for the treatment of allergic disorders. Several VEGF-A isoforms have been identified in human basophils (Marone et al. 2005a) and mast cells (Boesiger et al. 1998; Grützkau et al. 1998). In addition, immunologic activation of human basophils induces the release of VEGF-A (de Paulis et al. 2006), which is chemotactic for human basophils (de Paulis et al. 2006). Therefore, the antagonism of VEGF and their receptors by different strategic approaches may serve to interrupt an autocrine loop affecting tissue remodeling and angiogenesis.

Conclusions and implications The ability of basophils to migrate to sites of chronic inflammation, their powerful effector repertoire, their different activating ligands, and their plasticity in response to various signals suggest that these cells have a central role in several allergic disorders. Mast cells differ from basophils immunologically, biochemically, and pharmacologically and therefore have distinct roles in the orchestration of inflammation. It is quite possible that different subpopulations of basophils have different, in some cases even protective, roles in the appearance of the allergic phenotype. We have to continue working to define the cytokines and chemokines and their receptors acting on or released by human basophils. We must also elucidate the complex biochemical steps in human basophils activated by immunologic and nonimmunologic stimuli. Another area to explore is the paracrine and autocrine interactions of basophils on other immune cells (e.g., eosinophils, macrophages, Th2 cells, B cells). Finally, it remains to be elucidated whether basophils have role(s), in some cases even protective, in angiogenesis and tissue remodeling. Until a few years ago, partially degranulated basophils at sites of inflammation were hard to identify with conventional morphologic techniques. The availability of monoclonal antibodies that identify specific basophil epitopes (e.g., basogranulin) marks a breakthrough in clarifying the roles of these cells in allergic diseases. However, we still need more knowledge of the biological properties of human basophils to modify their “bad” behavior without compromising their homeostatic and protective roles in innate and acquired immunity.

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Acknowledgments This work was supported by grants from the Ministero dell’Università e Ricerca, the Istituto Superiore di Sanità (AIDS Project 40G.38), Ministero della Salute “Alzheimer Project” (Rome, Italy), and Regione Campania (Naples, Italy). We thank Giorgio Giannattasio for a critical reading of the manuscript and Francesco Granata for the artwork. We also wish to acknowledge the important contribution of colleagues whose work has not been included due to space constraints.

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Leukocyte Adhesion in Allergic Inflammation Michelle J. Muessel and Andrew J. Wardlaw

Summary

Introduction

The pattern of inflammation in chronic allergic disease is controlled by selective expression of adhesion molecules and chemoattractants that cause the preferential migration, tissue localization and activation of eosinophils, mast cells, and Th2 cells. The adhesion receptors predominantly involved are the selectins and their ligands, in particular P-selectin glycoprotein ligand (PSGL)-1 and the leukocyte integrins and their ligands intercellular adhesion molecule (ICAM)-1–3 and vascular cell adhesion molecule (VCAM)-1. The function of the leukocyte integrins is controlled mainly by conformational changes in the receptor via a process called “inside-out” signaling, whereas expression of endothelial adhesion receptors is controlled largely by increased expression as a result of inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-4. One of the most striking features of allergic disease is the increased numbers of tissue eosinophils. This results from a combination of increased eosinophilpoiesis, increased migration through the endothelium controlled by a combination of P-selectin/PSGL-1 and VCAM-1/VLA-4, and prolonged survival in tissue under the influence of IL-5 and granulocyte– macrophage colony-stimulating factor (GM-CSF). Mast cells migrate into tissue as precursor cells and their activation and localization in tissue is modulated by their integrin-mediated interaction with matrix proteins such as collagen and fibronectin as well as their ability to adhere avidly to structural cells through novel adhesion pathways. There does not appear to be any specific adhesion pathway for Th2 cells, with selective Th1 versus Th2 migration controlled principally by chemokines and their receptors. Our understanding of the adhesion receptors controlling leukocyte migration into tissue has yet to be translated into new therapies. Potent yet safe adhesion receptor antagonists have been difficult to develop and in the case of VLA-4 antagonists have been disappointing in allergen challenge studies. Nonetheless this remains a fruitful area for future drug development.

In the last decade, dramatic progress has been made in our understanding of the structural and functional properties of the molecules involved in leukocyte adhesion. The migration of leukocytes from the vasculature across the endothelial barrier to sites of allergic inflammation, known as transendothelial migration (TEM) or diapedesis, remains central to the immune response. This multistep sequential process involves (i) leukocytes rolling along the endothelium, (ii) arrest and firm adhesion, and (iii) subsequent transmigration through the endothelial barrier (Springer 1994). The adhesion molecules utilized during this process also work in a sequential fashion. Selectins and their counterstructures slow the velocity of leukocytes leading to rolling along the endothelial wall (Luscinskas et al. 1994; Alon et al. 1995). Firm adhesion is mediated by leukocyte integrins such as αLβ2 (LFA-1) or α4β1 (VLA-4) binding to members of the immunoglobulin superfamily such as ICAM-1 on endothelium (Lawrence & Springer 1991; Carlos & Harlan 1994). Subsequently, cells transmigrate through the endothelium either via transcytosis of the endothelial cell or via the predominant paracellular route between adjacent cells (Feng et al. 1998; Muller 2003; Zen et al. 2005; Dejana 2006). Transmigration is an active process engaging other members of the immunoglobulin superfamily, including junctional adhesion molecules (JAMs), platelet endothelial cell adhesion molecule (PECAM)-1, and CD99 (Muller 2003). Orchestrating these events is a variety of signaling molecules unique to each of these steps. Proinflammatory mediators such as TNF-α and IL-1 prime the endothelial cells, increasing the surface expression of various adhesion molecules that mediate rolling. As leukocytes roll along the endothelial surface, they encounter chemotactic factors such as chemokines that activate integrins, thus mediating firm adhesion (von Andrian & Mackay 2000). The selective recruitment of eosinophils and T cells to sites of allergic inflammation attest to the exquisite regulation of the immune response. The diversity of cytokines, adhesion molecules, and chemokines utilized in specific combinations allows a high level of specificity in recruitment of selective subpopulations of leukocytes. The selectivity can be modulated at each step in the process, thus offering the possibility of


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inhibiting leukocyte migration at each step. Indeed, blockade of β2 or α4 integrins has been shown to be a promising route of therapeutic intervention for a variety of inflammatory and autoimmune disorders (Gottlieb et al. 2000; Jackson et al. 2002).

Structure and function of leukocyte adhesion receptors Selectins and their counterstructures Selectins Selectins and their counterstructures regulate the first step in the process of TEM, the slow rolling of leukocytes along the surface of endothelial cells (Luscinskas et al. 1994; Alon et al. 1995; Berlin et al. 1995). There are three members of the selectin family, L-, E- and P-selectin, which mediate cell contact by binding weakly to carbohydrate structures typified by sialyl Lewis x (sLex)-like glycans and with high affinity to specific glycoprotein counterstructures (covered later in the chapter) (Somers et al. 2000). Recent studies have demonstrated that selectins form “catch bonds” where dissociation rates decrease with increasing applied force (Marshall et al. 2003; Sarangapani et al. 2004; Yago et al. 2004; Hammer 2005; Pereverzev et al. 2005). The rolling/tethering step is a prerequisite for firm adhesion (Lawrence & Springer 1991; Milstone et al. 1998), allowing leukocytes to sense their local environment for proinflammatory stimuli such as cytokines and chemokines presented on the endothelial surface. L-selectin is constitutively expressed on most types of leukocytes, implying that the specificity of extravasation into tissue must be regulated by the inducibility of its endothelial ligands. L-selectin was first identified by the monoclonal antibody MEL 14, which blocks the binding of lymphocytes to lymph node high endothelial venules (HEV) (Gallatin et al. 1983). E-selectin is specific to endothelial cells. E-selectin was also found using the monoclonal antibody technique while searching for cytokine-inducible endothelial surface molecules (Bevilacqua et al. 1987). Proinflammatory stimuli such as TNF-α or IL-1β induce de novo gene expression and protein synthesis of E-selectin on endothelium through NF-κB and JNK/p38 mitogen-activated protein kinase (MAPK) pathways (Bevilacqua et al. 1987; Read et al. 1997). Surface expression of E-selectin is maximal at 4– 6 hours after stimulation and rapidly downregulated to baseline levels after 12–16 hours (Weller et al. 1992; Hahne et al. 1993). P-selectin was identified in platelet storage granules as a protein of unknown function and was later detected in endothelial cells (Hsu-Lin et al. 1984; McEver & Martin 1984). P-selectin is found on endothelium as well as platelets, where its expression is regulated by proinflammatory mediators such as TNF-α and lipopolysaccharide (LPS) (Weller et al. 1992; Hahne et al. 1993). However, the mechanism of regulation for P-selectin is different from other selectins. P-selectin protein is stored in

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cytosolic storage granules called Weibel–Palade bodies (Birch et al. 1992; Hannah et al. 2002). These intracellular vesicles then fuse with plasma membrane to release P-selectin in response to various stimuli such as histamine and thrombin. P-selectin is rapidly downregulated by internalization (Green et al. 1994) but can be recycled from endosomes to storage granules (Subramaniam et al. 1993). The extracellular domains of all three lectins are very similar. The N-terminus is a C-type lectin, followed by an epidermal growth factor-like domain, then a series of short consensus repeats that are variable in number, with L-selectin having two repeat units, E-selectin six, and P-selectin nine (Fig. 15.1). Soluble forms of all three selectins exist due to shedding or proteolytic cleavage (Kishimoto et al. 1990; Gearing & Newman 1993). The soluble form correlates with membrane expression of selectins (Leeuwenberg et al. 1992) and is increased in asthma and atopic dermatitis (Kobayashi et al. 1994; Wuthrich et al. 1995). Blockade of L-selectin shedding decreases leukocyte rolling in vivo, leading to the hypothesis that shedding may be important to prevent an excessively strong interaction mediated by L-selectin (HafeziMoghadam & Ley 1999). The C-type lectin domain binds to the carbohydrate family of sialylated fucosylated glycosaminoglycans typified by the sugar moiety sLex (Springer & Lasky 1991) (Fig. 15.2). The importance of fucosylation is Endothelium

Leukocyte

GlyCAM-1 L-selectin

CD34 MadCAM-1

E-selectin

ESL-1

P-selectin

PSGL-1

Lectin domain Transmembrane region EGF domain Cytoplasmic domain Short consensus repeats

Ig domain N-linked glycosylation O-linked glycosylation

Fig. 15.1 Schematic representation of the structure of selectins and their ligands. EGF, epidermal growth factor. (From Vestweber & Blanks 1999, with permission). (See CD-ROM for color version.)

NeuAca2

3Galb1

Sialylated Lewis x

4GlcNAcb1 3 Fuca1

3Galb1

4GlcNAcb1 3 Fuca1

Fig. 15.2 Structure of the carbohydrate moieties recognized by selectins including the structure of sialyl Lewis x. NeuAc, sialyl group; Gal, galactose; Fuc, fucose; GlcNac, N-acetylglucosamine.

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demonstrated by the genetic disorder leukocyte adhesion deficiency (LAD) II, in which patients are immunodeficient due to the inability of leukocytes to bind through selectins (Etzioni et al. 1993). LAD II has recently been shown to involve a genetic defect that results in a malfunctioning nucleotide sugar transporter (Luhn et al. 2001). All three selectins contain a single transmembrane region followed by a short cytoplasmic tail that consists of 17 amino acids in L-selecin, 32 in E-selectin, and 35 in P-selectin (Huang 1997). Selectin structure is most diverse in the transmembrane and cytosolic tail regions, implying that they are either differentially regulated or transduce different signals. Some intracellular binding partners have been identified for selectins, which give us some clues as to function. The cytoplasmic tail of selectins has been shown to bind the cellular cytoskeleton (Pavalko et al. 1995; Yoshida et al. 1996; Ivetic et al. 2002) and leukocytes show reduced tethering and rolling in vitro on deletion of this region in L-selectin (Dwir et al. 2001). Calmodulin binds to the L-selectin cytoplasmic tail and may negatively regulate shedding (Kahn et al. 1998; Diaz-Rodriguez et al. 2000). The cytoplasmic tail of selectins is also important for signaling, which may depend on selectin clustering. The intracellular signaling molecule, Rho A, a member of the small GTPase family, regulates E-selectin localization and clustering (WojciakStothard et al. 1999) and ligand-induced activation of phospholipase C-γ is dependent on clustering in lipid rafts (Kiely et al. 2003). Clustering also induces increases in intracellular free Ca2+, cytoskeletal shape changes, and F-actin distribution (Kaplanski et al. 1994; Lorenzon et al. 1998). The increase in F-actin filament polymerization has been shown to be dependent on the small GTPase Rac2 (Brenner et al. 1997). The signaling pathway has been further studied in L-selectin that activates p56lck, Grb2/sos, Ras, Rac2, and MAPK to increase synthesis of reactive oxygen species (Waddell et al. 1995; Brenner et al. 1996). In addition, E-selectin has also been shown to stimulate MAPK and Erk1/2 (Hu et al. 2000, 2001). Most importantly, L-selectin ligation has been shown to upregulate the surface expression of the αMβ2 integrin (Mac-1) (Crockett-Torabi et al. 1995), providing an important ingredient for the next step in the process of leukocyte extravasation into tissue.

Leukocyte Adhesion in Allergic Inflammation

1 (Berg et al. 1993), Sgp200 (Hemmerich et al. 1994), and podocalyxin-like protein (Sassetti et al. 1998). GlyCAM-1 and CD34 are both sialomucins displaying large sialic acidrich O-linked carbohydrate side chains that are necessary for binding to L-selectin. CD34 is a heavily O- and N-linked glycosylated transmembrane receptor expressed on most endothelial cells and hematopoietic progenitor cells (Greaves et al. 1992). GlyCAM-1 does not have a transmembrane region and is a secreted protein (Brustein et al. 1992) found in cytoplasmic granules (Kikuta & Rosen 1994). Sulfation is essential for GlyCAM-1 binding to L-selectin (Imai et al. 1993). Vascular MAdCAM-1 also contains a sialomucin domain and carries O-linked carbohydrate modifications recognized by L-selectin (Berg et al. 1993). Interestingly, MAdCAM-1 is unusual in that it also serves as a ligand for the integrin α4β7 and contains two immunoglobulin domains that are important for this interaction. The P-selectin counterstructure PSGL-1 is also a sialomucin, identified through expression cloning using a P-selectin– immunoglobulin fusion protein as bait (Sako et al. 1995). A 250-kDa protein identified earlier by affinity isolation using P-selectin an affinity probe was described on neutrophils (Moore et al. 1992) and was subsequently demonstrated to be identical to PSGL-1 (Moore et al. 1994). The carbohydrate modifications of sialylation and fucosylation are required for PSGL-1 binding (Larsen et al. 1992; Lenter et al. 1994) as are branched carbohydrate side chains generated by the core-2 enzyme (Wilkins et al. 1995; Kumar et al. 1996). Furthermore, sulfation at one of the three tyrosine residues at its N-terminus is necessary for binding to P-selectin (Sako et al. 1995; Wilkins et al. 1995) and probably also to L-selectin (Spertini et al. 1996) (Fig. 15.3). The recently elucidated co-crystal structure of PSGL-1 with P-selectin demonstrated that tyrosine sulfation and sialylated and fucosylated sugars within the terminal 19 amino acids of PSGL-1 bind separately to P-selectin adjacent to the calcium atom (Somers et al. 2000). PSGL-1 was found to be the major ligand for P-selectin on both neutrophils (Moore et al. 1995) and T cells (Vachino et al. 1995), although PSGL-1 is also recognized by E- and L-selectins (Li et al. 1996; Goetz et al. 1997). PSGL-1-deficient mice exhibit deficits in P-selectin-mediated rolling and neutrophil recruitment,

Selectin counterstructures The ligands for selectins are composed of a scaffolding protein modified by various carbohydrates and other posttranslational modifications such as sulfation. The protein core is important for increased specificity and affinity of binding while the cellular background itself is important for providing the necessary repertoire of glycosylation enzymes to confer the selectivity of selectin binding. Five ligands for L-selectin have been identified using soluble recombinant protein as affinity probes: glycosylated cell adhesion molecule (GlyCAM)-1 (Lasky et al. 1992), CD34 (Baumhueter et al. 1994), mucosal addressin cell adhesion molecule (MAdCAM)-

15/16 Decamer repeats NH2

Y

N-glycosylation sites

COOH

Ser/Thr/Pro rich region

Transmembrane region

Y Three sulfated tyrosine amino acids essential for ligand binding Fig. 15.3 Schematic representation of the structure of PSGL-1 showing the position of the sulfated tyrosines essential for high-affinity binding to P-selectin. (See CD-ROM for color version.)

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Inflammatory Cells and Mediators cell-surface counterreceptors and other extracellular ligands (Plow et al. 2000; Van der Flier & Sonnenberg 2001). They transmit signals both into cells regarding their environment (“outside-in”) and out of cells (“inside-out”) (Hynes 1992, 2002). Originally termed integrins due to the fact that they are integral membrane proteins linking extracellular contacts to the intracellular cytoskeleton, they regulate and modulate actin filaments through a complex array of submembrane linker proteins (Hynes 1992; Van der Flier & Sonnenberg 2001; Zamir & Geiger 2001). Integrins are heterodimers of two subunits, α and β. The combination of different α and β subunit partners makes each integrin unique, with each subunit contributing to ligand specificity. Most of each polypeptide subunit, greater than 1600 amino acids, resides in the extracellular space. The two short cytoplasmic domains usually comprise 20–50 amino acids. In mammals, there are known to be eight β and 18 α subunits comprising a total of 24 distinct integrins (Fig. 15.4). The uniqueness of each integrin is displayed by the specificity of its ligand binding partner, an arrangement reinforced by the specific defects in leukocyte function present in the phenotype of knockout mice: knockout of αL leads to impaired leukocyte recruitment (Schmits et al. 1996); αM deficient mice are defective in phagocytosis and apoptosis of neutrophils and mast cells (Coxon et al. 1996; Dong et al. 1997; Tang et al. 1997); knockout of αE leads to reduced numbers of intraepithelial lymphocytes (Schon et al. 1999); mice deficient in β2 have leukocytosis, impaired inflammatory responses, skin infections, and T-cell proliferation defects (Scharffetter-Kochanek et al. 1998); mice deficient in β6 display inflammation in skin and airways as well as impaired lung fibrosis, probably due to the failure to activate transforming growth factor (TGF)-β (Huang et al. 1996; Munger et al. 1999); and, finally, mice deficient in

demonstrating its in vivo relevance (Yang et al. 1999). PSGL-1 binding to E-selectin is also dependent on the same carbohydrate modifications as required for binding to P-selectin, although sulfation of tyrosine residues is unnecessary (Somers et al. 2000). Studies on PSGL-1 provide insights into two general points for selectin counterstructures. Firstly, while only 15% of lymphocytes bind P-selectin, the majority of lymphocytes express PSGL-1, emphasizing the importance of carbohydrate modification to binding specificity. Secondly, despite the fact that PSGL-1 represents only 1% of the sLex on the cell surface, it is a requirement for leukocyte binding to P-selectin (Norgard et al. 1993; Li et al. 1996), emphasizing the importance of a single glycoprotein to high-affinity binding and specificity. ESL-1 is a selective E-selectin ligand found on leukocytes that requires N-linked carbohydrate modifications for binding. The 150-kDa glycoprotein ESL-1 was identified using affinity isolation using an E-selectin–immunoglobulin fusion protein as bait (Levinovitz et al. 1993). ESL-1 is not a sialomucin and furthermore has been identified as a variant of the fibroblast growth factor receptor (Steegmaler et al. 1995). In contrast to these other structures, which are concentrated at the tips of microvilli, ESL-1 is expressed on the surfaces of microvilli and may have a function after the tethering step. Finally, another high-affinity ligand for E-selectin is L-selectin itself (Kishimoto et al. 1991; Lawrence et al. 1994). Affinity isolation experiments using E-selectin–immunoglobulin fusion protein provided direct evidence of this interaction (Jones et al. 1997; Zollner et al. 1997).

Integrins Integrins are major leukocyte receptors for mediating adhesion to a wide array of extracellular matrix proteins as well as

Alpha Chain

Beta Chain b1

a1 a2 a3 a4 a5 a6 a7 a8 a9

b2

b3

b4

b5

VLA-1 (L:C) VLA-2 (Fn:L:C) VLA-3 (L:C) VLA-4 (VCAM-1:Fn) VLA-5 (Fn) VLA-6 (L) a7b1(L) a8b1 a9b1

b6

b7

a4b6

a4b7:VCAM-1 MAdCAM-1:Fn

aEb7 E-cadherin

aE aL aM aX aD aIIb aV

340

b8

aVb1(Vn)

LFA-1 (ICAM-1-3) Mac-1 C3bi:ICAM-1:Fb p150,95 (Fb,ICAM-1, VCAM-1) aDb2 (VCAM-1) aIIbb3 (Fn:Fb) aVb3(Vn:Fn:Ln)

aVb5(Fn)

aVb6(Vn)

aVb8

Fig. 15.4 Structure of the integrin family showing the pairing of the a and b chains and, where known, the main ligands recognized by the integrin. Fn, fibronectin; Fb, fibrinogen; Vn, vitronectin; L, laminin; C, collagen.

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Leukocyte integrins Each leukocyte subtype expresses one or more members of the β2 (CD18) family of integrins; further, β2 and β7 are restricted in their expression to leukocytes. β2 is a common subunit of four different binding partners (αL, αM, αX, and αD), while β7 has just one binding partner, αE, making a total of five integrins unique to leukocytes (Fig. 15.5). The first leukocyte integrin, αMβ2 (Mac-1/Mo-1/CR3/ CD11b), was identified by using a monoclonal antibody as a marker for myloid cells (Springer et al. 1979). The expression of αMβ2 is restricted to myloid cells such as monocytes and neutrophils, large granular lymphocytes, and a subset of B and T cells (Arnaout & Colten 1984). αMβ2 has a wide array of binding partners including ICAM-1, -2, -3, -4 and -5 (Diamond et al. 1990; Xie et al. 1995; Ihanus et al. 2003), JAM-C (Santoso et al. 2002; Chavakis et al. 2004; Aurrand-Lions et al. 2005), “inactivated” opsonic C3b (iC3b) component (Beller et al. 1982), fibrinogen (Wright et al. 1988), vitronectin (Kanse et al. 2004), Leishmania gp63 (Russell & Wright 1988), the LDL receptor-related protein (Spijkers et al. 2005), matrix metalloproteinase (MMP)-9 (Stefanidakis et al. 2003), and Thy-1 (Wetzel et al. 2004). In addition, αMβ2 binds to yeast, zymosan,

Endothelium

MAdCAM-1 VCAM-1

a4b7 a4b1 aDb2

ICAM-1 ICAM-2

aXb2 Mac-1

Leukocyte

β7 show defects in gut-associated lymphocytes and reduced intraepithelial lymphocytes (Wagner et al. 1996). Leukocyte-specific α subunits have an inserted I/A domain. The crystal structure of this domain was elucidated by Lee et al. (1995) from αMβ2 (CD11b/CD18). There is a metal ion coordination site at the “top” of the I/A domain of αM, which explains the necessity of divalent cations for ligand binding. Additional structural and biochemical experiments show that ligand binding is regulated through coordination of the metal ion site in the I/A domain which causes confirmational changes allowing the integrin head to adopt either an open highaffinity “active” form or a closed low-affinity “inactive” form (Shimaoka et al. 2000; Xiong et al. 2000). However, there is some evidence to show that this “active” form may not be generally true of all leukocyte integrin α subunits (Walters et al. 2005). The recent elucidation of the crystal structure of one integrin, αVβ3, has led to advances in our understanding of integrin structure and function (Xiong et al. 2001, 2002). In agreement with a large body of evidence from other techniques, it was confirmed that the β subunit also contains an I/A domain (or β I-like domain) and together with the propeller domain from the α subunit forms the ligand-binding head of the integrin, which is attached to two legs, one from each subunit. However, the configuration of unliganded crystal structure yielded a surprise: an integrin bent over at an angle of 135°, which was presumed to be inactive. Crystal structure of the ligand-bound integrin confirmed that, on activation, presumably either through ligand binding or via “inside-out” signaling, straightening and separation of the legs allows the integrin to become fully functional.


LFA-1

Fig. 15.5 Schematic structure of the leukocyte integrins and the endothelial adhesion receptors they recognize.

plastic, and glass (Anderson et al. 1986; Wallis et al. 1986). Phosphorylation of the cytoplasmic tail of αMβ2 is important for activation (Fagerholm et al. 2006) as is selective recruitment of the src family kinase Hck (Tang et al. 2006). αLβ2 (LFA-1/CD11a) was identified as a surface antigen involved in cytotoxic T cell-mediated killing (Davignon et al. 1981). αLβ2 is expressed by virtually all leukocytes (Krensky et al. 1983). Binding partners for αLβ2 include ICAM-1, -2, -3 and -4 as well as JAM-A (Luscinskas et al. 1991; Ostermann et al. 2002; Ihanus et al. 2003; Fraemohs et al. 2004; Yang et al. 2005). Phosphorylation of the cytoplasmic tail of αLβ2 is important for adhesion (Fagerholm et al. 2005; Nurmi et al. 2007). Α third member of the family, αXβ2 (P150,95/CR4/CD11c), was identified shortly thereafter (Lanier et al. 1985; Springer et al. 1986). Expression of αXβ2 is restricted to monocytes, macrophages, and CD8− dendritic cells with only weak expression on neutrophils (Springer et al. 1986; Shortman & Liu 2002). Ligands for αXβ2 include collagen type I, iC3b, fibrinogen, LPS, ICAM-1 and -4, as well as Thy-1 (Loike et al. 1991; Diamond et al. 1993; Bilsland et al. 1994; Choi et al. 2005; Ihanus et al. 2006). Additionally, αXβ2 has been identified as a marker for hairy cell leukemia (Schwarting et al. 1985), as well as interacting with the rotavirus protein VP7 during viral cell entry (Graham et al. 2004). αXβ2 is more resistant to activation than other leukocyte integrins and recent evidence shows that this is due to activation restraints imposed by structural changes in the cytoplasmic domain (Zang & Springer 2001). αDβ2 (CD11d) is expressed on monocytes, macrophages, natural killer (NK) cells, neutrophils, eosinophils, dendritic cells, and a subset of T and B cells (Danilenko et al. 1995; Grayson et al. 1998; Mabon et al. 2000). The principal ligand for αDβ2 is VCAM-1 (Van der Vieren et al. 1999; Mabon et al. 2000). Administration of antibodies directed against αDβ2 significantly inhibited macrophage as well as neutrophil recruitment in vivo (Mabon et al. 2000). Phorbol esters increase αDβ2 surface expression as well as eosinophil αDβ2-mediated adhesion to VCAM-1 (Kikuchi et al. 2003). αEβ7 is expressed by dendritic cells (Pribila et al. 2004), mast cells (Tegoshi et al. 2005), and T cells in the lung and gut including regulatory T cells (Lehmann et al. 2002; Uss et al. 2006). Expression

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of αEβ7 is induced by TGF-β (Kilshaw 1999; Robinson et al. 2001). αEβ7 mediates lymphocyte adhesion to epithelial cells through its only known ligand, E-cadherin (Cepek et al. 1994; Kilshaw 1999). Other members of the integrin family are variably expressed on leukocytes depending on the activation state. αIIbβ3 is only expressed on platelets and megakaryocytes (Wee & Jackson 2005). αVβ3 is expressed by tissue macrophages and lymphocytes (Savill et al. 1990; Legler et al. 2004). Most members of the β1 family bind to extracellular matrix proteins such that α4β1 (VLA-4) binds to fibronectin, although it also recognizes immunoglobulin superfamily cell-surface counterparts such as VCAM-1 (Luscinskas et al. 1991) as well as JAM-B (Cunningham et al. 2002). α4β1 is expressed by T lymphoblastoid cell lines and mononuclear leukocytes (Hemler et al. 1984). The integrin α4β7 is expressed by lymphocytes, mast cells, eosinophils, and NK cells and recognizes MAdCAM-1 and VCAM-1 (Erle et al. 1994; Gurish et al. 2001). Although it is well documented that integrins mediate firm adhesion, it is becoming increasingly clear that, like selectins, several integrins also participate in the rolling and arrest of leukocytes under flow conditions including α4β1, α4β7, and αLβ2 (Berlin et al. 1995; Dunne et al. 2003; Rosenthal-Allieri et al. 2005; Pendu et al. 2006).

Regulation of integrin function Spatial and temporal regulation of leukocyte adhesion is an absolute necessity in many leukocyte functions including migration. It is important that integrins are inactive on resting leukocytes in order to avoid inflammation. In contrast, it is important that they are able to rapidly activate to facilitate leukocyte function. Elucidation of the crystal structure of αVβ3 integrin, as mentioned above, has allowed integration of the functional data with a large body of experimental work involving the regulation of integrin function. It has been known for many years that integrins on resting leukocytes are unable to bind ligand and do not signal. For example, resting T cells and granulocytes bind to purified ICAM-1 very weakly. It had been proposed that the changes in integrin function are due to a conformational change in the ligand-binding domain. It has now been shown that the I/A domains of αM can adopt either an open or closed position (Xiong et al. 2000). The open state is a high-affinity or “active” state while the closed position is low affinity or “inactive.” Ligand binding or stimulation by other known activating agents such as antibodies or divalent cations induces a conformational switch beween the two states. Integrins are also capable of regulating each other, either inhibiting or activating function. For instance, the major platelet integrin αIIbβ3 can be activated by collagen signaling through its receptors GPVI and the integrin α2β1. Conversely, it has been shown that antibodies to αVβ3 can inhibit the role of α5β1 in cell migration and phagocytosis (Simon et al. 1997; Scharffetter-Kochanek et al. 1998; Blystone et al. 1999).

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Another important aspect of integrin regulation is the coordination of adhesion molecule signaling with more traditional signaling pathways such as growth factor receptors. Integrin signaling that regulates many aspects of cell behavior such as proliferation, apoptosis, differentiation, migration, and gene expression is intimately coupled with pathways triggered by growth factor receptors to yield coordinated functional responses. Similarly, integrin attachment to substrate is necessary for many cellular responses to soluble growth factors such as thrombin, platelet-derived growth factor, and lysophosphatidic acid (LPA). Again, dysregulation of leukocyte integrins in both mice and humans attest to the importance of their tight regulation. For instance, as discussed earlier, knockout of β2 integrins leads to defects in leukocyte function (Scharffetter-Kochanek et al. 1998). The importance of β2 integrins is also demonstrated by the human disease LAD I, a genetic mutation in the gene for β2. These patients suffer from leukocytosis and failure to recruit leukocytes to sites of infection, leading to early death (Etzioni et al. 1999). A more recently described disease, LAD III, also demonstrates activation defects in integrin function possibly through signaling defects in the small GTPase Rap1, a key component in inside-out signaling (Etzioni & Alon 2004; Kinashi et al. 2004). These patients suffer from defects in platelet aggregation, recurrent bacterial infections, and a severe bleeding tendency.

Immunoglobulin family members ICAM-1 ICAM-1/CD54 is a cell-surface glycoprotein of the immunoglobulin superfamily that is critical for trafficking of leukocytes through leukocyte–leukocyte, leukocyte–endothelial, and leukocyte–epithelial interactions (Rothlein et al. 1986; Springer 1990). ICAMs are cellular binding partners for β2 integrins on leukocytes, for example ICAM-1 binds αLβ2 on lymphocytes or αMβ2 on neutrophils to facilitate TEM (Springer 1990; Staunton et al. 1990; Vonderheide et al. 1994). There are five members of the ICAM family, which show variable expression patterns. The most extensively studied member of this family, ICAM-1, is expressed constitutively at low levels on endothelial and epithelial cells as well as on some leukocytes, dendritic cells, and fibroblasts (Rothlein et al. 1986; Shappell et al. 1990; Kelly et al. 1992; Dippold et al. 1993). ICAM-2 is constitutively expressed on endothelial and mononuclear leukocytes, but not on neutrophils (de Fougerolles et al. 1991), ICAM-3 is restricted to leukocytes where it is constitutively expressed (de Fougerolles & Springer 1992), ICAM-4 is restricted to erythrocytes (Bailly et al. 1994), and ICAM-5 is strongly expressed in the brain (Mizuno et al. 1997). While ICAM-1 and ICAM-2 are both constitutively expressed on endothelium, surface expression of ICAM-1 but not ICAM-2 (Staunton et al. 1989) is upregulated in response to the inflammatory cytokines IL-1, interferon (IFN)-γ, and TNF-α (Rothlein et al. 1986; Rice et al. 1990; Min

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et al. 2005). ICAM-1 is a variably glycosylated, 76–114 kDa glycoprotein with a 55-kDa peptide backbone (Dustin et al. 1986). The extracellular portion of all five ICAMs possesses variable numbers of immunoglobulin-like domains. ICAM-1 consists of five immunoglobulin-like extracellular domains, with the first domain responsible for binding to αLβ2 (LFA-1) and the third domain responsible for binding to αMβ2 (MAC-1) (Staunton et al. 1990; Vonderheide et al. 1994). In addition to binding β2 integrins, ICAM-1 is also a receptor for Plasmodium falciparum (Berendt et al. 1989) and the major group of rhinovirus (Greve et al. 1989; Tomassini et al. 1989). Alternative splicing of ICAM-1 produces multiple isoforms (King et al. 1995) including a soluble form (Rothlein et al. 1991) that binds the human rhinovirus, inhibiting infection (Marlin et al. 1990). The soluble extracellular domain may inhibit leukocyte–endothelial interactions by acting as a decoy receptor (Kusterer et al. 1998). ICAMs possess one transmembrane segment and a short cytoplasmic tail, which interacts with the cytoskeletal protein vinculin and is important for intracellular signaling events as well as diapedesis (Carpen et al. 1992; Lyck et al. 2003; Yang et al. 2005; Nieminen et al. 2006). ICAM-1 mediates granulocyte extravasation and antibodies to ICAM-1 inhibit leukocyte binding to endothelium as well as extravasation (Smith et al. 1988, 1989; Barton et al. 1989). In light of this, it is unsurprising that ICAM-1deficient mice display impaired neutrophil trafficking (Sligh et al. 1993). Studies on how ICAM-1 mediates firm adhesion have recently demonstrated that ICAM-1 (and to a lesser extent VCAM-1) concentrates in an actin-rich, cup-like docking structure that not only binds leukocytes to the endothelium but is important for TEM (Barreiro et al. 2002; Carman & Springer 2004; Yang et al. 2005). Additionally, a recent study has proposed a more specific role for ICAM-1 in mediating TEM via the transcellular route (through single endothelial cells). Millan et al. (2006) have demonstrated that leukocyte adhesion induces clustering of ICAM-1 and caveolin which may form a channel through which the leukocyte migrates across individual endothelial cells.

VCAM-1 VCAM-1 (CD106) is a type I transmembrane glycoprotein member of the immunoglobulin superfamily that mediates TEM by binding to α4β1 (VLA-1) on leukocytes (Dustin & Springer 1988; Rice & Bevilacqua 1989; Elices et al. 1990; Cybulsky & Gimbrone 1991). It is expressed by a variety of cell types including endothelium, epithelium, fibroblasts, dendritic cells, and bone marrow stromal cells (Koopman et al. 1991; Miyake et al. 1991; Osborn et al. 1992; Salomon et al. 1997). Expression of VCAM-1 is upregulated by TNF-α, IL-1 and IL-4, as well as by nucleotides released by endothelial cells and detected by the P2Y2 receptor (Iademarco et al. 1992; Seye et al. 2004). Two alternatively spliced forms of VCAM-1 exist in humans (Cybulsky et al. 1991; Osborn et al. 1992; Chuluyan et al. 1995). The predominant form expressed


on the cell surface (7D VCAM-1) contains seven extracellular immunoglobulin-like domains, while the alternatively spliced form (6D VCAM-1) lacks immunoglobulin-like domain 4 (Hession et al. 1991). There is increasing evidence that the soluble form of 6D VCAM-1 augments leukocyte migration (Rose et al. 2001) possibly by binding α4β1 with higher affinity than 7D VCAM-1 (Woodside et al. 2006).

PECAM-1 PECAM-1/CD31 contains intracytoplasmic immunoreceptor tyrosine inhibitory motifs (ITIMs) that mediate an inhibitory function through recruitment of protein-tyrosine phosphatases (Jackson et al. 1997; Hua et al. 1998; Henshall et al. 2001; Newman et al. 2001). This has led to its subclassification into the immunoglobulin-ITIM superfamily, a subset of the conventional immunoglobulin superfamily (Hua et al. 1998; Newman 1999; Henshall et al. 2001). It is a 150-kDa glycoprotein consisting of six extracellular immunoglobulin domains, a transmembrane region, and a cytoplasmic domain. It has a number of O- and N-linked glycosylation sites as well as splice variants expressed in different cell types (Baldwin et al. 1994). PECAM-1 also has a soluble form that is generated by splicing out exon 9, which encodes the transmembrane region (Goldberger et al. 1994). PECAM-1 is expressed at high density on the lateral junctions of endothelial cells and on the surface of most leukocytes (Newman 1997; Mamdouh et al. 2003). Surface expression of PECAM-1 is regulated by shedding due to matrix metalloprotease and caspase activity (Ilan et al. 2001). The ligands for PECAM-1 include homophilic binding with itself on other cells, αVβ3, and CD38 as well as proposed interactions with proteoglycans (Piali et al. 1995; Sun et al. 1996; Deaglio et al. 1998). In addition, phosphorylation at tyrosine residue Y686F on the cytoplasmic tail results in ligand specificity switching from heterophilic to homophilic binding (Famiglietti et al. 1997). PECAM-1 has several important functions in both endothelial cells and leukocytes that contribute to leukocyte migration and TEM. PECAM-1 has been shown to be important for regulation of endothelial barrier function (Ferrero et al. 1995). This importance was reinforced by recent studies demonstrating that endothelial cells from PECAM-1-deficient mice show enhanced permeability in response to histamine (Graesser et al. 2002). PECAM-1 is required for leukocyte migration and TEM both in vitro and in vivo (Muller et al. 1993; Bogen et al. 1994; Nakada et al. 2000; Wang et al. 2005). Anti-PECAM-1 antibodies arrest leukocyte migration at the apical surface of endothelium prior to proceeding through the junction, demonstrating that PECAM-1 mediates the initial phase of diapedesis (Schenkel et al. 2002). There are several studies indicating that PECAM-1 may regulate these functions by regulating integrin activation (Piali et al. 1995; Chiba et al. 1999) and PECAM-1-deficient mice have impaired “outsidein” signaling by the platelet integrin αIIbβ3 (Wee & Jackson 2005).

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CD99 CD99 is a small, heavily O-glycosylated transmembrane glycoprotein expressed on most leukocytes, erythrocytes, and at endothelial cell junctions (Aubrit et al. 1989; Muller 2002; Schenkel et al. 2002). It shows no resemblance structurally or at amino acid level to any known protein family and its functions are poorly understood, although along with PECAM-1 and JAMs it is involved in diapedesis. The gene for CD99 encodes two alternatively spiced products that appear to have opposite effects on lymphocyte adhesion as well as on the expression of LFA-1 and ICAM-1 (Hahn et al. 1997; Byun et al. 2006). Anti-CD99 antibodies have demonstrated lymphocyte adhesion mediated by homotypic interactions (Kasinrerk et al. 2000; Imbert et al. 2006). In addition, ligation of CD99 on lymphocytes has been shown to induce adhesion by regulating expression of α4β1 binding to VCAM-1 (Bernard et al. 2000). Antibodies directed against CD99 block T-cell recruitment in vivo (Bixel et al. 2004). Recent work indicates that antibodies against CD99 arrest monocyte migration but, importantly, only when the cells are located deep within the paracellular pathway and close to completion of transmigration into tissue, suggesting that PECAM-1 and CD99 work in a sequential fashion to mediate paracellular diapedesis (Aurrand-Lions et al. 2002; Schenkel et al. 2002).

JAMs JAMs are recently discovered members of the immunoglobulin superfamily. JAM-A was first reported as the ligand for platelet activating antibody and thus was referred to as F11 receptor (Kornecki et al. 1990). Sequencing of JAM-A from epithelial and endothelial cells led to its initial description in the formation of tight junctions (Martin-Padura et al. 1998; Palmeri et al. 2000). JAM proteins are type I transmembrane receptors consisting of an extracellular domain containing two immunoglobulin-like domains, a transmembrane domain, and a short cytoplasmic tail (Malergue et al. 1998; Martin-Padura et al. 1998; Sobocka et al. 2000; Arrate et al. 2001). Two related JAMs were subsequently found, JAM-B and JAM-C, which were also localized primarily at cell–cell junctions on endothelial cells (Cunningham et al. 2000; Arrate et al. 2001; Aurrand-Lions et al. 2005). In addition, two more JAMs have been recently identified: JAML in humans (Moog-Lutz et al. 2003) and JAM4 in mice, although it is unknown in the latter whether a human homolog exits (Hirabayashi et al. 2003). In addition, JAM4 and JAML appear to be less closely related to JAM-A, JAM-B or JAM-C (Mandell & Parkos 2005). Subsequently, the expression pattern of JAM proteins has been found to be much wider than previously thought and these proteins have been implicated in a variety of events important to leukocyte function including tight junction assembly (Martin-Padura et al. 1998; Liang et al. 2000; Itoh et al. 2001; Ebnet et al. 2003; Mandicourt et al. 2007), platelet activation (Kornecki et al. 1990; Ozaki et al. 2000; Sobocka et al. 2000), and leukocyte transmigra-

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tion (Martin-Padura et al. 1998; Johnson-Leger et al. 2002; Ostermann et al. 2002). Indeed, antibodies directed against JAM-A and JAM-C inhibit leukocyte migration both in vitro and in vivo (Martin-Padura et al. 1998; Del Maschio et al. 1999; Chavakis et al. 2004). JAM-A is expressed on the surface of lymphocytes, monocytes, neutrophils, platelets, and erythrocytes (Martin-Padura et al. 1998; Williams et al. 1999; AurrandLions et al. 2001). JAM-A binds itself (Bazzoni et al. 2000) as well as reovirus protein (Barton et al. 2001). In addition, JAM-A on endothelial cells binds to αVβ3 (Naik et al. 2003; Naik & Naik 2006), as well as αLβ2 on neutrophils and T cells, facilitating TEM (Ostermann et al. 2002; Fraemohs et al. 2004). Furthermore, soluble JAM-A has been used to inhibit TEM of T cells under flow conditions (Ostermann et al. 2005). Inflammatory mediators such as TNF-α and IFN-γ cause junctional disassembly of JAM-A and relocalization to the apical surface of endothelial cells (Ozaki et al. 1999; Shaw et al. 2001). The JAM-A–αLβ2 interaction mediates tight adhesion or transmigration depending on the apical or junctional localization of JAM-A on endothelial cells (Ostermann et al. 2002). JAM-B has restricted expression to endothelial cells where it mediates leukocyte adhesion by binding to α4β1 (VLA-4) as well as JAM-C (Cunningham et al. 2002; Liang et al. 2002) and promotes lymphocyte TEM (Johnson-Leger et al. 2002). JAM-C is expressed on endothelial cells, leukocytes, and fibroblasts (Cunningham et al. 2000; Palmeri et al. 2000; Aurrand-Lions et al. 2001; Morris et al. 2006). JAM-C binds to JAM-B (Arrate et al. 2001) as well as αMβ2 (Mac-1/ CD11b/CD18) on neutrophils and mediates TEM both in vitro and in vivo (Santoso et al. 2002; Chavakis et al. 2004; AurrandLions et al. 2005; Zen et al. 2005; Mandicourt et al. 2007). JAML is found on human granulocytes (Moog-Lutz et al. 2003), and coxsackie and adenovirus receptor (CAR) has been found to be an epithelial counterreceptor that mediates neutrophil migration across tight junctions (Zen et al. 2005). Consequently, JAMs appear to mediate leukocyte adhesion through both homophilic interactions as well as heterophilic interactions with integrins and other JAMs (Bazzoni et al. 2000; Arrate et al. 2001; Cunningham et al. 2002; Santoso et al. 2002; Lamagna et al. 2005; Mandell et al. 2005). Like other leukocyte adhesion molecules, cytokines seem to be important for JAM regulation. However, to date, these studies demonstrate that, rather than regulating expression levels, cytokines regulate JAM localization from intercellular junctions to the apical membrane (Ozaki et al. 1999; Shaw et al. 2001) and internalization of JAMs (Bruewer et al. 2003, 2005; Utech et al. 2005). Phosphorylation of the cytoplasmic tail appears to be a molecular mechanism for regulation of intracellular binding partners, adhesive function, and localization (Ozaki et al. 2000; Ebnet et al. 2003; Dejana 2006; Mandicourt et al. 2007).

MAdCAM-1 MAdCAM-1 is an endothelial cell glycoprotein that specifies lymphocyte homing to mucosal sites (Sampaio et al. 1995).

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MAdCAM-1 is expressed at high density on HEV of lymphoid tissues such as Peyer’s patches and mesenteric lymph nodes and to a lesser extent in lamina propria (Picker & Butcher 1992; Briskin et al. 1997). Recent evidence indicates that MAdCAM-1 may be more widely expressed than previously thought, with evidence of expression on fibroblasts and melanoma cells (Leung et al. 2003) and in the brain (Kanwar et al. 2004). Cloning of human MAdCAM-1 revealed a multifunctional type I transmembrane adhesion molecule comprising two extracellular immunoglobulin-like domains, an extracellular mucin-like domain, one transmembrane region, and a short cytoplasmic tail (Shyjan et al. 1996; Leung et al. 1997, 2003). Its unique structure allows it to bind to lymphocytes and mediate homing through both α4β7 and L-selectin (Berg et al. 1993; Berlin et al. 1993; Shimizu & Shaw 1993; Hamann et al. 1994). Thus, MAdCAM-1 is uniquely able to participate in two phases of diapedesis: lymphocyte rolling with L-selectin and the firm adhesion step by binding with the integrin α4β7. The importance of MAdCAM-1 leukocyte trafficking is shown by studies demonstrating enhanced expression of MAdCAM-1 in patients with inflammatory disease (Souza et al. 1999; Arihiro et al. 2002; Ohara et al. 2003). Blocking antibodies to MAdCAM-1 have been shown to reduce leukocyte extravasation in vivo (Matsuzaki et al. 2005; Farkas et al. 2006). Numerous studies indicate that MAdCAM-1 expression can be induced by TNF-α and IL-1β (Sikorski et al. 1993; Takeuchi & Baichwal 1995; Ando et al. 2005). The cytoplasmic tail is involved in signaling via phosphorylation of cytoskeletal proteins (Murata et al. 2002).

Adhesion molecules in allergic disease Adhesion molecule expression Endothelial adhesion molecule expression Adhesion molecule function is regulated in a number of ways, including increased expression as with E- and P-selectin, ICAM-1 and VCAM-1, shedding as with L-selectin, and conformational changes in the binding affinity of the receptor as seen with many integrins. A number of groups have studied expression of E-selectin, ICAM-1, and VCAM-1 in asthma and other allergic inflammatory conditions. P-selectin expression has been less widely studied, partly because of the difficulty in distinguishing between intracellular and luminal staining. Studies of adhesion receptors expressed following allergen challenge have been generally consistent with observations in vitro with cytokine-stimulated human umbilical vein endothelial cells (HUVECs). In the skin, low background expression of ICAM-1 is seen with absent expression of E-selectin and VCAM-1. After allergen challenge, increased endothelial expression of all three receptors has been reported (KyanAung et al. 1991; Leung et al. 1991). In the airway, Montefort et al. (1994a) found increased expression of ICAM-1 and E-selectin 6 hours after local allergen challenge with no


increase in VCAM-1 expression. Bentley et al. (1993) reported a trend toward increased VCAM-1 expression (significance was lost through one outlier), with a good correlation between VCAM-1 expression and eosinophil infiltration 24 hours after aerosol allergen challenge. In sensitized lung explants, allergen challenge increased ICAM-1, E-selectin, and VCAM-1 expression in a manner similar to that seen in HUVECs. Upregulation was mediated by a combination IL-4 and TNF-α (Hirata et al. 1998). In clinical asthma, findings have been more variable, perhaps reflecting the inherent problems in accurately quantifying small changes in expression using immunohistochemistry. Montefort et al. (1992b) were unable to detect changes in adhesion receptor expression in atopic asthma compared with normal subjects. In a study of atopic and nonatopic asthma, Bentley et al. (1993) could only detect a modest increase in ICAM-1 and E-selectin expression in nonatopic asthmatics compared with normal subjects, with relatively high expression in normals. However, the E-selectin antibody cross-reacted with P-selectin, which makes the data on E-selectin difficult to interpret. In contrast, Gosset et al. (1995) found low expression in normal subjects and could detect increases in adhesion molecule expression in atopic but not nonatopic asthmatics. Ohkawara et al. (1995) agreed with these findings in six atopic asthmatics but Fukuda et al. (1996) detected no increase in ICAM-1 or E-selectin staining over controls. However, the E-selectin antibody they used also cross-reacted with P-selectin. Nevertheless, this group did find an increase in VCAM-1 expression that correlated with eosinophil counts but only in those subjects with detectable IL-4 in bronchoalveolar lavage (BAL) fluid. In nasal endothelium, generally weak expression of VCAM-1 has been observed, although increased over normal controls, in both perennial rhinitis and nasal polyps (Montefort et al. 1992a). Increased expression of ICAM-1, VCAM-1, and E-selectin was also observed after allergen challenge (Braunstahl et al. 2001). We have found E-selectin expression to be weak in nasal polyps, lung resection tissue, and perennial rhinitis. We have found that P-selectin is widely expressed in both nasal and lung tissue (Symon et al. 1994; Ainslie et al. 2002). Strong expression was seen in nasal biopsies from both normal controls and patients with perennial rhinitis, with little difference between the two groups after fixation with either acetone or paraformaldehyde (both of which favor surface as opposed to intracellular staining). Similarly, in endobronchial biopsies good expression was observed in sections from both asthmatic and control subjects. Consistent with this observation, P-selectin was well expressed in lung resection tissue from patients with lung cancer. Expression was seen on both bronchial and pulmonary venules but not the pulmonary capillaries. As mentioned above it is difficult to distinguish conclusively between intracellular and luminal staining using standard immunohistochemistry. However, the strong expression in the airway does

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suggest P-selectin rather than E-selectin is the major selectin involved in leukocyte migration into the lung in humans. ICAM-1 expression on epithelial cells is consistently increased on bronchial epithelium in asthma (Vignola et al. 1993). The role of ICAM-1 as a receptor for the major group of rhinoviruses means that the epithelium in asthmatics may be more vulnerable to viral infection. Expression of CD44, a receptor for the matrix protein hyaluronate, is increased on the bronchial epithelium in asthma, although it is also found on normal epithelium (Lackie et al. 1997).

Soluble adhesion molecules Several adhesion molecules can be detected in soluble form circulating in the plasma. Montefort et al. (1994b) found that concentrations of E-selectin, ICAM-1, and VCAM-1 were not elevated in stable asthma, but there was a significant increase compared with normal controls in concentrations of sE-selectin and sICAM-1 in patients with acute severe asthma. However, concentrations of these molecules did not correlate with disease severity and were therefore not thought useful in clinical management. In another study of 45 atopic and nonatopic asthmatics serum concentrations of sICAM-1, sE-selectin, and sVCAM-1 were increased during “asthma attacks” compared with stable periods (Kobayashi et al. 1994; Koizumi et al. 1995). Modest increases in concentrations of sICAM-1 and sE-selectin have also been detected in BAL fluid after segmental allergen challenge (Georas et al. 1992; Takahashi et al. 1994). Zangrilli et al. (1995) measured sVCAM-1 concentrations in BAL fluid 24 hours after segmental allergen challenge in 27 ragweed allergic asthmatics and 18 atopic nonasthmatics. A marked increase in sVCAM-1 concentrations was observed in BAL fluid that correlated with increased numbers of eosinophils and concentrations of IL-4 and IL-5. Most of the increase occurred in the late responders. In atopic dermatitis in children, plasma E-selectin was raised and correlated with disease severity during exacerbations but did not fall after treatment (Wolkerstorfer et al. 2003). Therefore as yet no clear-cut correlations between disease severity and concentration of soluble adhesion receptors have been found that would allow these molecules to be used as biomarkers of diagnosis or disease activity.

Leukocyte migration and adhesion Eosinophil migration and adhesion The mechanisms controlling eosinophil migration into tissue have been the subject of intensive study for over four decades and there is a wealth of literature on the subject summarized in several recent reviews (Weller 1997; Wardlaw 1999; Bochner 2000; Rothenberg & Hogan 2006). The eosinophil expresses a number of adhesion receptors that are involved in cell trafficking and effector function (Table 15.1). In the normal individual eosinophils make up only 1–5% of the white cell count and in the normal lung there are 50 neutrophils for every eosinophil. In severe asthma there is an average of four eosinophils for every neutrophil in the bronchial sub-

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Table 15.1 Eosinophil adhesion receptors and the ligands they recognize. Ligand Receptor Integrins a4b1(VLA-4) a4b6 a4b7 LFA-1(aLb2) Mac-1(aMb2) P150,95(axb2) adb2 Selectins and ligands PSGL-1 L-selectin Other CD44 ICAM-3 PECAM Siglec-8

Endothelial

Matrix protein

VCAM-1

Fibronectin Laminin Fibronectin

MAdCAM-1 ICAM-1-3 ICAM-1 VCAM-1(ICAM-3?)

P-selectin(E-selectin) GlyCAM-1,CD34,Podocalyxin

Hyaluronate PECAM Sialic acid

mucosa, representing a 200-fold enrichment of eosinophils over neutrophils in the process of accumulation. Even in mild asthma there is an up to 30-fold enrichment (Azzawi et al. 1992; Brightling et al. 2003). Historically this was thought to be due to a selective chemoattractant. A factor termed “eosinophil chemotactic factor of anaphylaxis” (ECF-A) was detected in supernatants from anaphylactically challenged guinea-pig lung that appeared to be selectively chemotactic for eosinophils (Kay et al. 1971). This was subsequently found to consist of a combination of leukotriene (LT)B4, which is active on guinea-pig eosinophils but less so on human eosinophils, and 15-hydroxyeicosatetraenoic acid (Sehmi et al. 1991). ECF-A from human lung was later identified and characterized as two tetrapeptides, Val-Gly-Ser-Glu and AlaGly-Ser-Glu (Goetzl & Austen 1975). However, in comparison with platelet-activating factor, these peptides were found to have negligible activity (Wardlaw et al. 1986). More recently it has become clear that eosinophil migration is the result of an interaction between selective expression of adhesion molecules that bias toward eosinophil recruitment and eosinophil-selective chemoattractants, particularly chemokines. Accumulation of eosinophils is not therefore the result of any single event but occurs because of selective pressure at every stage in the life cycle of the eosinophil, including eosinophilopoiesis and egress from the bone marrow, adhesion mechanisms, chemotaxis and prolonged survival under the influence of locally generated growth factors (Fig. 15.6).

Eosinophilopoiesis and egress from the bone marrow Eosinophils differentiate from bone marrow precursors under the influence of growth factors, especially IL-5. Increased

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IL-5 eotaxin 1

IL-4/ IL-13

PSGL-1/P-selectin VLA-4/VCAM-1 Chemotaxis CCL26/CCR3

Survival IL-5/GM-CSF IL-4 IL-5 IL-13

4

2


Tether/roll Flow

Activation Firm arrest

3

In situ Diff/IL-5 5 CCL26/CCR3

Th2

6 Apoptosis

Fig. 15.6 Selective accumulation of eosinophils is a multistep process directed by Th2-associated cytokines. Step 1 involves selective eosinophilopoiesis under the influence of IL-5 and egress from the bone marrow promoted by chemoattractants such as eotaxin 1. Step 2 involves selective migration through venular endothelium that is promoted by a4b1/VCAM-1, PSGL-1/P-selectin, and (CCL26) eotaxin 3. After transmigration (step 3), tissue accumulation is controlled by a balance between prolonged survival and retention in tissues as a result of adhesion to matrix proteins such as fibronectin (step 4), local differentiation from precursors within tissue (step 5), and signals for egress into the lumen provided by epithelial-derived chemoattractants such as CCL26 (step 6). All this is orchestrated by IL-4, IL-13 and IL-5 released by Th2 lymphocytes. (See CD-ROM for color version.)

amounts of IL-5 generated at sites of allergic inflammation act hormonally to increase eosinophil production. There is on average about a fourfold increase compared with normal subjects in the number of circulating eosinophils in allergic individuals. There is also evidence for increased numbers of circulating eosinophil precursors in the peripheral blood of allergic patients. This increase is the consequence of both increased production and egress from the bone marrow. In guinea pigs IL-5 selectively promoted the egress of eosinophils from the marrow (Collins et al. 1995). This was enhanced by eotaxin, an eosinophil-selective chemokine (Palframan et al. 1998a), and controlled by the adhesion molecules VLA-4, inhibition of which accelerates egress, and Mac-1, whose inhibition prevents egress (Palframan et al. 1998b). In this contex IL-5 is probably acting as a chemokinetic factor as well as priming eosinophils for chemotactic responsiveness.

Migration through vascular endothelium Adhesion to and migration through vascular endothelium is an important control point in the migration of all leukocytes including eosinophils. As for other leukocytes, migration in most situations occurs through the postcapillary venular endothelium and follows the well-established multistep paradigm of a capture step, often followed by rolling along the surface of the vessel until the leukocyte becomes activated, which results in arrest of the cell and transmigration (Fig. 15.7). An exception to this is migration into the peripheral lung

a4/b1/VCAM-1 PSGL-1/P-selectin L-selectin

Gai, PT sensitive 7TM receptors

CD18/ICAM-1 a4b1/VCAM-1

Fig. 15.7 Schematic illustration of the multistep paradigm of leukocyte migration through endothelium in the high-flow systemic circulation, which involves a capture step mediated by selectins and VCAM-1, an arrest step mediated by activating signals such as chemoattractants expressed on the endothelial surface, and a transmigration step mediated largely by b2 integrins and ICAM-1. (See CD-ROM for color version.)

compartment through the capillary bed and venules of the low-pressure pulmonary circulation, which is less dependent on adhesion receptors, being predominantly controlled by chemoattractant stimuli (Fig. 15.8). Eosinophils adhere in much greater numbers than neutrophils to vascular endothelium stimulated with the Th2 cytokines IL-4 and IL-13 as a result of selective adhesion to VCAM-1 and P-selectin, which work in a synergistic manner to capture and then arrest these cells on vascular endothelium (Patel 1998; Woltmann et al. 1999). Human endothelial cells stimulated for over 24 hours with IL-4 or IL-13, but not IL-1β or TNF-α, induced low levels of expression of both VCAM-1 and P-selectin (Yao et al. 1996). Eosinophils can adhere to VCAM-1 through VLA-4 whereas neutrophils that do not express this receptor are unable to do so (Walsh et al. 1991). Eosinophils adhere more avidly to low levels of P-selectin than neutrophils as a result of increased expression of the P-selectin receptor PSGL-1 and possibly because of differences in the pattern of O-glycosylation on the eosinophil PSGL-1 receptor (Symon et al. 1996; Edwards et al. 2000). P-selectin is well expressed on airway vascular endothelium and in an ex vivo assay of eosinophil adhesion to vascular endothelium in a nasal polyp model of eosinophilic inflammation P-selectin/PSGL-1 was the primary adhesion pathway involved (Symon et al. 1994). Eosinophil migration to the lung was reduced in a P-selectin gene-deleted mouse (Broide et al. 1998). In contrast, neutrophils bind more avidly to E-selectin, which is not well expressed on the airway vascular endothelium in asthma (Sriramarao et al. 1996; Kitayama et al. 1997a). VLA-4/VCAM-1 can also support selective transmigration through HUVECs and a number of animal models have shown that eosinophil migration into the lung is inhibited by anti-VLA-4 (Nakajima et al. 1994; Lobb et al. 1996; Cortijo et al. 2006). However, VCAM-1 expression is not particularly marked on the airway mucosal endothelium in asthma or nasal polyps; in both the nasal polyp model and in adhesion to IL-4-stimulated

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Bronchial artery and vein

Pulmonary vein Systemic circulation

Pulmonary artery

endothelium, inhibition of VLA-4/VCAM-1 on its own had a relatively minor effect (Bentley et al. 1993; McNulty et al. 1999; Woltmann et al. 2000). Antagonists of VLA-4 only partially blocked eosinophil adhesion to TNF-α- and IL-4-stimulated endothelium (Sedgwick et al. 2005). In addition VLA-4 antagonists had only a minor effect at best on eosinophil accumulation in the airways after allergen challenge, although it is possible that the inhaled route used in these studies was not optimal (Diamant et al. 2005; Norris et al. 2005). In summary, therefore, in vitro studies of adhesion to endothelium under conditions of Th2 cytokine generation have shown that selective adhesion is directed by the combined actions of VCAM-1/VLA-4 and P-selectin/PSGL-1, with the latter playing a dominant role in the capture of the cells. Under more Th1-like conditions where cytokines such as TNF-α and IL-1 are generated, adhesion will be through E-selectin, VCAM-1/ VLA-4, and ICAM-1/β2 integrins, but will not have a selective component (Bochner et al. 1991). Eosinophils express all four members of the β2 integrin family (Hartnell et al. 1990; Grayson et al. 1998). As is the case for neutrophils, αΜβ2 (Mac-1) and αLβ2 (LFA-1) have been shown to mediate eosinophil transmigration through TNFα- and IL-1β-stimulated endothelium mainly by binding to ICAM-1 (Yamamoto et al. 1998), although both αDβ2 and Mac-1 as well as VLA-4 have been shown to bind to VCAM-1 (Barthel et al. 2006a). Eosinophils bind VCAM-1 through podosomes (Johansson et al. 2004). Following the established paradigm for leukocyte adhesion to endothelium, after the capture and rolling step the eosinophil requires activation to mediate arrest and engagement of β2 integrins in particular. This then allows transmigration. Eosinophil activation in this context is complex and still relatively poorly understood. Although CCR3-binding chemokines can participate in this process, they have only a relatively minor effect on adhesion to cytokine-stimulated endothelium under flow conditions (Kitayama et al. 1998). Avidity for β1 and β2 integrins is differentially regulated depending on the timing and type of stimulus and eosinophils have possibly three distinct signal transduction pathways that regulate responses to chemo-

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Fig. 15.8 Migration into the lung differs between the large airways and the peripheral compartment. The bronchi are supplied by bronchial arteries that are part of the systemic circulation and therefore migration follows the multistep paradigm. Migration into the peripheral lung is through the capillary bed of the low-pressure pulmonary circulation and is relatively adhesion receptor independent. (See CD-ROM for color version.)

attractants (Weber et al. 1996; Kitayama et al. 1997b). More work is required in this area to understand the precise signals that control the activation step in eosinophil adhesion to endothelium. As well as VLA-4, eosinophils also express α4β7 and there is evidence that this receptor is involved in normal homing of eosinophils to the small intestine through binding to MAdCAM-1, although ICAM is the main receptor involved in migration into the large intestine (Mishra et al. 1999; Forbes et al. 2006). Transmigration of eosinophils is shear dependent and under flow conditions occurs on IL-4- but not TNF-α-stimulated endothelium. Under these conditions the CCR3-binding chemokine eotaxin 3 (CCL26) appeared to the main chemoattractant involved (Cuvelier & Patel 2001). Eosinophil adhesion to endothelium under shear flow resulted in endothelial activation of Rho-associated proteins and calpain, which are necessary for transmigration of eosinophils (Cuvelier et al. 2005).

Eosinophil migration within tissue Once eosinophils have migrated into tissue, as is the case with all leukocytes, they need to be able to integrate the various adhesive and chemotactic signals derived from structural cells and the extracellular matrix in order to direct their microlocalization within the tissue environment. This includes maintaining residence within the tissue or migrating through epithelium into the gut or airway lumen (Fig. 15.9). Eosinophils express the β1 integrins α4β1 and α6β1, which have been shown to play a role in binding to fibronectin and laminin respectively. These matrix proteins support eosinophil survival by triggering release of GM-CSF, an event inhibited by glucocorticoids (Walsh & Wardlaw 1997). With other aspects of eosinophil activation more variable effects with fibronectin have been observed, with some authors reporting enhancement and others inhibition of eosinophil degranulation (Neeley et al. 1994; Kita et al. 1996). Tissue eosinophils express a more activated phenotype than their blood counterparts, with increased expression of CD69, ICAM-1, and a number of other receptors (Hartnell et al. 1993). They also express an activated form of Mac-1 that results in a hyperadhesive phenotype to a

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Lymphatics

Airway lumen Fig. 15.9 Once through the endothelium, leukocytes undergo a process of microlocalization within the mucosa. This is controlled by integration of various adhesive and chemoattactant signals provided by the extracellular matrix, structural cells, and other leukocytes. Microlocalization is likely to be important in the effector role of leukocytes, which probably exert their effects across only a few micrometers. In particular mast cells colocalize with structural cells including airway smooth muscle in asthma, whereas T cells migrate into the epithelium or into the lymphatics on their way back to the blood. Eosinophils migrate into the lumen where they appear in induced sputum. (See CD-ROM for color version.)

number of matrix proteins, as well as VCAM-1 and ICAM-1 (Barthel et al. 2006b). The tissue phenotype can be mimicked by culturing blood eosinophils in IL-5 or GM-CSF for 24 hours. For eosinophils to migrate through epithelium to the lumen they need to migrate to and through the epithelium. Eosinophils adhere relatively weakly to cultured airway epithelial cells, which presumably allows them to escape into the lumen. Such adhesion is mediated by CD18/ICAM-1 interactions and is stimulated by IL-5 (Sanmugalingham et al. 2000). Migration through IL-4-stimulated epithelium, as is the case for endothelium, is primarily mediated by cell-bound eotaxin 3 (Yuan et al. 2006). Thus although there are a large number of chemoattractants involved in eosinophil migration under conditions of Th2 cytokine stimulation, the in vitro evidence points to eotaxin 3 binding to CCR3 as playing a central role. In summary, therefore, eosinophil trafficking into tissue involves a multistep process, with selective pressure on eosinophil recruitment occurring through selective expression of eosinophil-biased adhesion receptors and chemokines promoted by the Th2 cytokines IL-4 and IL-13. Once in tissue, adhesion to matrix proteins and structural cells plays an important role in survival, microlocalization, and eosinophilmediated tissue damage.

Basophil migration and adhesion Basophils, like eosinophils, are generated in the bone marrow and circulate in blood as mature granulocytes before migrating into tissue. Basophils express a similar but not identical pattern of adhesion and chemokine receptors to eosinophils.


They express PSGL-1 but at lower levels than eosinophils, and like neutrophils (but not eosinophils) express high levels of sLex, which suggests they preferentially bind E-selectin compared with P-selectin. They express L-selectin (which is shed on activation), all four β2 integrins, and α4β1 and α4β7. In addition they express α5β1 and αVβ3, but not α6β1 (Bochner & Schleimer 2001). Blocking antibodies against α4β1 have been shown to inhibit basophil binding to endothelium under static conditions (Bochner et al. 1996) as well as TEM through IL-3-stimulated endothelium, although β2 integrins binding to ICAM-1 was more important in TEM through IL-1-stimulated endothelium (Iikura et al. 2004). Under flow conditions basophil adhesion to IL-4-stimulated endothelium was mediated by both E-selectin and P-selectin, with α4β1 only playing a role if the basophils were activated, possibly through an Src-dependent pathway (Andrews et al. 2001; Kepley et al. 2002). Again under flow conditions, IL-3stimulated endothelium was able to bind basophils (as well as eosinophils but not neutrophils) through a PSGL-1/VLA-4 dependent pathway in a manner similar to IL-4- and IL-13treated endothelium. Of particular interest is the observation that the basophil arrest step appeared to be pertussis toxin sensitive and mediated by CCR7, whereas eosinophil arrest was not pertussis toxin sensitive as was also the case in the ex vivo nasal polyp model (McNulty et al. 1999; Lim et al. 2006).

Mast cell migration and adhesion Mast cells are released from the bone marrow and circulate in the blood as precursors (pMC) before fully differentiating in tissue. In blood they are found among a subset of mononuclear cells that express CD34, CD13, and the stem cell factor (SCF) receptor c-kit. Immature peripheral blood mast cells, or cells derived from human cord blood as a model of pMC, express α4β1, α4β7, αMβ2, αVβ2, and PSGL-1 (Inamura et al. 2001; Tachimoto et al. 2001; Boyce et al. 2002). They can therefore bind VCAM-1, MAdCAM-1, ICAM-1/2, E-selectin, and P-selectin. They do not express L-selectin. The patterning of binding of pMC to vascular endothelium is similar in some respects to basophils rather than eosinophils, with a relatively low affinity for P-selectin and therefore an inability to bind to IL-4-stimulated endothelium under flow conditions. They utilize predominantly E-selectin and VLA-4/VCAM-1 for binding to TNF-α-stimulated endothelium (Boyce et al. 2002). Interestingly, the β2 integrins and ICAM-1 do not play a major role in adhesion under these conditions despite these receptors being involved in IL-4-mediated homotypic adhesion (Toru et al. 1997). The chemoattractants involved in migration of pMC across the endothelium have not been well defined. pMC express CCR3 and may therefore be able to respond to eotaxin 3, which is secreted by endothelial cells (Romagnani et al. 1999; Juremalm & Nilsson 2005). They also express CXCR4 and transmigrate across endothelium in response to CXCL14 (SDF-1) (Juremalm et al. 2000; Lin et al. 2000).

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When pMC migrate into tissue they differentiate into tryptase-expressing mast cells (MCT) or chymase/tryptaseexpressing mast cells (MCTC) depending on local developmental signals. During this process they change their pattern of chemokine receptors, for example increasing expression of CXCR3 and reducing expression of CCR3 (Brightling et al. 2005a), a change that may promote their migration toward airway smooth muscle (ASM) (Brightling et al. 2005b). Mature mast cells can also respond to fractalkine, another chemokine released by ASM (Papadopoulos et al. 2000; El-Shazly et al. 2006). An important feature of mast cell biology is their anatomic proximity to the structural cells within the tissue, presumably in order to modulate the function of these structures. This includes vascular and neural tissue, mucous glands, epithelium, and ASM where colocalization has been shown to be related to presence of airway hyperresponsiveness (Costello et al. 1997; Brightling et al. 2002). Whether it is the pMC, mature mast cells, or both that migrate is not known but there is evidence that mature cells can move from the submucosa to the epithelium during allergen challenge (Viegas et al. 1987). Adhesion to matrix proteins also plays an important role in mast cell activation, acting as a priming stimulus for mediator release. Mast cells are able to adhere to fibrinogen and von Willebrand factor via αIIbβ3 after stimulation with SCF and cross-linking of FcεRI. Binding to fibrinogen has a marked effect on function, with enhanced proliferation, cytokine production, and migration (Oki et al. 2006). Similarly, binding to fibronectin and vitronectin primes the mast cell line HMC-1 for mediator release stimulated by phorbol myristate acetate and ionomycin (KrugerKrasagakes et al. 1999). Human intestinal mast cells are able to adhere to fibronectin, laminin and collagens I, III, VI, and XIV. Adhesion was enhanced fivefold by SCF and was mediated by a combination of α2β1–α5β1 and αVβ3. The effects of SCF were reduced by cotreatment with IL-4 and by blockade of phosphatidylinositol 3-kinase and MAPK (Lorentz et al. 2002). Adhesion to fibronectin and vitronectin was also inhibited by an antibody against CD63, a member of the tetraspanin family that forms multimolecular complexes with a variety of membrane proteins including β integrins. In addition, anti-CD63 inhibited FcεRI-mediated degranulation of adherent but not nonadherent mast cells (Kraft et al. 2005). Activation of Toll-like receptor (TLR)3 on mast cells had a similar effect to blockade of CD63. Mast cells express TLR1–7 and TLR9 and when stimulated through TLR3 mast cell adherence to fibronectin and vitronectin as well as FcεRI-mediated degranulation was reduced as a result of inactivation of CD29 (β1) (Kulka & Metcalfe 2006). The close proximity of mast cells to structural cells may also be the result of avid cell–cell adhesion. A striking feature of the cell differential from bronchial washings and induced sputum is the paucity of mast cells (and T cells) compared with neutrophils and (in asthma) eosinophils, despite these cells being present in similar numbers in the submucosa. This

350

is likely to be due to the firmer anchoring of mast cells in the tissue as a result of binding to matrix proteins and structural cells including epithelium. In support of this hypothesis, mast cells bind much more firmly to bronchial epithelial cells than eosinophils (60% vs. 10%) (Sanmugalingam et al. 2000). Similar levels of mast cell adhesion have been observed to ASM and fibroblasts (Trautmann et al. 1997; Yang et al. 2006a). Interestingly this adhesion pathway, although partly calcium dependent, is not mediated by any of the classical adhesion receptors including the β1 or β2 integrins and appears to involve in part novel pathways, with tumor suppressor in lung cancer-1 mediating adhesion to ASM and a galactosebearing carbohydrate pathway adhesion to epithelial cells. Unlike matrix, adherence to epithelial cells attenuates mast cell degranulation stimulated by FcεRI (Yang et al. 2006b). Mast cell adherence to matrix proteins and structural cells is therefore of central importance to the function of these cells in allergic disease and dissection of the receptors involved and the consequences of their inhibition it likely to be a fruitful area of future research.

T-cell migration T cells, especially those of the Th2 subset, are thought to play a central part in allergic disease. The role of adhesion in T-cell migration and function is considerably more varied and complex than granulocytes and this topic can only be touched on in this section. The reader is referred to recent reviews of the subject for a detailed background (Campbell et al. 2003; Mora & von Andrian 2006). Unlike other leukocytes T cells migrate from blood into tissue and then back to the blood in a directed manner depending on their state of antigenic stimulation. Naive cells migrate only into lymph nodes whereas memory cells can migrate into both lymphatic tissue and organs which make up a tertiary lymphoid structure. T cells preferentially migrate back to the organ where they first encountered their cognate antigen, a process called lymphocyte homing. T cells expressing high levels of α4β7, which therefore bind MAdCAM-1, are thought to be preferentially gut homing whereas T cells that express CLA, a glycoform of PSGL-1 which preferentially binds E-selectin, are thought to be skin homing (Campbell & Butcher 2000). Lymphocyte homing may be important in directing allergen-sensitized T cells to the airway or skin. For example, caesin-reactive T cells from patients with milk-induced eczema had higher expression of the CLA antigen than Candida albicans-reactive T cells from the same patients or caesin-reactive T cells from nonatopic controls (Abernathy-Carver et al. 1995) . When house-dust mite-sensitive patients with asthma and atopic dermatitis were compared, the house-dust mite-responsive T cells from the eczema patients, but not from the asthma group, were in the CLA-positive T-cell subset (Santamaria Babi et al. 1995). Lung T cells are distinct from those of the gut and skin, being CLA negative and α4β7low (Campbell et al. 2001). CXCR6 is potentially involved in lung homing (Wardlaw et al. 2005)

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CLA/E-selectin CCR4/CCL17 CCR10/CCL27 CCR8/CCL1 Fig. 15.10 Lymphocytes migrate in a controlled manner, with naive lymphocytes only able to enter the secondary lymphoid tissue and memory lymphocytes preferentially returning to the organ where they encountered their cognate antigen. This process of lymphocyte homing is controlled by differential expression of a combination of chemoattractant and adhesion receptors and their ligands. (See CD-ROM for color version.)


P-selectin/PSGL-1 CXCR6/CXCL16

Skin

Lung

L-selectin/PNAd CCR7/CCL21 CCR7/CCL19 Lymph node

(Fig. 15.10). Migration during inflammatory states is superimposed on this homeostatic process. The number of T cells in tissue at any point in the normal state varies considerably. T cells are numerous in the gut and lung submucosa where many are likely to be long-term residents. However in the normal skin and in nonmucosal organs such as the heart and kidney, T cells are scanty. As with other leukocytes, T cells can be found in different compartments within tissue. For example in the lung there is the bronchial submucosa, epithelium, airway lumen, alveolar compartment, and interstitium. It is likely that the migration pathway into each compartment is different. As discussed above, in the lung there is a major difference between migration into the alveolar compartment through the low-pressure capillary bed, which is likely to be largely chemoattractant dependent, and migration into the bronchial mucosa via the systemic highpressure bronchial circulation, which involves the established multistep paradigm (Wardlaw 2002). There is a general assumption that Th2 cells are present in greater numbers in the tissue in allergic disease and there has therefore been considerable interest in the mechanisms driving this process, largely focused on the role of chemokines and their receptors. Most studies, the majority of which examined those with mild asthma, have not shown any increase in the numbers of T cells in the submucosa between asthmatics and normal subjects. There is also no consistent increase in T-cell numbers in BAL fluid or bronchial mucosa after allergen challenge (Aalbers et al. 1993; Frew et al. 1996). Again, at least in mild asthma, there is only very limited evidence for a major increase in IL-4-secreting T cells in the asthmatic lung (Morgan et al. 2005). Surprisingly, therefore, there is no strong signal for T-cell recruitment into the lung in asthma, although this picture may differ in more severe disease. T-cell capture by endothelium is mediated by a combination of P-, E- and L-selectin depending on the organ involved. L-selectin mediates adhesion to HEV in lymph nodes, and

a4b7/MAdCAM-1 CCR9/CCL25

Small intestine

L-selectin-negative T cells cannot migrate though HEV. As histamine stimulates endothelial expression of P-selectin in the short term and IL-4 and IL-13 in the long term, it might be considered that Th2 cells would preferentially bind to this receptor. In support of this, T-cell adhesion to nasal polyp endothelium was P-selectin/PSGL-1 dependent (Symon et al. 1999). However in vitro polarized Th1 cells preferentially bound P- and E-selectin compared with Th2 cells as a result of IL-4 inhibition of expression of FucTVII enzyme, which is necessary for the function of PSGL-1, although whether this is relevant in vivo is less clear (Wagers et al. 1998; Bonder et al. 2005a). T cells express both α4β1 and αLβ2 (LFA-1) and arrest and migration through endothelium is mediated by these receptors binding to VCAM-1 and ICAM-1 and -2 respectively, with no obvious differences between Th1 and Th2 cells despite the association of VCAM-1 expression with Th2 cytokines. Indeed in liver sinusoids, α4β1 mediated Th1 recruitment whereas vascular adhesion protein (VAP)-1 mediated Th2 recruitment (Bonder et al. 2005b). Once in the tissue T cells will integrate adhesive and chemoattractant signals in order to orientate themselves within various compartments. How this occurs is poorly understood. Unlike mast cells, T cells do not associate with ASM cells at least in mild asthma, but a subset do migrate into the epithelium where they remain anchored, appearing in the lumen in only small numbers as evidenced by the relatively small numbers of T cells in induced sputum in both asthmatics and normal controls (Brightling et al. 2002). Lung T cells express a variable pattern of activation receptors that probably directs their localization. About 30% of cells coexpress αEβ7, CD69, VLA-1, CXCR3, CCR5, and CXCR6 and it is likely that these cells are retained within the mucosa for prolonged periods. αEβ7-expressing cells (a receptor for E-cadherin) may become anchored within the epithelium, whereas VLA-1-expressing cells adhere to collagen within the airway mucosa. In contrast, cells that do not express any of these receptors but which

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tend to have increased expression of CCR7 are able to migrate into the lymphatics and recirculate back into the blood (Debes et al. 2005). ICAM-1 and VCAM-1 are expressed on ASM cells after stimulation with TNF-α and these receptors together with CD44 have been shown to mediate adhesion of activated T cells to ASM cells (Lazaar et al. 1994; Brightling et al. 2002). However, the lack of any association between T cells and ASM cells in bronchial biopsies from asthmatics raises questions about the physiologic relevance of this interaction.

In vivo studies of adhesion receptor function in allergic inflammation In vivo models of allergen challenge in a variety of animal species have been used extensively to investigate the role of adhesion receptors, mainly in terms of cell recruitment and airway hyperresponsiveness, using either monoclonal antibodies or other adhesion receptor antagonists or gene-deleted mice. These studies are detailed in Table 15.2. Inhibitory effects have been demonstrated using antibodies against a

Table 15.2 In vivo models of adhesion receptors in allergen challenge. Receptor

Species

Findings

Reference

E-selectin

Monkey

In this neutrophil-dependent model of late-phase response to Ascaris challenge, both cell migration and bronchoconstriction were inhibited

Gundel et al. (1991)

ICAM-1

Monkey

In this multiple allergen (Ascaris) challenge model of asthma, both airway eosinophilia and development of BHR were inhibited

Wegner et al. (1990)

Mac-1

Monkey

In the same multiple antigen challenge model, anti-Mac-1 inhibited development of BHR and ECP concentrations in BAL but not eosinophil counts

Wegner et al. (1993)

a4

Sheep

Late response to Ascaris challenge inhibited when antibody given both intravenously and by inhalation but no effect on BAL eosinophils

Abraham et al. (1994)

a4/Mac-1/LFA-1

Rat

Antibodies against all three receptors inhibited early and late response to ovalbumin challenge without any effect on cell counts in BAL at 8 hours

Rabb et al. (1994)

a4

Rat

Antibody against VLA-4 inhibited eosinophil and T lymphocyte infiltration at 24 hours

Richards et al. (1996)

a4

Guinea pig

Inhibition of eosinophil infiltration into the skin in both PCA reaction and chemoattractant induced

Weg et al. (1993)

a4

Guinea pig

Inhibition of BHR, EPO release, and eosinophil infiltration into the airway of ovalbumin-challenged animals

Pretolani et al. (1994)

a4/VCAM-1/ ICAM-1/LFA-1

Mouse

Eosinophil and T-cell infiltration inhibited by anti-a4 and anti-VCAM-1 but not by anti-ICAM-1 or anti-LFA-1

Nakajima et al. (1994)

a4

Mouse

Inhibition of eosinophil recruitment after ovalbumin challenge with antibody given either by inhaled or peritoneal route but inhibition of BHR, mucus and cytokine production only when inhaled

Henderson et al. (1997)

ICAM-1

Mouse

Eosinophil and T-cell influx inhibited by anti-ICAM-1 monoclonal antibody

Chin et al. (1998)

P-selectin KO

Mouse

Eosinophil and T-cell recruitment reduced in gene-deleted mouse after ovalbumin challenge

De Sanctis et al. (1997)

P-selectin/ICAM-1 KO

Mouse

Reduced migration of eosinophils into the lung after ovalbumin challenge in both P-selectin and ICAM-1 deficient animals

Broide et al. (1998)

Mac-1 KO

Mouse

Complex effect with increased eosinophilia in Mac-1 KO (ovalbumin challenge) and inhibitory effect with anti-Mac-1 monoclonal antibody

Kanwar et al. (2001)

ICAM-2 KO

Mouse

Increased eosinophil adhesion after allergen challenge

Gerwin et al. (1999)

ICAM-1/L-selectin KO

Mouse

Reduced inflammation and AHR in ICAM-1 KO. Reduced AHR in L-selectin KO

Tang & Fiscus (2001)

ICAM-1 KO

Mouse

Inhibition of inflammation in a TDI model of asthma with ICAM-1 gene deletion

Furusho et al. (2006)

AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BHR, bronchial hyperresponsiveness; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; KO, knockout; PCA, passive cutaneous anaphylactic; TDI, toluene diisocyanate.

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number of receptors including α4β7, Mac-1, LFA-1, ICAM-1, VCAM-1, and E-selectin, although differences in degree and pattern of inhibition have been observed depending on the species used and the exact conditions. Most support has been gained for an important role of α4β1 and VCAM-1 in both eosinophil and T-lymphocyte migration into the airways after allergen challenge and this has given impetus to the development of α4β1 antagonists in clinical trials. However, several studies have shown a clear dissociation between effects on leukocyte migration and effects on airway physiology. This mirrors the picture in asthma where there is only a weak relationship between the severity of airway inflammation and the severity of bronchial hyperresponsiveness (Wardlaw et al. 2002). Some studies with gene-deleted mice have given unexpected results, for example in an ICAM-2 knockout mouse there was increased eosinophilia after allergen challenge. With Mac-1 contrasting results were seen with an antibody and with gene deletion, which emphasizes the complexity of these models.

Adhesion receptor antagonists as treatments for allergic disease Chronic allergic disease is caused by persistent inflammation characterized by infiltration of tissues with eosinophils and activation of mast cells, basophils, and Th2 lymphocytes. As detailed above, recruitment of these cells into tissue is dependent on adhesion receptor interactions and an impetus for research in this area has been the hope that adhesion receptor antagonists will be effective forms of antiinflammatory treatment. From the above review of the literature it can be seen that both VLA-4 and P-selectin antagonists might be expected to selectively and effectively inhibit eosinophil migration. However, despite potential adhesion receptor targets having been identified for well over a decade, no such treatments have yet reached the clinic. The reasons for this include difficulty in designing potent low-molecular-weight antagonists, concern about redundancy on the one hand and immunosuppression on the other, and potential side effects of treatment. Most effort appears to have gone into the development of α4β1 antagonists, which have also been used for a number of other diseases such as multiple sclerosis and Crohn disease. Natalizumab is a humanized antibody against α4β1 that has recently been licensed for the treatment of multiple sclerosis despite being associated with the occurrence in three patients of progressive multifocal leukoencephalopathy, an opportunistic infection of the brain with polyoma virus JC (Khalili et al. 2007). A number of low-molecular-weight antagonists of α4β1 have been shown to have activity in animal models of asthma (Huryn et al. 2004; Singh et al. 2004; Lawson et al. 2006; Okigami et al. 2007). However, the early results of both inhaled and oral treatment with these compounds in humans in allergen challenge models has so far proved disappointing, with no effect on lung physiology and minimal effect on eosinophilic inflammation (Diamant et al. 2005; Norris


et al. 2005; Ravensberg et al. 2006). Potent selectin antagonists have been difficult to develop because of the low affinity/highavidity nature of selectin-based carbohydrate interactions. The development of selectin antagonists has recently been reviewed (Kneuer et al. 2006). The only candidate that has been explored in asthma is the pan-selectin antagonist TBC1269 (bimosiamose) originally developed by Texas Biotechnology Corporation (Kogan et al. 1998). Intravenous TBC1269 had no effect on the early and late asthmatic response after allergen challenge, although inhaled TBC1269 did inhibit the late response to allergen challenge by about 50% without any effect on the early response, airway hyperresponsiveness, or exhaled nitric oxide (Avila et al. 2004; Beeh et al. 2006). This compound was unable to inhibit eosinophil adhesion to vascular endothelium under flow conditions (Davenpeck et al. 2000), and is probably not sufficiently potent to be an effective treatment, but may be a model for more potent compounds (Kranich et al. 2007). It is perhaps surprising that a humanized monoclonal antibody against P-selectin has not been investigated in severe asthma. The only other adhesion receptor that has been targeted in allergic disease is LFA-1. An antibody against LFA-1 significantly inhibited sputum eosinophilia and there was a trend toward inhibition of the late response after allergen challenge (Gauvreau et al. 2003). However, there was a high rate of adverse effects, particularly with flu-like symptoms, and this approach has not so far been pursued.

Conclusion There is a wealth of data concerning both the biology of adhesion receptors and their role in the pathogenesis of allergic disease, particularly in the context of leukocyte trafficking. So far this information has not been translated into promising new therapies with a frustrating lack of potent and safe compounds for human use. However, this remains a potentially fruitful approach to the treatment of allergic disease and further studies are eagerly anticipated.

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16

Airway Epithelium Pedro C. Avila and Robert P. Schleimer

Summary The airways form conduits to allow ambient air to reach alveoli in terminal airways, where gas exchange occurs, followed by elimination of exchanged air. Although apparently simple, this function requires the airways to condition inspired air, to maintain patent conduits, and to protect the lungs against airborne threats. Airway epithelium performs all these functions: it conditions and filters air in the upper airways, provides an important barrier, is intimately involved in gas exchange, and plays a key role in host defense because of its position at the boundary between the environment and the internal tissues. The epithelium responds to microbes and noxious stimuli that overcome the mucociliary barrier by releasing inflammatory mediators that recruit immune cells, triggering immune responses. This chapter reviews the anatomy, physiology, and pathophysiology of the airway epithelium as they relate to allergic diseases and asthma. Additional information on innate immunity (Chapter 9), the mucociliary system (Chapter 39), airway remodeling (Chapter 55), and airway pathology (Chapters 62, 68, 78 and 79) is reviewed elsewhere in this book.

Anatomy and physiology The upper airway consists of the nasal cavities, paranasal sinuses, pharynx, and larynx, namely all airways above the vocal cords. The nasal cavities filter, warm and humidify inhaled air. Airflow is made turbulent in the nasal airways and is ultimately diverted 90° as it flows into the nasal cavity (Fig. 16.1), causing impact of airborne particles against the mucus layer overlaying the epithelium. The 10–15 μm deep mucus layer traps particles, filtering the air. Larger particles are more efficiently filtered than smaller ones. Particles with


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aerodynamic equivalent diameters of 30, 10, and 2.5 μm are 50%, 70%, and 90% respirable (able to reach lower airways) respectively (Eccles 2003). Once trapped in the mucus layer, particles are moved by the mucociliary escalator toward the pharynx and ultimately eliminated into the digestive tract. The abundant blood vessels in the nasal submucosa warm inhaled air to about 30–32ºC so that it reaches 37ºC after passing through a few bronchial branches (McFadden et al. 1985). Likewise, in the proximal airways, water vapor from the mucus layer humidifies the inhaled air before it reaches the distal airways in the lungs. The lower airway (below the vocal cords) consists of the trachea, right and left main bronchi, and the remainder of the bronchial tree, which undergoes a total of 18 branching generations before reaching bronchioles, all kept patent by their cartilaginous support (McFadden 1998). After about 18 generations, bronchial branches become bronchioles, which are 1 mm or less in diameter and remain patent because of the elastic fiber recoil of the lung parenchyma (McFadden 1998). The proximal bronchioles are called terminal bronchioles and they branch five additional generations before becoming respiratory bronchioles, at which point gas exchange begins to take place. Altogether, after a total of about 23 generations from the trachea, the respiratory bronchioles finally branch into alveolar ducts, which then end in the alveoli (Fig. 16.1). Diseases of the airways that affect epithelium include, among others, rhinitis (upper airways), sinusitis (sinuses), asthma (mostly medium-size bronchi), bronchitis (larger airways), and emphysema (small airways and alveoli). There are more than 50 cell types in the airways, of which 12 are considered to be epithelial cells (Harkema et al. 1991; Albertine et al. 2000). The epithelium in the conducting airways, which does not exchange air, is ciliated pseudostratified and columnar, except in the anterior tip of the lower turbinate, pharynx, and larynx, where it is squamous. In the bronchioles, the ciliated epithelium becomes thinner and cuboid (Fig. 16.2). The main epithelial cell types in the bronchial airways are the ciliated, goblet, and basal cells. Clara cells are present in the terminal bronchioles among ciliated cuboid cells. At the bronchioles the epithelium suddenly changes from ciliated pseudostratified to the alveolar epithelium, demarcating the transition from terminal to respiratory

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BRAIN

FS

ST SS

MT

Nasopharynx

LT 1 AIR FLOW

Oral cavity Tongue

Uvula Oropharynx Epiglottis Larynx Glottis

Vocal cords Trachea

Esophagus

2 3 Bronchial tree

Respiratory bronchioles

4

Terminal bronchioles

I II

Alveoli

Alveolar ducts

Fig. 16.1 Anatomy of the airways. Stratified squamous epithelium (insert 1) lines pharynx and larynx. Pseudostratified ciliated columnar epithelium (insert 2) lines nasal and sinus cavities as well as lower airways where it becomes thinner and cuboid in the small peripheral airways (insert 3). Cartilaginous support maintains patency of trachea and bronchial tree and ends before terminal bronchioles. Bronchioles and alveoli are kept patent by parenchymal elastic recoil. The sudden transition from ciliated to alveolar

epithelium marks the change from terminal to respiratory bronchioles (insert 3). Alveolar epithelium (insert 4) carries out gas exchange and is composed of alveolar type I (flat cells in insert 4, marked I) and round type II (gray cell, marked II) cells. Pores of Kohn (not shown) present between adjacent alveoli allow further air circulation other than that via airways. FS, frontal sinus; SS, sphenoid sinus; LT, MT, ST, lower, middle and superior turbinates respectively. See text for further details. (See CD-ROM for color version.)

bronchioles. Gas exchange occurs from respiratory bronchioles to alveoli, the so-called respiratory airways, all lined by the single layer of alveolar epithelium, which is composed of alveolar type I and II cells (or type I and II pneumocytes) and form the lung parenchyma. Finally, mucous and serous cells form submucosal glands, which are present from the upper airways to distal bronchial branches.

Ciliated epithelial cells The ciliated epithelial cells move mucus toward the pharynx, conduct vectorial ion transport conferring electrical properties to the epithelium, form the epithelial permeability barrier through tight junctions, and play an important role in innate immunity and inflammation. Ciliated cells are rectangular, measuring up to 20–25 μm in length, but become short and

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Large airways Mc Ne

Eo

Mucus Cilia Small airways

Go

Ci

De Cla

PB Ly

PN

Ba

Ms

Basement membrane

Pore Vagus nerve

Sensory nerve

cuboid in the terminal bronchioles (Albertine et al. 2000; Shebani et al. 2005) (Fig. 16.2). Each cell has 200–300 cilia on its apical (luminal) surface. Cilia are 5– 6 μm high and 0.2– 0.3 μm wide. Each cilium contains a microtubule arrangement (axoneme) composed of a central pair and nine peripheral doublets of microtubules (9 + 2 axoneme) (Satir & Christensen 2007; Zariwala et al. 2007). The peripheral doublets are connected by dynein arms and nexin links, whereas radial spokes connect the peripheral with the central microtubules (Fig. 16.3, insert top right). At baseline, cilia beat at about 10 Hz in the sol phase of the mucus layer but can beat as fast as 100 Hz, moving the entire mucus layer at 2–20 mm/min (Harkema et al. 1991; Widdicombe 1991) (see Chapter 41). Ciliated cells are able to perform vectorial ion transport, polarizing the epithelium (Widdicombe 1991). Airway epithelium actively secretes Cl− into the lumen, mainly via cystic fibrosis transmembrane conductance regulator (CFTR) located in the apical membrane, which is followed by water diffusion, hydrating the mucus. On the other hand, active absorption of Na+ from the lumen, via the epithelial sodium channel (a Na+/K+-ATPase) located in the basolateral membrane, dehydrates the mucus layer. Thus, epithelial ion transport plays a role in the homeostasis of mucus hydration, maintaining the depth of the mucus sol layer similar to the height of the cilia so that cilia can beat freely and their tips can push the mucus gel layer. Ion transport electrically polarizes the epithelium, which is negatively charged at its luminal surface and is about –5 to –10 mV compared with the skin (Knowles et al. 1995; Chung et al. 2003; Schuler et al. 2004). This polarization is also possible because of the permeability barrier of the tight junctions (zona occludens, see below), which seal

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Fig. 16.2 Pseudostratified columnar epithelium lines upper and lower airways from trachea to terminal bronchioles. Epithelial cells include basal (Ba), parabasal (PB), ciliated (Ci), goblet (Go), and Clara (Cla) cells. Several types of immune cells traffic through the airway epithelium in normal conditions. Although overly represented in the figure for normal conditions, they may dramatically increase in numbers in disease processes. These cells include lymphocytes (Ly), dendritic cells (De), neutrophils (Ne), eosinophils (Eo), mast cells (Ms), macrophages (Mc), and monocytes (not shown). In addition, there are sensory nerve fibers and pulmonary neuroendocrine cells (PN). See text for further details. (See CD-ROM for color version.)

the apicolateral regions between superficial epithelial cells, restricting passive diffusion of water and ions between cells (paracellular space). This barrier also creates a transepithelial electrical resistance of more than 100 Ω cm2.

Apical intercellular adhesion complex Adhesion among epithelial cells is maintained by complex structures. Superficial epithelial cells adhere to each other via the apical intercellular adhesion complex (Fig. 16.3), which comprises three zonulae: the zonula occludens (or tight junctions), zonula adherens (or intermediate junctions), and the macula adherens (or desmosomes). The tight junctions are located in the apicolateral membrane closest to the lumen in the superficial epithelial cells (Shin et al. 2006; Godfrey 1997). They consist of interconnections of transmembrane proteins including occludins, claudins, junctional adhesion molecules, and crumbs 3. Claudins, a family of more than 20 members, form the calcium-independent tight interconnecting strands between cell membranes seen in freeze-fracture electron micrographs. They seal the epithelial cells at its apical–lateral areas and regulate paracellular permeability (Van Itallie & Anderson 2006). The transmembrane proteins interact with intracellular scaffold proteins such as zonula occludens protein (ZO)-1, ZO-2, ZO-3, and cingulin, which in turn interact with the actin cytoskeleton. In addition, tight junction proteins interact with signaling molecules so that disruption of the junction signals epithelial cells to undergo cell proliferation and initiate repair processes (Matter et al. 2005). The zonula adherens consists of epithelial cadherins (Ecadherin or cadherin 1, one of more than 20 members of the

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Cilium

Airway Epithelium

Plasma membrane Peripheral microtubule doublet Outer dynein arm Inner dynein arm Radial spoke Nexin link Central microtubule pair

Cilia Tight Junction

Occludin

Z0-1 Cing Z0-2 Z0-3 Actin

Claudin

Z0-1 Cing Z0-2 Z0-3

JAM

Cing

CRB3

Z0-3 Go Intermediate Junction

E-Cadherin

Ci Fig. 16.3 Ciliated epithelium: structure of cilium and apical intercellular adhesion complexes. Adhesion complex comprises three zonulae: the zonula occludens (or tight junctions), zonula adherens (or intermediate junctions), and macula adherens (or desmosomes). In addition, hemidesmosomes adhere basal cells to the basement membrane. See text for details and Fig. 18.2 for cell types. Actin, actin cytoskeleton; Cat, catenin; Cing, cingulin; CRB3, crumbs 3; DPK, desmoplakin; DSC, desmocollins; DSG, desmogleins; JAM, junctional adhesion molecules; KIF, keratin intermediate filaments; P120, catenin d; PKG, plakoglobin; PKP, plakophilin; ZO, zonula occludens protein. See text for further details. (See CD-ROM for color version.)

a-cat PB

b-cat p120

Actin PB

Ba

Desmosome PKG KIF DPK

PKP

DSG

Basement membrane

cadherin gene family), which form transmembrane homodimers that mediate calcium-dependent adhesion between epithelial cells throughout the epithelial layer (Lilien et al. 2002; McGuire et al. 2003; Lilien & Balsamo 2005). The intracellular domain of E-cadherin homodimers interact with β-catenin, catenin δ1 (p120ctn), and α-catenin. The latter catenin interacts with the actin cytoskeleton. Disruption of E-cadherin adhesion between cells augments epithelial permeability. Catenins also interact with protein tyrosine kinases and phosphatases mediating intracellular signaling (e.g., proliferation). Cadherins also serve as a substrate for adhesion of T lymphocytes or mast cells to the epithelium via the adhesion molecule αEβ7 (Taraszka et al. 2000). The macula adherens or desmosomes, although present in airway epithelium (Shebani et al. 2005), are best studied in the skin (Kottke et al. 2006). Desmosomes also consist of

Hemidesmosome

DSC

transmembrane proteins that connect with the cytoskeleton through scaffold proteins. In this case, the transmembrane proteins desmogleins and desmocollins mediate calciumdependent adhesion between cells throughout the epithelial layer. Their intracytoplasmic domains interact with desmoplakin, plakophilin, and plakoglobin, which form the characteristic paramembranous plaques of desmosomes seen in electron micrographs. These protein plaques interact with a dense bundle of filamentous proteins (actin and keratin intermediate filaments) of the cytoskeleton. Hemidesmosomes adhere basal epithelial cells to the extracellular matrix of the epithelial basement membrane. Integrins on the epithelial cells also contribute to the adhesion of basal epithelial cells to the basement membrane. Integrins are transmembrane heterodimeric adhesion molecules. The integrins α6β4, present in hemidesmosomes, and α3β1,

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present throughout the epithelium, are receptors for laminin 5 (Sheppard 2003), present in the basement membrane of conducting airways (Coraux et al. 2002). These integrins also bind to laminins 10 and 11. Integrin α2β1 is expressed on basal cells, binds to collagens I and IV, and tenascin C (expressed in injury), and functions as the receptor for echoviruses. In asthma, there seems to be an enlargement of the intervening space between basal cells and between basal cells and basement membrane, suggesting a defect in the integrity of desmosomes and hemidesmosomes (Shebani et al. 2005). Epithelial barrier function is regulated by a host of factors intrinsic to the epithelium (see above) as well as external factors such as proteases and inflammatory mediators. Although most studies have been performed with keratinocytes, recent data suggest that a family of cytokines related to interleukin (IL)-10 influences barrier function. These cytokines, which include IL-19, IL-20, IL-22, IL-24, and IL-26, modulate the expression of a host of molecules involved in regulating the protease–antiprotease balance and the expression of structural genes involved in maintaining epithelial barrier function (Hor et al. 2004; Boniface et al. 2005; Sa et al. 2007). Exogenous proteases from microorganisms or allergens can alter this balance. For example, Der p1, a major allergen of Dermatophagoides pteronyssinus and a cysteine proteinase, cleaves occludin and claudin 1, disrupting tight junction integrity and increasing airway epithelial permeability (Wan et al. 1999).

Goblet cells Goblet cells are nonciliated mucus-secreting cells interspersed between ciliated cells in the superficial layer of the epithelium (Albertine et al. 2000). They decrease in number toward the peripheral airways, disappearing in the terminal bronchioles. Their characteristic numerous, mucous, electron-lucent granules at the apical surface are loaded with acidic mucin, particularly Muc5AC, but also Muc1 and Muc5B (Ordonez et al. 2001). Together with mucins from submucosal glands, goblet cell mucins form the gel phase of airway mucus. Goblet cell exposure to airborne insults such as chemical irritants, pollutants (ozone, sulfur dioxide), allergens, tobacco smoke, and microbes can stimulate mucin secretion (Harkema et al. 1991). These insults can also induce goblet cell hyperplasia with repeated exposures. In airways of individuals with mildto-moderate asthma, there are twice as many goblet cells and twice the stored mucus content than in airways of healthy individuals (Ordonez et al. 2001) (see Chapter 41). An increased number or function of goblet cells is also observed in chronic obstructive pulmonary disease (COPD) and rhinitis.

Clara cells Although Clara cells resemble goblet cells, they are present in the terminal and respiratory bronchioles (Harkema et al. 1991; Albertine et al. 2000) and have less abundant apical granules. In contrast to goblet cell granules, Clara cell granules are electron-dense and have little if any mucin (glycoproteins).

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Clara cell granules contain proteins such as surfactant protein D, antileukoproteases, and uteroglobin (also called Clara cell secretory protein), among others. In addition, Clara cells have abundant agranular endoplasmic reticulum, glycogen stores, and mitochondria occupying 40% of the cytoplasm. Clara cells also possess cytochrome P450 monooxygenases for metabolism of xenobiotic substances. In animals, Clara cells are also precursors of ciliated cells (Harkema et al. 1991) and goblet cells (Hayashi et al. 2004). In mice, ciliated epithelial cells can also transdifferentiate into goblet cells via epidermal growth factor receptor and IL-13 signaling (Tyner et al. 2006).

Basal epithelial cells Basal epithelial cells are very small and closely attached to the basement membrane via hemidesmosomes. This attachment prevents loss of basal cells during processes that cause epithelial damage and desquamation. Basal cells are precursors for all epithelial cells, including parabasal cells, ciliated cells, and goblet cells (see Fig. 16.2). In normal airways, about 0.8% of the epithelial cells are proliferating at baseline, and among the proliferating cells are basal cells, parabasal cells, and Clara cells (Harkema et al. 1991; Boers et al. 1998, 1999). Proliferation rate can increase 10–20 fold, as shown in airways of rats exposed to nitrogen dioxide (Harkema et al. 1991). Basal cells are also affected by airborne insults and produce inflammatory mediators. Serous and mucous epithelial cells (Wine & Joo 2004) line submucosal glands, although occasional serous cells can be seen in the mucosal epithelium as well. Glands provide an estimated 95% or more of the mucus content of the upper airways. There is on average one gland every square millimeter of mucosa in the trachea down to the 10th generation of bronchial branches (airway diameter 1–2 mm), where they disappear. From the airway lumen, the gland duct (containing ciliated epithelium) penetrates into the submucosa and branches into numerous tubules, where they become lined with mucous cells proximally and with serous cells in distal acini. Normal glands are composed of 60% serous and 40% mucous cells by volume. Serous cells secrete water, ions, antimicrobials (e.g., lysozyme, lactoferrin, sIgA), and antiinflammatory and antioxidant agents, whereas mucous cells secrete sialomucins and sulfomucins. Glandular secretion is stimulated by acetylcholine released from the parasympathetic innervation, which does not affect goblet cell secretion.

Alveolar epithelium Alveolar epithelium lines the respiratory epithelium, from the respiratory bronchioles to the alveoli, and consists of alveolar type I and II cells. Type I cells are “fried egg”-like cells that line respiratory airways as a thin single-cell layer. They are adjacent to endothelial cells, with a very thin interstitium separating them so that the distance between the alveolar lumen and the capillary lumen is less than 1 μm, particularly when the alveoli are stretched at total lung

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capacity. Type I cells express aquaporin 5 and the epithelial sodium channel and may play an important role in the transport of water and ions across the alveolar epithelium (Albertine et al. 2000). Alveolar type II cells are cuboidal cells attached to type I cells via tight junctions. Type II cells have electron-dense lamellar cytoplasmic inclusions, or lamellar bodies. These are granules containing several proteins including surfactant proteins A, B, C and D, lysosomal enzymes, and α-glucosidase. One of the most important functions of type II cells is the production of surfactant lipids, which are required for the proper assembly and function of surfactant proteins (Ridsdale & Post 2004). Type II cells can also synthesize chemokines, cytokines, matrix proteins, and transporters for water and ions. They also participate in epithelial regeneration and act as stem cells, differentiating into type I cells. Although type II cells outnumber type I cells by 2 : 1, type I cells cover over 90% of the alveolar surface due to their attenuated morphology.

Pulmonary neuroendocrine cells Pulmonary neuroendocrine cells (PNECs) are scattered along the airways from trachea to bronchioles (Johnson 1991). They occur just above the basement membrane, typically in contact with afferent nerve fibers. They are more numerous in proximal than distal airways, and in airways of newborns, where they occur in clusters (neuroepithelial bodies). In adults, they occur as single cells, accounting for 0.41% of all epithelial cells (Boers et al. 1996). They have characteristic small dense-cored cytoplasmic vesicles and cytoplasmic projections that extend between epithelial cells toward the lumen and in parallel to the basement membrane (Johnson 1991). These vesicles contain amine hormones (serotonin, dopamine, norepinephrine) and peptide hormones (calcitonin, leu-enkephalin, bombesin, somatostatin, endothelin) (Johnson 1991). The adjacent sensory nerve fibers contain substance P and vasoactive intestinal peptide. One of the functions of the PNEC is believed to be as a chemoreceptor for sensing irritants (e.g., tobacco smoke), pollutants (e.g., ozone), hypoxia, and hypercapnia, all stimuli that increase the number of PNECs in the airways. Hyperplasia of PNECs also occurs in many lung diseases including, but not limited to, asthma, cystic fibrosis, COPD, and bronchiectasis (Johnson 1991; Joad et al. 2006). PNEC secretory products are believed to play a role in bronchospasm, vasospasm, inflammation, airway hyperreactivity, and airway remodeling (Johnson 1991).

Olfactory epithelium In the upper airways, the olfactory epithelium consists of specialized epithelial cells that interact with olfactory receptor neurons and two types of glial cells (sustentacular cells and olfactory ensheathing cells) that collectively mediate olfaction (Beites et al. 2005). Anosmia, in which the olfactory epithelium is depleted or fails, is not an uncommon occurrence in diseases of the upper airways, particularly nasal polyposis.

Airway Epithelium

Basement membrane The basement membrane separates epithelium from submucosa. It provides support and orientation for epithelial cell growth and repair, and serves as a semipermeable barrier. It has two layers, the more superficial lamina densa, and the deeper and thicker lamina reticularis. The lamina densa is 0.1 μm thick and is mainly composed of collagen type IV and laminin V, which are produced by epithelial cells. The lamina reticularis is 2–6 μm thick and is mostly composed of fibronectin and collagen type III and V, which are secreted by subepithelial fibroblasts and myofibroblasts (Paulsson 1992). In asthma, the lamina reticularis is thickened 40–50% (Shahana et al. 2005), and its thickness correlates with the number of subepithelial myofibroblasts (Brewster et al. 1990). Although some researchers have detected a 30% thinning of the reticularis membrane after inhaled corticosteroid therapy (Laitinen et al. 1997; Hoshino et al. 1998; Sont et al. 1999), others have failed to confirm this effect (Jeffery et al. 1992; Barnes et al. 2000; Boulet et al. 2000). Pores are also randomly scattered throughout the basement membrane, possibly from immune cell migration (Howat et al. 2002). In healthy airways the pores number about 740/mm2, are ovoid in shape, 2–4 μm long, and 0.4–2.7 μm in diameter with a corresponding cross-sectional area of 1–30 μm2.

Other cell types In addition to epithelial cells, the airway epithelium contains numerous other cell types (see Fig. 16.2). Sensory nerve fibers can penetrate far into the epithelium in close proximity to the airway lumen. All immune cells, including lymphocytes, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and macrophages, can traffic through the airway epithelium and are present in histologic sections of normal airways and in luminal secretions. The biology of these cells is discussed elsewhere in this book. Most importantly, epithelial cells can chemoattract and interact with these cells via contact through surface membrane molecules and/or via soluble molecules to alter the function of one another. Underneath the basement membrane, fibroblasts and myofibroblasts can interact with epithelial cells in health and airway diseases such as asthma (Holgate et al. 2004).

Models used to study epithelial cells The function of epithelial cells can be studied in several different models (Table 16.1). In vitro, cancer or virustransformed epithelial cell lines and primary culture of freshly isolated airway epithelial cells have been used to study their function. These cells are often used in their undifferentiated state, as a monolayer of flattened cells resembling the basal epithelial cells, which lack epithelial polarization. Some transformed cell lines and primary airway epithelial cells can be grown on a porous membrane bathed by medium underneath and

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Table 16.1 Common models used to study human airway epithelial cells. Cell line or model

Characteristics

Model of

A549 cells

Lung carcinoma cell line from a 58-year-old white male. Chromosomal abnormalities include two X and two Y chromosomes, and about 20% of cells with 64, 65 or 67 chromosomes

Alveolar type II cells

BEAS-2B

Normal bronchial epithelial cells transformed by infection with adenovirus 12 and simian virus 40

Bronchial epithelial cells

CALU-3

Lung adenocarcinoma cell line grown from pleural effusion of a 25 year-old white male. Abnormal chromosomes 1, 13, 15, 17 and Y

Bronchial gland epithelial cells

NCI-H292

Lung mucoepidermoid carcinoma isolated from a lymph node metastasis in a 32 year-old African female. About 36% of cells have 47 chromosomes

Mucus/mucin-producing bronchial epithelial cells

16HBE14o–

Normal tracheal epithelial cells transformed by infection with simian virus 40 that can differentiate in air–liquid interface cultures forming tight junctions and microvilli

Somewhat differentiated bronchial epithelial cells

Primary epithelial cells

Normal nasal, tracheal, and bronchial cells can be grown for a few passages. Early passages (P0 to about P2) can be differentiated in air–liquid interface cultures to form ciliated and polarized epithelium with vectorial ion transport and adhesion complex structures that resemble native pseudostratified ciliated epithelium

Normal differentiated pseudostratified ciliated epithelium to study mucociliary function, ion transport, and electrical properties

Tissue engineering

Airway fibroblasts are grown in a gel matrix and airway epithelial cells grown in air–liquid interface overlaying the gel. Leukocytes may be added to the system

Interactions between epithelium, fibroblasts, and leukocytes

Tissue explants

Segments of airways (e.g., trachea) or mucosal biopsies are directly cultured ex vivo to study their responses to different stimuli

Response of minimally manipulated tissues to stimuli

Human xenograft

Human bronchial epithelial cells grown inside rat trachea denuded of its own epithelium and implanted in the subcutaneous tissue of T-cell-immunodeficient mice (prevents rejection). Epithelium remains exposed to air

Normal human airway epithelium to study innate response and regeneration

Source: American Type Culture Collection (www.atcc.org).

exposed to air above, so-called air–liquid interface culture, which in combination with several hormones induces differentiation (Van Scott et al. 1991). Differentiated epithelial cultures resemble the natural ciliated pseudostratified columnar epithelium, forming three- to five-cell layers, a mucociliary system, polarization (tight junctions), vectorial ion transport, and normal electrical properties (Wu et al. 1985; Yamaya et al. 1992, 1993; Gruenert et al. 1995; Lopez-Souza et al. 2003; Widdicombe et al. 2003, 2005). It has also been possible to produce an engineered human bronchial mucosa in which bronchial fibroblasts are grown in a collagen gel on top of which primary epithelial cells are grown at air–liquid interface. This system allows study of the complex interactions between epithelial cells, leukocytes, and fibroblasts (Chakir et al. 2001). In addition, tracheal segments and biopsies from airway mucosa, so-called tissue explants, have been placed in culture medium ex vivo to study their response to stimuli (Warner & Azen 1984; Lundgren et al. 1991; Roat et al. 1993; Roca-Ferrer et al. 2000; Morin et al. 2005). In vivo models of the airway epithelium include studies

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in animal models, in humans, and human xenograft transplants in immunodeficient mice. In the latter model, human bronchial epithelial cells are grown inside rat trachea denuded of its own epithelium and implanted in the subcutaneous tissue of immunodeficient mice (e.g., nude mice, severe combined immunodeficient mice). The ends of the trachea are connected to ambient air via sterile polyethylene tubing (Puchelle & Peault 2000). The immunodeficient mice do not reject the rat trachea or human airway epithelial cells, allowing studies of innate immunity and regeneration processes of human cells in vivo.

Pathophysiology In asthma, the airway epithelium undergoes several pathologic changes even in mild disease (see also Chapters 56, 63, 69 and 80). As mentioned above, the airway epithelium of asthmatic individuals manifests goblet cell hyperplasia, thickening of the reticular basement membrane, and loosen-

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ing of epithelial intercellular adhesion. In addition, there are areas of desquamation, i.e., loss of superficial epithelial cells exposing basal cells to the lumen, and areas of epithelial denudation, in which all epithelial cells slough off and basement membrane is exposed to the lumen. These changes are more often seen in severe disease (Laitinen et al. 1991; O’Byrne 1991), and may not occur in mild asthma (Ordonez et al. 2000a). The lost airway epithelium can form Creola bodies, which are round clusters of epithelial cells seen in sputum cytology in asthma exacerbations (Naylor 1962; O’Byrne 1991). Also seen in sputum are corkscrew-shaped strings, so-called Curschmann’s spirals, caused by mucus hypersecretion and drying of mucus in the lumen of small airways. Tenacious mucus plugs occlude the airways of patients who die of asthma (Jeffery 1992). The epithelial damage is a consequence of inflammation. For example, major basic protein (MBP) derived from eosinophil degranulation can desquamate epithelium in guinea pigs (Motojima et al. 1989) and epithelial denudation is observed in areas containing large numbers of eosinophils in human asthma (Gleich et al. 1987). Other inflammatory cell products are also likely involved in epithelial damage, such as those from eosinophils (Hirata et al. 1996), the main inflammatory cell infiltrating the asthmatic airway lumen in asthma (see Chapters 67, 79 and 80), and products from neutrophils, the main infiltrating cells in asthma exacerbations (Fahy et al. 1995; Ordonez et al. 2000b).

Role of epithelium in innate immunity The first line of defense provided by the epithelium is the mucociliary system, present from the upper airways to the terminal bronchioles (see Chapter 41). As mentioned above, sudden changes in airflow direction in the upper airways impacts air against the mucosa, where particles are trapped in the mucus. Ciliary beating then moves mucus toward the pharynx where it is eliminated by the digestive system. Mucus is composed of long chains of glycoproteins that determine its viscosity, elasticity, and adhesive properties. There is population diversity in the carbohydrate composition of mucus glycoproteins. For example, different sugar residues decorate the H antigen, giving rise to different ABO blood group antigens. These antigens are also synthesized by epithelial cells. People who have functional fucosyltransferase 2 (FUT2) can add fucose to the H antigen enabling secretion of ABO blood group antigens in body fluids (so called secretors). Individuals with loss-of-function mutations in FUT2 can not secrete ABO antigens in their secretions (nonsecretors) (D’Adamo & Kelly 2001). Among several features of these phenotypes is the fact that secretors are more susceptible to respiratory virus infections (Raza et al. 1991), whereas nonsecretors are more susceptible to Helicobacter pylori infections and peptic ulcer disease (D’Adamo & Kelly 2001). The susceptibility mechanism seems related either to the ability of different sugars in mucus glycoproteins to bind and coat microbes in the lumen

Airway Epithelium

or to the ability of different sugars on cell-surface glycoproteins (glycocalyx) to allow adhesion of microbes. Therefore, the types of sugars in mucus influences susceptibility to infections, including to viral respiratory infections (Raza et al. 1991); which are known to play a role in the development of asthma (Friedlander et al. 2005) and precipitate asthma exacerbations (Johnston et al. 1995). Mucus composition can change as a result of several processes. The mucus is derived from products of airwayresident and immune cells, which can alter the content or volume of their secretions on stimulation by microbes, irritants, and inflammatory mediators. Airway-resident cells that contribute to mucus production include ciliated cells, goblet cells, Clara cells, and serous and mucous cells. Mucus also has components derived from plasma transudation and from immune cells such as mast cells, dendritic cells, neutrophils, eosinophils, monocytes, macrophages, and lymphocytes, which are normally present in the airways in small numbers. In addition, during inflammation, newly formed mediators and infiltrating cells alter mucus production and constituents (see Chapter 41). The important influence of epithelial cells on the ionic content of mucus is illustrated by abnormalities in cystic fibrosis, in which several antimicrobial substances fail to kill microorganisms as a result of alterations of the ionic properties of airway secretions (Travis et al. 1999). That mucus possesses antimicrobial properties was first recognized by Sir Alexander Fleming, who discovered the ability of lysozyme in saliva and nasal secretions to kill Micrococcus lysodeikticus (Fleming 1922). Mucus contains numerous other antimicrobial products secreted by epithelial cells and immune cells. The following text, and Table 16.2, list several antimicrobial products found in the airways, most produced by airway epithelial cells.

Secreted antimicrobials Lysozyme Lysozyme is a 14-kDa enzyme that cleaves the 1–4 β-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid residues in peptidoglycan, a cell wall component of Gram-positive bacteria (Ganz 2004). It can also kill bacteria by a nonenzymatic mechanism. It is ineffective against Gramnegative bacteria unless the outer envelope is damaged (e.g., as a result of the action of lactoferrin) and allows penetration of lysozyme. Lysozyme is decreased in patients with recurrent sinusitis and perennial allergic rhinitis (Kalfa et al. 2004).

Lactoferrin Lactoferrin is an 80-kDa glycoprotein related to transferrin that acts by chelating iron, an essential nutrient for respiration in microbes (Ganz 2004). This action also prevents biofilm formation (Singh et al. 2002). The cationic N-terminal of lactoferrin can also directly kill bacteria.

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Table 16.2 Antimicrobials produced in the airways. Antimicrobial

Action

Affected microbes

Sources

Lysozyme

Cleavage of peptidoglycan

Gram-positive bacteria

Serous epithelial cells, monocytes, macrophages

Lactoferrin

Chelation of iron, prevention of biofilm formation

Bacteria

Serous epithelial cells, neutrophils

Secretory leukocyte proteinase inhibitor

Direct antimicrobial activity

Bacteria, fungi

Serous epithelial and Clara cells, alveolar type II cells, monocytes, macrophages, neutrophils

Elafin


Bacteria, fungi

Serous epithelial and Clara cells, alveolar type II cells, macrophages

Phospholipase A2


Bacteria

Serous epithelial cells

Cathelicidins


Bacteria, fungi, enveloped viruses

Mucosal, serous and mucous epithelial cells, neutrophils, monocytes

b Defensins

Permeabilization of microbial membranes

Bacteria, fungi, enveloped viruses

Mucosal epithelial cells, monocytes, macrophages, dendritic cells

Collectins (SP-A, SP-D)

Opsonization, neutralization

Bacteria, fungi, viruses

Clara cells, alveolar type II cells

Collectin (MBL)

Opsonization, complement activation, neutralization

Bacteria, fungi, viruses, protozoa

Hepatocytes (leaked from plasma into airways)

Pentraxin-3

Opsonization, complement (C1q) activation

Bacteria, fungi

Mucosal epithelial cells, alveolar type II cells, dendritic cells, monocytes, macrophages, fibroblasts, endothelial cells

C-reactive protein


Phosphorylcholineexpressing bacteria

Hepatocytes and nasal epithelial cells

Serum amyloid A


Bacteria

Naso-sinusal epithelial cells

Complement components

Opsonization, complement activation, chemoattraction

Bacteria, fungi, viruses, parasites

Mucosal epithelial cells, liver, mononuclear phagocytes (monocytes, macrophages)

Reactive oxygen species


Bacteria, fungi, parasites, viruses

Mucosal epithelial cells, mononuclear and polymorphonuclear (eosinophils, neutrophils) phagocytes

Reactive nitrogen species


Bacteria, fungi, parasites, viruses

Mucosal epithelial cells, alveolar type II cells, macrophages, neutrophils, mast cells, endothelial cells among others (see text)

SP, surfactant protein; MBL, mannose-binding lectin, also known as mannan-binding protein (MBP).

Secretory leukocyte proteinase inhibitor Secretory leukocyte proteinase inhibitor (SLPI) is a 12-kDa polypeptide that contains two whey acid protein (WAP)/fourdisulfide core domains. Its N-terminal domain has modest antibacterial activity, whereas its C-terminal domain inhibits neutrophil elastase (Ganz 2004) as well as cathepsin G, trypsin, chymotrypsin, and chymase (Sallenave 2002). It also inhibits nuclear factor (NF)-κB activation (Hiemstra et al. 2004) and attenuates tumor necrosis factor (TNF)-α secretion by lipopolysaccharide (LPS)-stimulated monocytes (Sallenave 2002). These antiinflammatory effects may provide SLPI with the

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ability to inhibit the allergic late response to allergen in animal models of asthma (Wright et al. 1999). Elafin (6 kDa), or elastase-specific inhibitor, is another serine protease inhibitor that is structurally similar to the WAP/four-disulfide core domains of SLPI. Its precursor, pre-elafin (9.9 kDa), contains an N-terminal domain (cementoin) that is a substrate for the enzyme transglutaminase and is a member of the trappin family; “trappin” is an acronym for TRansglutaminase substrate and wAP domain containing ProteIN, and refers to its functional property of “getting trapped” in tissues by covalent cross-linking (Williams et al. 2006). Like SLPI, it is upregulated

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by LPS, IL-1, TNF-α, neutrophil elastase, and neutrophil αdefensins. Elafin exerts antibacterial effects on Gram-positive and Gram-negative bacteria and fungi (Simpson et al. 1999). Like SLPI, elafin also inhibits NF-κB activation and is chemotactic for neutrophils. The N-terminus of elafin forms polymers that bind to extracellular matrix proteins, making it a tissue-bound elastase inhibitor (Sallenave 2002).

Phospholipase A2 Phospholipase (PL)A2 has been described in nasal lavage (Stadel et al. 1994; Aho et al. 1997). It increases after nasal challenge with methacholine, histamine, and allergen. It has direct calcium-dependent antimicrobial activity against Gram-positive and Gram-negative bacteria (Cole et al. 1999). The PLA2 superfamily is large, and only selected members are antibacterial (Schaloske & Dennis 2006). Cathelicidins Cathelicidins comprise a family of antimicrobial polypeptides of 110–214 amino acids containing a variable C-terminal cationic antimicrobial domain (12–100 residues) that becomes active after being enzymatically freed in the extracellular space from the N-terminal conserved cathelin domain (98–114 residues) of the holoprotein (Zanetti 2004). Both domains have antimicrobial activity. The most studied human cathelicidin is the human cationic antimicrobial protein (hCAP)-18, which can be cleaved into LL-37 and cathelin by neutrophil elastase, proteinase 3, and prostate fluid pepsin C. hCAP-18 is produced by mucosal epithelial cells and submucosal glands and kills Gram-negative and Gram-positive bacteria by itself and in synergism with lactoferrin and lysozyme (Bals et al. 1998). LL-37 is a chemoattractant for neutrophils, monocytes, CD4 T cells, and mast cells. It also activates macrophages and epithelial cells, degranulates mast cells, and stimulates angiogenesis (Hiemstra et al. 2004). The mRNA for cathelicidin LL-37 is increased in nasal polyps from patients with chronic rhinosinusitis (CRS) (Chen & Fang 2004). Defensins Defensins are small antimicrobial polypeptides (3–5 kDa) of 30– 45 amino acids characterized by a three-dimensional β-sheet conformation due to three disulfide bonds (six cysteine residues) (Ganz 2004; Hiemstra et al. 2004). Defensins are classified into two groups, α and β defensins. The α defensins are present in the azurophil granules of neutrophils [human neutrophil defensins (DEFA)1–4] and in the secretory granules of Paneth cells (human α defensins DEFA5 and DEFA6). Paneth cells are involved in host defense and are found at the bottom of small intestinal epithelial crypts. DEFA5 is also produced by airway epithelium (Frye et al. 2000), and DEFA1 induces bronchial epithelial secretion of IL-1β and IL-8 via activation of NF-κB (Van Wetering et al. 1997; Sakamoto et al. 2005). The β defensins differ from their α counterparts by the position of the disulfide bonds

Airway Epithelium

and cysteine residues. Four human β defensins (DEFB1–4) are well described, although a whole-genome search reveals up to 28 genes possibly encoding additional DEFBs (Hiemstra et al. 2004). DEFB1–4 are present in mucosal epithelial cells of various organs and in skin. DEFB1 is constitutively expressed, whereas DEFB2 (also known as DEFB4) is inducible (Hiemstra et al. 2004). In the lungs, the conducting airway superficial and submucosal gland epithelial cells produce DEFB2 on stimulation via Toll-like receptor (TLR)4 with LPS and/or cytokines (e.g., IL-1) (Ganz 2004). Defensins kill Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses by permeabilizing membranes rich in anionic phospholipids and sparing the neutral and cholesterol-rich membranes of human cells. Defensins also stimulate mitogenesis of fibroblasts and bronchial epithelial secretion of IL-8 and SLPI, and they also inhibit fibrinolysis (Ganz 2004). DEFB2 is a chemoattractant for dendritic cells and memory T cells via CCR6, providing a link between innate and adaptive immunity.

Collectins Collectins are a small family of glycoproteins containing collagenous regions and C-type lectin domains (Hickling et al. 2004). The lectin domain (“head”) binds to a variety of monossacharides, conferring binding specificity to targets, whereas the collagen portion (“stalk”) mediates effector function. They trimerize to form a subunit, which in turn polymerizes via disulfide and noncovalent bonds into a cruciate or sertiform structure with up to six subunits. The human collectins include surfactant proteins (SP-A and SP-D), mannose-binding lectin (MBL) or mannan-binding protein, CL-L1 (liver, collectin 10), and CL-P1 (placenta, collectin 12). All four surfactant proteins are produced by alveolar type II cells (Albertine et al. 2000) and Clara cells (Hickling et al. 2004; Kishore et al. 2006), of which SP-A and SP-D are major collectin constituents of surfactant. SP-B and SP-C function as true surfactants, helping maintain the patency of terminal airways by their action on surface tension, whereas SP-A and SP-D participate in host defense by agglutinating and opsonizing microbes, facilitating their clearance by phagocytosis. Most collectins can activate the complement protein C1q and mediate their opsonizing effects partly through complement receptors on phagocytes. They can also bind to allergens and impair allergic response in animal models of allergy to fungal and dust mite allergens, presumably by inhibiting activation of basophils and mast cells and altering allergen presentation to T cells (Hickling et al. 2004; Kishore et al. 2006). The amount of SP-A is reduced in lungs of asthmatic individuals. SP-A decreases lymphocyte proliferation to allergens and attenuates allergic airway inflammation in mouse models of asthma. MBL is produced in the liver and found mainly in blood, but is also present in upper airways (Hickling et al. 2004; Dommett et al. 2006). It binds to several microbes, attenuating infections through neutralization, opsonization, and activation

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of complement. About 5% of the population can be deficient in MBL, as defined by low serum levels (< 50 ng/mL), a condition that is most often asymptomatic but which may predispose to recurrent infections in infants (Hickling et al. 2004; Dommett et al. 2006). Deficiency of MBL may also predispose adults to infections caused by mycoplasma, severe acute respiratory syndrome (SARS)-coronavirus, and to more severe forms of hepatitis B and C virus infections. MBL also facilitates macrophage clearance of apoptotic cells (Hickling et al. 2004).

Pentraxins Pentraxins are a superfamily of interspecies-conserved acutephase proteins that also facilitate opsonization and complement activation (Bottazzi et al. 2006). They are produced early during inflammation on stimulation by TLRs (Doni et al. 2006), IL-1β, or TNF-α (Bottazzi et al. 2006). They form multimers with 5–10 units. Structurally, they share a 200amino acid residue C-terminal pentraxin domain and have an N-terminal domain that varies in length between the short and long forms (Garlanda et al. 2005). For example, C-reactive protein is a short pentraxin produced in the liver and nasal epithelial cells (Gould & Weiser 2001), whereas pentraxin-3 (PTX-3) is a long pentraxin secreted by mononuclear phagocytes, alveolar type II cells, and mucosal airway epithelial cells (Han et al. 2005). PTX-3, a 45-kDa glycoprotein, binds to C1q to activate complement, binds to the extracellular matrix component TNF-α-induced protein 6 to facilitate assembly of hyaluronic acid-rich structures, and binds to some microbes such as Aspergillus fumigatus and Gram-negative bacteria including Pseudomonas aeruginosa (Garlanda et al. 2005; Bottazzi et al. 2006). PTX-3-deficient mice are more susceptible to invasive pulmonary aspergillosis (Garlanda et al. 2002). Serum amyloid A Serum amyloid A (SAA) binds directly to Gram-positive bacteria and is probably an important opsonin. mRNA for SAA is expressed in sinus mucosa and is increased in patients with recalcitrant CRS with polyps as compared to those with treatment-responsive CRS (Lane et al. 2006a). Experiments in bronchial epithelial cell lines indicate that SAA and complement components are expressed as part of a local epithelial acute-phase response reminiscent of the hepatic acute-phase response (Sha et al. 2004). Complement components Several complement components play roles in airway diseases. Anaphylatoxin receptors C3aR and C5aR are expressed on airway epithelial cells, smooth muscle cells (Drouin et al. 2001), endothelial cells, and leukocytes, more so in fatal asthma (Fregonese et al. 2005). In animal models of asthma, C3a and C5a mediate several features of Th2 inflammation in airways of mice sensitized and challenged to allergens

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(Sarma et al. 2006; Kohl & Wills-Karp 2007). However, during the sensitization phase of animals, C5a protects against generation of Th2 effector adaptive response (Drouin et al. 2006), probably via C5aR on dendritic cells and induction of tolerance by T regulatory cells (Kohl et al. 2006; Kohl & Wills-Karp 2007). Sinonasal mucosa can also produce C3, which is observed in inflamed mucosa of CRS (Vandermeer et al. 2004; Lane et al. 2006b). Cleavage products of C3 are potent chemoattractants for granulocytes and act to opsonize particulates for removal by phagocytes. Sinus mucosal epithelial cells also produce alternative complement proteins such as factors B, H and I and properdin (Vandermeer et al. 2004). In the airways, these proteins may thus be expected to derive from plasma exudation as well as local production by epithelium.

Reactive oxygen species Enzymes involved in the production of reactive oxygen species (ROS) participate in several biological processes. Production of ROS starts with formation of superoxide (O2–) from oxygen (O2) catalyzed by NADPH oxidase. Next, superoxide dismutase (SOD) transforms 2H+ and 2O2– molecules into one hydrogen peroxide (H2O2) molecule. Then peroxidases metabolize H2O2 and Cl– into H2O and hypochlorate (OCl–). These ROS damage nucleic acids, lipids and proteins (Valko et al. 2007). Similarly to phagocytes, airway epithelial cells also express these enzymes. The NADPH oxidase activity in epithelial cells is mediated by dual oxidases 1 and 2 (Duox 1 and 2), high-molecular-weight enzymes that also have peroxidase domains (Forteza et al. 2005). Epithelial Duox activity is important for epithelial production of mucin (Shao & Nadel 2005), epithelial repair (Wesley et al. 2007), epithelial production of acid (Schwarzer et al. 2004), innate immunity (Forteza et al. 2005), and for epithelial responses to ozone (Salmon et al. 1998), LPS (Koff et al. 2006), and cigarette smoke (Lavigne & Eppihimer 2005). Regarding innate immunity, epithelial Duox and SOD produce H2O2, which in the lumen is used by airway lactoperoxidase to oxidize thiocyanate anions (SCN–) to hypothiocyanite (OHSN), a potent antimicrobial (Wijkstrom-Frei et al. 2003; Forteza et al. 2005). Eosinophil peroxidase also converts SCN– into OHSN. Hypothiocyanite is an important antimicrobial that selectively kills microorganisms and spares host cells, unlike hypohalous acids that are often generated in the absence of a source of thiocyanate. Epithelial transport of thiocyanate is defective in cystic fibrosis (Moskwa et al. 2007). Upregulation of epithelial Duox activity in airway epithelial cells occurs in response to ATP and increased intracellular calcium (Forteza et al. 2005; Wesley et al. 2007). In alveolar type II cell lines, Duox activity increases as a result of mechanical stretching (Chapman et al. 2005). On the other hand, NADPH oxidase activity in neutrophils plays a role in transepithelial migration on chemoattraction by leukotriene (LT)B4 (Woo et al. 2003), and NADPH oxidase activity intrinsic

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to pollens (e.g., ragweed, plantain, birch, oak, grass pollen) can amplify Th2 sensitization and the allergic response to challenge in animal models of asthma (Boldogh et al. 2005; Ritsick & Lambeth 2005). Nonhematopoietic NADPH oxidase (e.g., in endothelial cells) regulates transendothelial migration of eosinophils into the lung (Abdala-Valencia et al. 2007). Inhalation of pollutants such as ozone (O3), sulfur dioxide (SO2), nitrogen dioxide (NO2), and particulate air pollutants (e.g., diesel exhaust particles) induce oxidative stress in the airways (Li et al. 2003). Airways resist endogenous and exogenous ROS damage through several mechanisms. The epithelial lining fluid contains antioxidants such as vitamins C and E, urate, and reduced glutathione. Intracellular ROS scavenger enzymes and antioxidants in airway cells include heme oxygenase I, glutathione-S-transferase, glutathione peroxidase, SOD, catalase, and thioredoxin reductase, many of which use gluthatione and thyoredoxin as substrates. If the intensity of oxidative stress overcomes these protective mechanisms, bronchial epithelial cells initiate inflammation by secreting cytokines and chemokines via activation of the transcription factors activator protein (AP)-1 and NF-κB. At high intensity of oxidative stress, however, the epithelium undergoes apoptosis via caspase activation or necrosis due to ROS cytotoxicity (Li et al. 2003).

Reactive nitrogen species Like ROS, reactive nitrogen species (RNS) participate in several biological processes. Indeed, ROS and RNS interact in disease, combining oxidative stress and nitrosative stress to cause tissue damage (Ricciardolo et al. 2006; Valko et al. 2007). Increased nitric oxide in exhaled air is a hallmark of asthma (Kharitonov & Barnes 2006). Nitric oxide (NO, or NO· because it has one unpaired electron) is produced in all organ systems by nitric oxide synthase (NOS), which use NADPH to transform the semi-essential amino acid L-arginine and oxygen into NO– and L-citrulline via a five-electron oxidative reaction (Ricciardolo et al. 2006). NOS has two domains, a C-terminal reductase domain with binding sites for NADPH, FAD and FMN (electron donors), and the N-terminal oxygenase domain with binding sites for L-arginine. Between these two domains is a consensus sequence that binds to calmodulin and which transfers electrons between the two domains. There are three members of the NOS family: constitutive neural NOS (NOS1 or nNOS, inducible NOS (NOS2 or iNOS), and constitutive endothelial NOS (NOS3 or eNOS). Airway epithelial cells express eNOS and iNOS. nNOS is expressed in airway nerves, where NO acts as a neurotransmitter and mediates airway smooth muscle relaxation via the inhibitory nonadrenergic, noncholinergic system. eNOS produces small amounts of NO and is constitutively expressed in airway epithelial cells, type II alveolar epithelial cells, and pulmonary vessel endothelial cells. iNOS is expressed in airway epithelial cells, endothelial cells, macrophages, airway and vascular smooth muscle cells,

Airway Epithelium

lung fibroblasts, mast cells, neutrophils, and chondrocytes. On stimulation, the iNOS gene is induced and large quantities of iNOS produced within hours; levels remain high for hours to days. Stimuli include, but are not limited to, TNF-α, IL-1β, interferon (IFN)-γ, chemokines, bacterial toxins, virus infections, allergens, pollutants (e.g., ozone, oxidative stress, silica), hypoxia, and the presence of tumors. Induction of iNOS expression is inhibited by glucocorticosteroids. iNOS produces large amounts of NO, which has a half-life of < 5 s in an aqueous environment because NO combines with oxygen donors to react with most biomolecules by nitration, nitrosation, and nitrosylation (addition of NO2, NO+, and NO, respectively). These reactions mediate the nitrosative stress that can result in inflammatory, antimicrobial, or antitumor effects, and in regulation of apoptosis. Physiologically, NO produced by constitutive NOS functions as an intracellular second messenger and neurotransmitter, inducing vasodilation and bronchodilation (Ricciardolo et al. 2006). As an antimicrobial (Mannick 2006), NO reacts with superoxide from the oxidative burst to form peroxynitrite (ONOO–), a potent oxidant. RNS deaminate DNA, inhibit DNA synthesis, and damage enzymes, which suppresses growth of, or kills, viruses, fungi, bacteria, and parasites. NO can inhibit apoptosis in low concentrations or cause apoptosis and necrosis in high concentrations by nitrosative stress in the context of inflammation and tumor suppression. NO inhibits apoptosis via a cGMP-induced increase in Bcl-2, via an increase in heat-shock protein 70 (which inhibits release of cytochrome c), via inhibition of caspase 3 by S-nitrosylation, and via an increase in the anti-apoptotic thioredoxin. In high concentrations, NO-induced nitrosylation leads to apoptosis by inhibition of NF-κB, reduction in Bcl-2, increase in p53, inhibition of the proteasome, and DNA damage (Mannick 2006). As with ROS induced-signaling and damage, the effects of RNS can be reversed by incompletely understood mechanisms. For example, the balance between nitrosylation and denitrosylation regulates apoptosis (Mannick et al. 1999; Mannick 2007) and enzyme activities such as that of tissue transglutaminase (Lai et al. 2001). In the airways of asthmatic individuals, increased oxidative and nitrosative stress (Misso & Thompson 2005) directly inhibits airway epithelial SOD, leading to increased epithelial apoptosis to an extent directly proportional to clinical severity of disease (Comhair et al. 2005). To counteract these stresses, the airway lumen of asthmatic individuals contains large amounts of glutathione peroxidase, a critical first-line antioxidant defense against ROS and RNS (Comhair et al. 2001). Inhibition of NOS by NG-monomethyl-L-arginine increases bradykinin-induced bronchospasm in asthmatic individuals, suggesting a bronchoprotective role for constitutive eNOS in asthma. On allergen challenge, however, eNOS is suppressed and iNOS upregulated, amplifying Th2 inflammation as suggested by asthma models in iNOS-deficient mice, which have attenuated eosinophil influx, vascular

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leakage, and airway obstruction compared with wild-type mice (Ricciardolo et al. 2006).

Epithelial receptors for sensing microbes Epithelial cells can detect the presence of microbes through several receptors, which recognize molecular patterns common to microbes and are designated pattern recognition receptors (PRRs). These receptors are located in the plasma membrane and in the intracellular compartments and can detect both extracellular and intracellular microbes. Among PRRs, there are 10 TLRs that recognize pathogen-associated molecular patterns (PAMPs) from viruses, bacteria, fungi, protozoa, and multicellular parasites (Table 16.3). TLRs were named for their homology to the Toll molecule in Drosophila melanogaster, which protects the fly against Aspergillus infection (Trinchieri & Sher 2007).

Toll-like receptors TLRs detect microbial products and transduce intracellular signals via adapter molecules (e.g., MyD88), leading to activation of the transcription factors NF-κB and interferonregulatory factor (IRF). These transcription factors activate innate immune responses such as the interferon response, expression of costimulatory molecules, and secretion of cytokines, chemokines and host defense molecules. Structurally, TLRs consist of three domains (Uematsu & Akira 2006). The extracellular domain is a leucine-rich repeat domain that accounts for specificity to PAMPs. The transmembrane domain anchors the molecule to the plasma membrane or intracellular membranes (e.g., endosomes). The cytoplasmic domain is similar to that of the IL-1 receptor, and is called the Toll/IL-1 receptor (TIR) domain. TIRs bind to adapter molecules (see Table 16.3) to transduce signal to cytosolic molecules including NF-κB, IRF, mitogen-activated protein kinases (MAPK), AP-1, and others (Hasan et al. 2005; West et al. 2006; Barton 2007; Trinchieri & Sher 2007). All 10 TLRs likely play roles in innate defense of the airways since they are expressed on epithelial and immune cells. mRNA for all 10 TLRs is present in sinonasal mucosa (Dong et al. 2005; Lane et al. 2006a). TLR2 mRNA is expressed at higher levels in the mucosa of subjects with CRS (Lane et al. 2006b), and TLR9 mRNA levels are higher in mucosal tissue from those with nasal polyps compared with mucosa from control subjects (Ramanathan et al. 2007). Bronchial epithelial cells express functional TLR1–6 and TLR9 (Sha et al. 2004; Guillot et al. 2005; Hewson et al. 2005; Ritter et al. 2005; Zhang et al. 2005; Groskreutz et al. 2006; Kato, A. et al. 2006; Mayer et al. 2007). Primary alveolar type II cells isolated from normal lungs express functional TLR2 and TLR4 (Armstrong et al. 2004). In BEAS-2B bronchial epithelial cells, IFN-γ increases expression of TLR3 and TLR4, whereas TNF-α increases expression of TLR2 (Homma et al. 2004; Ritter et al. 2005). Histamine increases TLR3 expression in the epithelial cell lines NCI-H292 and A549 (Hou et al. 2006).

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The glucocorticoid dexamethasone increases expression of TLR2 in epithelial cells (Shuto et al. 2002; Homma et al. 2004). Indeed, glucocorticoids seem to spare or increase expression of molecules related to innate immunity while inhibiting those involved in inflammation and/or adaptive immunity (Schleimer 2004). In addition to responding to microbial PAMPs, TLR2 can be involved in the response to particulate matter pollutants (Becker et al. 2005) and TLR2, TLR4, and TLR9 can also be involved in the response to dust mites (Boasen et al. 2005), although these environmental stimuli may also contain microbial components. TLR2 and TLR4 can also be activated by intrinsic ligands such as hyaluronan degradation products from extracellular matrix breakdown, resulting in inflammation and epithelial cell apoptosis (Jiang et al. 2005). Taken together, these studies demonstrate not only the presence of functional TLRs in airway epithelial cells, but also that their expression is modulated by mediators and hormones, which can modulate the ability of epithelial cells to sense and to respond to respiratory microbes and particles. In the context of an infection, more than one TLR can be simultaneously stimulated by different products of a single microbial species (Monick et al. 2003; Adamo et al. 2004; Morris et al. 2006; Trinchieri & Sher 2007). These TLR responses in epithelial and antigen-presenting cells (e.g., dendritic cells) result in increased expression of costimulatory molecules and secretion of cytokines and chemokines that can modulate the adaptive immune response. For example, stimulation of bronchial epithelial cells with ligands for TLR2, TLR8, and TLR9 (Lee & Ziegler 2007) and TLR3 (Kato et al. 2007) causes secretion of thymic stromal lymphopoietin (TSLP), an epithelial-derived cytokine that induces expression of OX40L on dendritic cells, which in turn induces differentiation of naive CD4 (Th0) cells into Th2 cells (Liu, Y.J. et al. 2007). Interestingly, rhinovirus synergized with IL-4 to induce TSLP in epithelial cells (Kato et al. 2007), providing a possible explanation for the enhanced allergic response in asthmatics undergoing rhinovirus infections (Lemanske et al. 2005).

Other microbe-sensing molecules In addition to TLRs, other recently recognized PAMP receptors are present in the cytosolic compartment and signal when microbes reach the intracellular compartment. Some of these intracellular receptors belong to two main families: the nucleotide-binding oligomerization domain (NOD)-like receptor (or NLR) family, and the helicase family (Trinchieri & Sher 2007). NLR family The NLR family consists of more than 20 members that are either NOD receptors, or NALPs (an acronym for NACHT-, leucine-rich repeat- and pyrin domain-containing proteins). NACHT is a common domain present in neuronal apoptosis inhibitory protein (NAIP), HLA class 2 transactivator (CIITA),

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Airway Epithelium

Table 16.3 Toll-like receptors (TLRs) on human airway epithelial cells*. TLR response in airway epithelial cells

TLR

Ligands

Adapter molecules

TLR1

TLR1/TLR2 binds to triacyl lipopeptides

See TLR2

TLR1 heterodimerizes with TLR2, which activates adapter molecules. See TLR2

See TLR2

TLR2

TLR2 binds to peptidoglycan, atypical LPS, modulin (Staphylococcus epidermidis), porin (Neisseria). Trypanosoma cruzi GPI anchors. Measles, CMV, HSV-1. See also TLR1 and TLR6

MyD88. TIRAP/Mal

TIRAP/Mal → MyD88. Myd88 → IRAK4, IRAK1 → TRAF6 → TAK1 → IKK (→ IkB → NF-kB,p65p50) and JNK,p38 (→ AP-1). Induce gene transcription of chemokines, cytokines, and surface adhesion and costimulatory molecules

IL-6, TNF-a, IL-8, b-defensin 2 (DEFB2 also known as DEFB4), MIP-3a, GROa, IP10, TSLP

TLR3

Double-stranded RNA formed during replication of RNA viruses such as respiratory viruses

TRIF

TRIF → TRAF6 (see MyD88), RIP1 (→ TAK1, see MyD88), and TBK1. TBK1 → IRF3 → interferon (IFN)-induced genes

IL-6, TNF-a, IL-8, IFN pathway (IFN-b1), MIP-3a, GROa, IP10, I-TAC, RANTES, MMP13, TSLP, BAFF

TLR4

Endotoxin/LPS, RSV F protein, fungal mannan, protozoal GIP

TRAM. TIRAP/Mal

TLR4 associates with CD14 and other proteins to form the LPS receptor. TRAM → TRIF (see TLR3). TIRAP/Mal → MyD88 (see TLR2)

IL-6, IL-8, MIP-3a, GM-CSF

TLR5

Flagellin (Pseudomonas)

MyD88

Myd88 → See TLR2

MIP-3a, GM-CSF, GROa, MMP13

TLR6

TLR6/TLR2 binds to diacyl lipopeptides, lipoteichoic acid. Fungal zymosan. T. cruzi glycolipids

See TLR2

TLR6 heterodimerizes with TLR2, which activates adapter molecules. See TLR2. CD36 associates with TLR2/6

See TLR2

TLR7

Viral single-stranded RNA

MyD88

Myd88 → . . . → IRF7 instead of IRF3 (see TLR9). IRF7 → IFN-induced genes

TLR8

Viral single-stranded RNA

MyD88

Myd88 → . . . → IRF7 instead of IRF3 (see TLR9). IRF7 → IFN-induced genes

TSLP (weak response)

TLR9

Bacterial unmethylated CpG DNA. T. cruzi DNA. Hemozoin from Plasmodium falciparum. HSV-1 and HSV-2 DNA

MyD88

In plasmacytoid dendritic cells: TLR7 and TLR9 → Myd88 → IRAK4, IRAK1 → TRAF6 → TAK1 → IKK (→ IkB → NF-kB, p65p50) and IRF7 instead of IRF3 → IFN-induced genes

TSLP (weak response)

TLR10

Homodimerizes and also heterodimerizes with TLR1 or TLR6. TLR10 ligands are unknown

MyD88

Myd88 → See TLR2

Intracellular signaling pathways

* TLRs are expressed on the plasma membrane, except for TLR3, TLR7, TLR8 and TLR9, which respond to nucleic acids and are mostly expressed in endosomal membranes inside the cells. Mice also have a TLR11 that binds to protein-like proteins from uropathogenic. Escherichia coli and from Toxoplasma gondii. Depicted signaling pathways relate to studies in several cellular systems, whereas the last column only focuses on reports of TLR stimulation effects on human airway epithelial cells. Different ligands for the same TLR can induce mildly different responses, which may result from binding of ligand to different TLR heterodimers, to proteins associated with TLRs (e.g., CD14, CD36), or from differential activation of adapter molecules. Microarray studies show that expression of a large number of genes is altered by TLR ligand stimulation, particularly TLR3 stimulation with synthetic dsRNA poly deoxyinosinic:deoxycytidylic acid (poly I:C). GPI, glycosylphosphatidylinositol; LPS, lipopolysaccharide; GIP, glycoinositolphospholipids; CMV, cytomegalovirus; HSV, herpes simplex virus; RSV F, respiratory syncytial virus fusion; TIRAP/Mal, Toll/IL-1R (TIR)-domain-containing adapter protein; MyD88, myeloid differentiation primary-response gene 88; IRAK, IL-1R-associated kinase; TRAF, tumor necrosis factor receptor associated factor; TAK, transforming growth factor b (TGF-b)-associated kinase; IKK, IkB kinase; IkB, inhibitor of NF-kB; NF-kB, nuclear factor kB; JNK, c-Jun N-terminal kinase (p38 is a mitogen-activated protein kinase); TRIF, TIR-domain-containing adapter protein inducing IFN-b; TRAM, TRIF-related adapter molecule; RIP, kinases receptor interacting protein; TBK, tumor necrosis factor receptor (TNFR)-associated NF-kB kinase (TANK)-binding kinase; IRF, interferon-regulatory factor; MIP, macrophage inflammatory protein; GM-CSF, granulocyte–macrophage colony-stimulating factor; GRO1/CXCL1, growth-regulated oncogene 1; IP10/CXCL10, IFN-g-inducible protein 10; I-TAC/IP9/CXCL11, interferon-inducible T-cell a-chemoattractant; MMP, matrix metalloproteinase; RANTES/CCL5, regulated upon activation, normally T-expressed, and presumably secreted; TSLP, thymic stromal lymphopoietin; BAFF, B-cell-activating factor of TNF family.

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heterokaryon formation gene locus E (HET-E), and transition protein (TP)-1 (Trinchieri & Sher 2007). NLRs have three domains: 1 a carboxy-terminal series of leucine-rich repeats that bind to PAMPs; 2 an NOD that mediates self-oligomerization; 3 an amino-terminal effector domain that can be either a pyrin domain or a caspase-recruitment domain (CARD). Signaling occurs either via NOD activation of receptorinteracting serine/threonine kinase followed by NF-κB activation, or via CARD activation of caspase-1. Both signaling pathways lead to transcription of inflammatory cytokines. Most of the NLR molecules have only recently been discovered and relatively little information is available for epithelial cells. Thus far, the NLR member NOD1 has been found in airway epithelial cells (Slevogt et al. 2007). NOD1 recognizes bacterial wall components. Together with TLR2, NOD1 recognizes Moraxella catarrhalis infection of airway epithelial cells and induces production of IL-8 (Slevogt et al. 2007). It is likely that other NLRs and NALPs will be found in airway epithelial cells because they are found in intestinal epithelial cells (Rosenstiel et al. 2006). Helicase family The helicase or RLR (RIG-I-like receptor) family consists of two members: retinoic acid-inducible gene (RIG)-I and melanoma differentiation-associated gene (MDA)5 (Uematsu & Akira 2006). They recognize intracellular double-stranded RNA (dsRNA) produced on replication of RNA viruses, such as all respiratory viruses except adenovirus (which is a DNA virus). Helicases have an amino-terminal helicase domain that binds to and unwinds dsRNA via an ATPase-dependent mechanism and activates the two tandem CARD domains in the carboxy-terminus (Uematsu & Akira 2006), which binds the CARD domain of IFN-β promoter stimulator (IPS)-1 and activates two signaling pathways involving: 1 FAS-associated via death domain (FADD), receptor interacting protein (RIP)-1, inhibitor of NF-κB kinase (IKK)α, IKKβ and IKKγ, inhibitor of NF-κB (IκB), and finally NF-κB; 2 IKKi, TANK-binding kinase (TBK)1, and activation of IRF7 and/or IRF-3, leading to type I IFN production (Uematsu & Akira 2006). Helicases therefore provide a TLR-independent mechanism for recognizing RNA virus infections and induce type I IFN and NF-κB responses. RIG-I in the alveolar type II cell line A549 recognizes dsRNA formed by respiratory syncytial virus (RSV), activates NF-κB, and induces production of IFN-β, interferon-inducible protein 10 (IP-10/CXCL10), CCL5/ RANTES, and TLR3 (Liu, P. et al. 2007). RIG-I expression is increased by IFN-γ in the bronchial epithelial cell lines BEAS2B and NCI-H292 cells (Imaizumi et al. 2005). Animal studies suggest that RIG-I is important in response to parainfluenza and influenza viruses, whereas MDA5 is critical for response to picornaviruses (e.g., rhinoviruses) (Kato, H. et al. 2006).

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Another well-known receptor for dsRNA is the dsRNAdependent protein kinase (PKR) (Garcia et al. 2006). PKR is a cytosolic serine/threonine kinase that dimerizes when it binds to dsRNA, leading to autophosphorylation and subsequent phosphorylation of the α subunit of eukaryotic translation initiation factor (eIF)-2α. Once phosphorylated, eIF-2α is unable to deliver Met-tRNAi to the 40S ribosome, preventing initiation of protein synthesis, which impairs virus replication and cell proliferation and induces apoptosis. PKR is upregulated by IFN and signals through the MAPKs JNK and p38, and through NF-κB, STAT1, and IRF-1. In airway epithelial cells, PKR participates in the response to rhinovirus by mediating production of CCL5/RANTES (Gern et al. 2003). In addition, PKR mediates IgE class-switching in the human Burkitt’s lymphoma B-cell line Ramos on rhinovirus infection (Rager et al. 1998). Together, TLR3, PKR, and helicases detect and initiate mucosal innate responses to RNA viruses such as most respiratory viruses. Recent evidence suggests that bronchial epithelial cells from asthmatic individuals are deficient in their production of antiviral IFNs. These cells secrete less type I IFN (IFN-β1) and have impaired induction of apoptosis on rhinovirus infection compared with cells from healthy individuals, which allows replication of the virus (Wark et al. 2005). Likewise, bronchial epithelial cells from asthmatic individuals are also deficient in production of type III IFNs such as IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B) on rhinovirus infection (Contoli et al. 2006). In addition, production of lymphocyte-derived type II IFN (IFN-γ) is also impaired in asthmatic individuals (Gern et al. 2000). These deficiencies in type I, II, and III IFNs correlate with severity of rhinovirus colds. Taken together, asthmatic individuals probably have IFN deficiencies that enhance susceptibility to respiratory infections. Whether this deficiency is primary (genetic susceptibility) or secondary to allergen-driven inflammation is not known.

Role of epithelium in inflammation In the 1980s, the airway epithelium was first noticed to produce inflammatory mediators, including arachidonic acid metabolites, NO and cytokines such as granulocyte– macrophage colony-stimulating factor (GM-CSF), IL-8, and IL-6 (Henke et al. 1988; Vanhoutte 1988; Denburg et al. 1991; Holtzman 1991; Marini et al. 1991; Churchill et al. 1992). Until then, the airway epithelium was mostly known for its mucociliary system, ion transport, and electrical properties. In the last two decades, numerous other inflammatory mediators have been found to be generated in airway epithelial cells in response to environmental agents and also on stimulation by immune cells.

Lipid mediators In airway epithelial cells, PLA2 metabolizes membrane phospholipids and releases arachidonic acid (AA), which can be

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further metabolized into prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids (HETE) and lipoxins (Henke et al. 1988; Holtzman 1991, 1992; Goetzl 2003; Schaloske & Dennis 2006). Cyclooxygenase (COX)-1 and COX-2 (Balzary & Cocks 2006) both transform AA into prostaglandin (PG)E2, one of the first inflammatory mediators found to be secreted by airway epithelial cells (Holtzman 1992). Other epithelial COX minor products are PGF2α and thromboxane (TX)B2. Exogenous PGE2, administered by nebulization immediately prior to bronchial allergen challenge, attenuates early and late airway bronchoconstriction responses, airway hyperresponsiveness, and influx of eosinophils (Gauvreau et al. 1999). PGE2 relaxes airway smooth muscle and also has several anti-inflammatory actions (Holtzman 1992; Gauvreau et al. 1999). 5-Lipoxygenase (5-LO) together with 5-lipoxygenaseactivating protein (FLAP) transforms AA into 5-HETE and leukotriene (LT)A4 (Holtzman 1991, 1992), which is unstable and is quickly transformed by LTA4 hydrolase (Bigby et al. 1989) into LTB4, a potent chemoattractant for neutrophil and cytotoxic CD8 lymphocytes (Goodarzi et al. 2003; Ott et al. 2003). LTA4 can also be transformed into LTC4 by LTC4 synthase, but this occurs in leukocytes. Epithelial cells also express 15-lipoxygenase (15-LO), which transforms AA into 15-HETE and other minor products such as 12-HETE and 8-HETE (Henke et al. 1988; Widdicombe 1991), whose fuctions are not completely understood. Epithelial 15-LO has been shown to be induced by IL-13 and has been implicated in both asthma and COPD (Chu et al. 2002; Kuperman et al. 2005). Recent studies indicate that 15-LO may inhibit epithelial transport of IgA into the airways (Kuperman et al. 2007). Products from lipoxygenases interact to form lipoxins (Bonnans et al. 2004). Interactions between LTA4 and 15HETE form lipoxin (LX)A4, while those between LXB4 and 5-epi-lipoxin form 15epi-LXA4 (Bonnans et al. 2004). These lipoxins have antiinflammatory and epithelium repair actions via inhibition of a number of processes, such as LTB4-mediated chemotaxis of neutrophils, release of IL-8 and elastase by neutrophils, platelet-activating factor-induced chemotaxis of eosinophils, and LTC4-induced bronchospasm (Bonnans et al. 2004).

Adhesion molecules Epithelial cells express adhesion molecules that not only maintain structural integrity of the normal epithelium but also interact with leukocytes, initiate intracellular signaling in inflammation and repair, and participate in lung morphogenesis (Sheppard 2003). Airway epithelial cells express integrins, intercellular adhesion molecule (ICAM)-1, and CD44 (Peroni et al. 1996) (Table 16.4). Integrins are heterodimeric transmembrane glycoproteins composed of α and β subunits. There are at least 18 α subunits and 8 β subunits that can form 24 integrin heterodimers.

Airway Epithelium

Virtually all cells in the body express integrins. There is redundancy because several integrins bind to the same ligand, and the same integrin–ligand pair can result in different responses in different tissues. On interaction, the conformational changes in the integrin–ligand complex can result in downstream intracellular signaling in both interacting cells. In epithelial cells, integrins regulate wound repair, establishment of polarity, differentiation into secretory cells, and inflammation. After binding to ligand in extracellular matrix or on the surface of adjacent cells, integrins change their conformation, initiating or repressing intracellular signaling through interactions between the short intracytoplasmic domains of their subunits and a multiprotein signaling complex. In this complex, focal adhesion kinase (FAK) has binding sites to interact with several signaling molecules, including ras-associated protein Grb2, the SH2 domains of src, phosphatidylinositol 3-kinase and PLC-γ, and adapter proteins (Sheppard 2003). In addition, some integrins (e.g., α2β1 and αVβ3) use another transmembrane protein called integrin-associated protein (IAP) for signaling. These proteins allow integrins to activate a broad number of downstream signaling pathways (see Table 16.4). Bronchial and nasal epithelial cells also express ICAM-1, a member of the immunoglobulin superfamily. ICAM-1 is increased in bronchial epithelial cells of asthmatic patients (Wegner et al. 1990) and its blockade in a primate asthma model attenuates airway eosinophilia and hyperresponsiveness (Wegner et al. 1990). Bronchial epithelial expression of ICAM-1 increases 24 hours after allergen challenge in asthmatic patients (Bentley et al. 1993), and also on stimulation with TNF-α, IFN-γ, IL-4, and IL-13 (Bianco et al. 1998; Striz et al. 1999). It reduces on treatment with corticosteroids (Papi et al. 2000) and histamine H1 receptor blockers (Ciprandi et al. 2003). Epithelial ICAM-1 mediates adhesion to eosinophils (Burke-Gaffney & Hellewell 1998) and neutrophils via Mac-1 (CD11b/CD18) and to lymphocytes via LFA-1 (CD11a/ CD18). Interaction between goblet cells and neutrophils via ICAM-1 mediates degranulation of goblet cells induced by neutrophil elastase (Nadel et al. 1999). Besides being expressed on the plasma membrane (mICAM-1), a soluble form of ICAM-1 (sICAM-1) can derive from either mRNA splicing or from protease-mediated cleavage of mICAM-1. The function of sICAM-1 is not fully established, but it could be a decoy inhibitory molecule interfering with ICAM-1-mediated adhesion. sICAM-1 is elevated in the serum of patients affected by many chronic inflammatory diseases including asthma (Witkowska & Borawska 2004). It increases further in acute asthma. ICAM-1 is also the receptor for major group rhinoviruses, which precipitate 30–50% of all asthma exacerbations. Rhinovirus further increases ICAM-1 expression by epithelial cells. The minor group rhinoviruses represent 10% of the serotypes and utilize the LDL receptor for cell entry (Edwards et al. 2006a).

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Table 16.4 Adhesion molecules on human airway epithelial cells. Molecule

Ligands

Localization

Function

Integrins a2b1

Collagen I and IV, tenascin C, echovirus

All epithelium, particularly basal cells

Adhesion of basal cells to basement membrane

a3b1

Laminins 5, 10, 11

All epithelium, particularly basal surface of basal cells


a5b1

Fibronectin

Expressed after injury on all epithelium

Binding to matrix and repair

a6b4

Laminins 5, 10, 11

Basal surface of basal cells


a9b1

Tenascin C, osteopontin, VCAM-1, L1-CAM, vWF, factor XIII, tissue transglutaminase, fibronectin, angiostatin, ADAMS 1, 2, 3, 9 and 15

All epithelium, particularly basal cells

Adhesion to basement membrane and leukocytes, coagulation, and repair

aVb5

Vitronectin, osteopontin, adenovirus

All epithelium, particularly on basal cells

Binding to matrix and repair

aVb6

LAP of TGF-b1 and -b3, fibronectin, tenascin C, osteopontin, vitronectin, foot and mouth virus

Highly expressed after injury on all epithelium

Binding to matrix, initiation of fibrosis and repair

aVb8

LAP of TGF-b1 and -b3, vitronectin


Binding to matrix, initiation of fibrosis and repair

Immunoglobulin superfamily molecule Eosinophils, neutrophils, lymphocytes, and ICAM-1 rhinovirus

Other adhesion molecule Hyaluronic acid, collagen and fibronectin CD44

Expressed on scattered epithelial cells. Increased in asthma

Mediates neutrophil-induced mucus secretion and adhesion to leukocytes

All epithelium, particularly on basal cells. Increased in asthma

Mediates neutrophil-induced mucus secretion

ICAM, intercellular adhesion molecule; LAP, latency-associated peptide; TGF, transforming growth factor; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor.

Cytokines, chemokines and neuropeptides In the past 20 years, airway epithelial cells have been found to secrete a vast number of cytokines and chemokines that recruit all types of leukocytes and regulate their functions. In this section, we focus on cytokines and chemokines secreted by the pseudostratified epithelium in the conducting airways as identified by airway biopsy studies using immunohistochemistry and in situ hybridization techniques and by cultures of bronchial or nasal epithelial cells (Tables 16.5 and 16.6). These tables do not include the biology of mediators secreted by other epithelial cell types such as alveolar epithelial cells, glandular epithelial cells, and squamous cells.

Cytokines Cytokines are secreted polypeptides that regulate growth, differentiation, and functions of leukocytes and structural cells. Epithelial cells can secrete pleiotropic cytokines such as TNF-α, IL-1β, IL-6, and IL-11 on respiratory virus infection

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and other stimuli. These cytokines initiate inflammation and the engagement of adaptive immune responses (Becker et al. 1991; Elias et al. 1994; Khair et al. 1995; Bitko et al. 1997; Terajima et al. 1997; Takizawa 1998; Nam et al. 2006). Epithelial cells can also secrete regulatory cytokines that affect monocytes (IL-10, IL-12p80, IL-19) (Bonfield et al. 1995; Walter et al. 2001; Bosson et al. 2003; Pathmanathan et al. 2003; Zhong et al. 2006), T cells (IL-15, IL16, TSLP) (Arima et al. 1999; Little et al. 2003; Ying et al. 2005; Kato et al. 2007; Regamey et al. 2007), and B cells (IL-6, IL-10, BAFF, APRIL) (Kato, A. et al. 2006). In addition, as part of the defense against respiratory viruses, epithelial cells release IFNs (Friedlander et al. 2005; Wark et al. 2005; Contoli et al. 2006; Edwards et al. 2006a). Epithelial cells also secrete growth factors that participate in injury repair, but can also be stimulated by inflammation and environmental agents (see Table 16.5) (Marini et al. 1991; Bedard et al. 1993; Coste et al. 1996; Hirst et al. 1996; Nakamura et al. 1996; Yi et al.

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Airway Epithelium

Table 16.5 Cytokines and other mediators produced by nasal and/or bronchial airway epithelial cells*. Molecule

Function

Stimuli for production

Interleukins IL-1b IL-6 IL-10 IL-11 IL12p80 IL-15 IL-16 IL-19 IL-28A/B, IL-29

Lymphocyte activation, endothelial expression of ICAM-1 and VCAM-1 Induces acute-phase reactants, stimulates T and B cells Inhibition of activated T cells and mononuclear phagocytes. Induction of Treg Stimulates hematopoiesis, myelofibroblast and collagen deposition IL12p40 homodimer attracts macrophages Stimulates T and NK cells, and differentiates monocytes into DCs Chemoattracts CD4+ T cells, eosinophils and monocytes Induces monocytes to secrete TNF-a Type III (lambda) interferons. Induce antiviral response similar to IFN-b

Virus infections, asbestos, TDI, HDM, DEP, CSE Virus and bacterial infections, O3, IL-17 TNF-a, IFN-l RSV, IL-1b, TGF-b1, and histamine Virus infections, allograft rejection IFN-l Histamine, TNF-a, TGF-b1, IL-9 Adenosine via A2B receptor Virus infections

Interferon type I. Induces epithelial antivirus defense Airway hyperresponsiveness, stimulates myofibroblast, mediates eosinophilia, production of SP and growth factors Promotes differentiation of Th0 to Th2 via dendritic cells and OX40 ligand Vasoconstriction, bronchospasm, mucus secretion, fibroblast proliferation, vascular leakage, production of PGD2 and LTD4 CGRP is a neuropeptide that induces potent arterial and venous vasodilatation

Virus infections IL-1a, TNF-a, TGF-b1

Other mediators IFN-b1 NGF TSLP Endothelin 1 CGRP

TNF superfamily† Endothelial expression of ICAM-1, VCAM-1 TNF-a Stimulates B-cell proliferation and differentiation into plasma cells including IgE BAFF switching Stimulates B-cell proliferation and differentiation into plasma cells including IgA APRIL switching Growth factors GM-CSF/CSF2 G-CSF/CSF-3 FGF-1, FGF-2 FGF-7 or KGF TGF-a EGFR pathway TGF-b1 TGF-b2 PDGF-b SCF Heregulin-a

Stimulates hematopoiesis for granulocytes and monocytes. Differentiation of DCs Stimulates hematopoiesis for granulocytes, particularly neutrophils Fibroblast proliferation and angiogenic Proliferation of keratinocytes and airway epithelial cells Surface bound. Released by TACE and induces mucin synthesis via EGFR Mucin synthesis, mucous cell metaplasia and fibroblast proliferation, differentiation, and extracellular matrix production Stimulates myofibroblast proliferation. Induces epithelial–mesenchymal transition. Induces IgA class switch Stimulates myofibroblasts to secrete extracellular matrix proteins Epithelial, fibroblast and smooth muscle cell proliferation and collagen deposition Mast cell proliferation and differentiation On injury, apically secreted heregulin can reach and interact with erbB-2, erbB-3 and erbB-4 receptors on the basolateral membrane to induce epithelial proliferation and wound repair

IL-1b, TNF-a, rhinovirus, stimulation of TLRs EPO, MBP Produced in epithelial and T cells during late-phase response to allergens

Virus and bacterial infections TLR3 stimulation via IFN-b1 TLR3 stimulation via IFN-b1

TNF-a, IL-1, IL-4, IL-13, virus infection, DEP, NO2, O3 TNF-a, IL-1, IL-17A, IL-17F, virus infection Increased in COPD. Virus infection Unknown EPO, MBP CSE, virus and bacterial infections, Th2 cytokines, neutrophil elastase via TGF-a, EGF EPO, MBP IL-4, IL-13 EPO, MBP, PAR-1 agonist Allergen exposure in sensitized individuals Mechanical disruption of epithelium integrity

* For more details on biology of cytokines, see Chapter 23. † Surface molecules that bind to their respective receptors during cell–cell contact and/or are cleaved by surface enzymes (i.e., TACE) to become soluble factors. APRIL, a proliferation-inducing ligand (also TNFSF13); BAFF, B-cell-activating factor (also TNFSF13B); CGRP, calcitonin-gene related peptide; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; DCs, dendritic cells; DEP, diesel exhaust particles; EGFR, epidermal growth factor receptor; EPO, eosinophil peroxidase; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colonystimulating factor; HDM, house-dust mite; ICAM, intercellular adhesion molecule; KGF, keratinocyte growth factor; MBP, major basic protein; NGF, nerve growth factor; NK, natural killer; PAR, protease-activated receptor; PDGF, platelet-derived growth factor; RSV, respiratory syncytial virus; SCF, stem cell factor; SP, substance P; TACE, TNF-a converting enzyme; TDI, toluene diisocyanate; TLR, Toll-like receptor; Treg, CD4+ T regulatory cells; TSLP, thymic stromal lymphopoietin; VCAM, vascular cell adhesion molecule.

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Table 16.6 Chemokines secreted by nasal and/or bronchial airway epithelial cells*. Molecule

Cells attracted

Stimuli for production

CC chemokines CCL1/I-309 CCL2/MCP-1 CCL3/MIP-1a CCL5/RANTES CCL11/eotaxin CCL13/MCP-4 CCL17/TARC CCL20/MIP-3a CCL22/MDC CCL24/eotaxin 2 CCL26/eotaxin 3

Eosinophils, monocytes Basophils, monocytes, activated T, NK, immature DC Eosinophils, monocytes, activated T, NK, immature DC Eosinophils, monocytes, activated T, NK, immature DC Eosinophils, basophils, activated T, NK, immature DC Eosinophils, basophils, activated T, NK, immature DC Th2, eosinophils Immature DC Eosinophils, basophils, activated T, immature DC Eosinophils, basophils, activated T, immature DC Eosinophils, basophils, resting T

Unknown Virus infections, a defensin 1, IL-4, IL-13, Der p1, TNF-a, IL-1b, IFN-l Virus infections IL-1b, virus infections, Pseudomonas, Der p1, O3, TNF-a, DEP, IL-4 Virus infections, IL-4, IL-13, TNF-a, Staphylococcus aureus TNF-a, IL-1b, IFN-l Der p1, IL-4, TGF-b1 Der p1, extracellular nucleotides, IL-17 Virus infection, IL-4, IL-13 Virus infection, IL-4, IL-13 Virus infection, IL-4, IL-13

CXC chemokines CXCL1/GROa CXCL2/GROb CXCL3/GROl CXCL5/ENA78 CXCL6/GCP2 CXCL8/IL-8

Neutrophils Neutrophils Neutrophils Neutrophils Neutrophils Neutrophils, DC

IL-17A, IL-17F, virus infections, IL-4, IL-13, DEP

CXCL9 /MIG CXCL10/IP-10 CXCL11/I-TAC

Activated Th1, NK Activated Th1, NK Activated Th1, NK

CX3C chemokines CX3CL1/fractalkine

DC–epithelial interaction

TNF-a Virus infection, O3 IL-17 Virus and bacterial infections, HDM, DEP, CSE, O3, IL-17, a defensin 1, TNF-a, IL-1b, IL-4 Virus infection, IFN-l Virus infection, IFN-l, Der p1 Virus infection, IFN-l

* For more details on names and biology of chemokines, see Chapter 24. T, CD3+ lymphocytes; Th, T helper CD3+CD4+ cells; Th1, CD3+CD4+ lymphocytes secreting IFN-l; Th2, CD3+CD4+ lymphocytes secreting IL-4, IL-13; NK, natural killer cells; DC, dendritic cells; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MIG, monokine induced by gamma interferon; MIP, macrophage inflammatory protein; TARC, thymus and activation-regulated chemokine; ENA, epithelial cell-derived neutrophilactivating peptide; GCP, granulocyte chemotactic protein; TECK/CCL25 and MEC/CCL28 have been described in mucosa of airways in animals. For definition of other abbreviations, see footnote of Table 16.3.

1996; Aoshiba et al. 1997; Ishibashi et al. 1998; Michelson et al. 1999; Gern et al. 2000; Shimizu et al. 2000; Nadel & Burgel 2001; Bosson et al. 2003; Pathmanathan et al. 2003; Sheppard 2003; Vermeer et al. 2003a; Al-Muhsen et al. 2004; Burgel & Nadel 2004; Holgate et al. 2004; Kowalski et al. 2005; Kranenburg et al. 2005; Shao & Nadel 2005; Takizawa 2005; Ingram & Bonner 2006; Koff et al. 2006; Pegorier et al. 2006; Tyner et al. 2006; Volonaki et al. 2006). In bronchial biopsies, epithelial cells from asthmatic subjects have increased expression of IL-12p80 (Walter et al. 2001), IL-16 (Laberge et al. 1997), eotaxins (Komiya et al. 2003), endothelin (Howarth et al. 1995), IL-11, IL-17, transforming growth factor (TGF)-β, (Chakir et al. 2003), TSLP (Ying et al. 2005), stem cell factor (Al-Muhsen et al. 2004), and nerve growth factor (Frossard et al. 2004). Epithelial cells are also a target for action of cytokines released by inflammatory cells and their own mediators

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(autocrine action). Most notably, in asthma, epithelial cells undergo mucous metaplasia driven particularly by IL-13 (Kuperman et al. 2002), but also by IL-4 (Iwashita et al. 2003), IL-9 (Vermeer et al. 2003b), IL-11, IL-25, and IL-33 (Tyner et al. 2006; Nakajima & Takatsu 2007). Indeed, in mice, tracheal administration of IL-13 or IL-4 causes airway hyperactivity within 6 hours without eosinophilic inflammation (Venkayya et al. 2002).

Chemokines As mentioned above, airway epithelial cells also secrete a large variety of chemokines that enable recruitment of all types of leukocytes on exposure to diverse stimuli including environmental agents and cytokines (see Table 16.5). Chemokines are small proteins of 8–12 kDa that act through chemokine receptors, with overlapping specificity constituting a redundant system. They are classified based on the

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number of amino acids between the closest cysteine residues forming disulfide bonds. The CC, CXC, and CX3C chemokine families have none, one or three intervening amino acids, respectively, between the cysteine residues forming the bonds. The C chemokine family has only one member, lymphotactin, not expressed by epithelial cells. Secreted chemokines are designated ligands, indicated by the letter L, and receptors, indicated by the letter R. For example, the chemokine RANTES is designated CCL5 and binds to three receptors, CCR1, CCR3, and CCR5. In general, CCL chemokines recruit lymphocytes, monocytes, eosinophils, basophils, and dendritic cells, whereas CXC chemokines recruit neutrophils. There is only one member of the CX3C chemokine category. There are 28 CCLs and 16 CXCs. Epithelial cells can secrete the CCL chemokines (see Table 16.6) CCL1/I-309 (Montes-Vizuet et al. 2006), CCL2/ monocyte chemoattractant protein (MCP)-1 (van der Velden et al. 1998; Ip et al. 2006; Liu, C.Y. et al. 2007), CCL5/RANTES (Teran 2000; Bayram et al. 2001), CCL11/eotaxin (Matsukura et al. 2001; Takamura et al. 2004; Escotte et al. 2006), CCL13/ MCP-4 (Stellato et al. 1997; Lamkhioued et al. 2000), CCL17 (Heijink et al. 2007), CCL20/macrophage inflammatory protein (MIP)-3α (Pichavant et al. 2005 and 2006; Marcet et al. 2007), CCL22/MDC (Lezcano-Meza et al. 2003), CCL24/ eotaxin 2 (Papadopoulos et al. 2001; van Wetering et al. 2007), and CCL26/eotaxin 3 (Komiya et al. 2003). CCL25/TECK (thymus-expressed chemokine) and CCL28/MEC (mucosaeassociated epithelial chemokine), B-cell attracting chemokines, have been described in mucosa of airways in mice (Meurens et al. 2006), where CCL28 was also involved in recruitment of eosinophils (John et al. 2005). Of the CXCL chemokines, airway epithelium can secrete CXCL1/GROα (Salvi et al. 2000; McAllister et al. 2005), CXCL5/ENA78 (Bosson et al. 2003; Edwards et al. 2006b), CXCL6/GCP2 (Prause et al. 2003; Rudack et al. 2006), and CXCL9–11 (MIG, IP10, I-TAC) (Sauty et al. 1999). In airways of asthmatics, the following chemokines are increased compared with nonasthmatic control subjects: CCL1/I309 (Montes-Vizuet et al. 2006), CCL2/MCP-1 (Sousa et al. 1994), CCL3/MIP-1α, CXCL8/IL-8 (Hamilton et al. 2003), CCL11/eotaxin (Pepe et al. 2005), CCL13/MCP-4 (Lamkhioued et al. 2000), and CX3CL1/fractalkine (Rimaniol et al. 2003).

Neuropeptides Neuropeptides are peptides released by nerve fibers that regulate function of nonneuronal tissue. The main neuropeptides secreted by nociceptive (pain sensation) nonmyelinated sensory nerve fibers (type C) in the airways are substance P (SP), neurokinin A (NKA), and calcitonin generelated peptide (CGRP). These neuropeptides induce bronchoconstriction (NKA > SP > CGRP) and vasodilatation (SP, CGRP), increase blood vessel permeability (SP), and induce glandular secretion (SP) (Casale & Baraniuk 1998). Among these neuropeptides, only CGRP increased in bronchial secre-

Airway Epithelium

tions and tissue in the late asthmatic response, i.e., 5 hours after inhalation challenge with cat allergen peptide (Kay et al. 2007). Immunostaining of bronchial biopsies revealed presence of CGRP in airway epithelium, smooth muscle, T cells, and macrophages. In this model, there is no early asthmatic response because peptides are too small to cross-link IgE and activate mast cells. Therefore, this model indicates that allergen stimulation of antigen-presenting cells and T cells leads to CGRP production, a potent arterial and venous vasodilator. This may be an important pathogenic event in late asthmatic responses to allergens because in the cat allergen peptide inhalation model these reactions are not mediated by histamine, leukotrienes, prostaglandins, or by influx of polymorphonuclear leukocytes.

Role of epithelium in adaptive immune responses Accumulating evidence indicates that epithelial cells play important roles in the initiation, maintenance, and regulation of adaptive immune responses in the airways. Epithelial cells can trigger and modify the activation and differentiation of dendritic cells (DCs), B cells, and T cells. They also play an important role in the formation of lymphoid structures in the airways. When the innate immune functions of epithelium discussed above fail, adaptive immune responses to potential pathogens are necessary and life-saving. In addition, restraint of inflammatory immune responses is essential to prevent excessive or unnecessary damage to the airways. While responses of cells armed with antigen-specific immunoglobulins (e.g., mast cells, basophils, eosinophils, neutrophils) are part of the adaptive response, they are discussed elsewhere in this volume and are not considered here. There is a highly meshed network of DCs within the respiratory epithelium. A subset of these DCs, referred to as intraepithelial dendritic cells, has a distinct phenotype, expressing αEβ7, Fc receptors, langerin, and tight junction proteins (claudin-1, claudin-7, and ZO-2) (Holt et al. 1989; Gong et al. 1992; Sung et al. 2006) Intraepithelial DCs extend processes into the airway lumen between epithelial cells, presumably to collect antigenic material from the mucosal surface. These cellular processes may interact with epithelial cells through the unusual membrane-associated chemokine CX3CL1/fractalkine, which is primarily expressed by epithelial cells (Lucas et al. 2001). Fractalkine and its receptor CX3CR1 have been shown to mediate DC–epithelial interactions in the gut (Rimaniol et al. 2003; Niess et al. 2005) and are elevated in asthmatic airways (Rimaniol et al. 2003). Mucosal M cells are a type of specialized epithelial cell found in the intestine that have high permeability and permit antigen tissue entry for access to subepithelial DCs. Analogous cells do not appear to be found in normal human airway or in bronchialassociated lymphoid tissue (BALT), which is generally only found in inflamed human airways (Richmond et al. 1993). There is growing evidence that epithelial cells play a role in the recruitment and local survival of DCs, as they produce

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the chemokine CCL20/MIP-3α and the cytokine GM-CSF, respectively, which promote these processes (Reibman et al. 2003; Sha et al. 2004). Epithelial IL-15 has been shown to transform monocytes into plasmacytoid DCs (Regamey et al. 2007). The nature of the immune response that occurs after DC exposure to antigen (e.g., Th1, Th2, Treg, Th17) is determined by the state of activation of DCs and the context in which they present antigen to T cells (level and type of costimulatory molecules expressed by the DC, production of IL-10, IL12, etc.). While factors associated with antigen, such as the presence of TLR ligands, can have a profound outcome on the nature of the DC response, it is now clear that epithelial cells can also influence subsequent DC activation status. Recent studies have focused on the epithelial-derived factor TSLP, which has been shown to skew DCs so that they activate formation of Th2 cells. TSLP is a four-helix bundle cytokine related to IL-7 and binds to a specific receptor composed of the IL-7R α chain and the TSLP receptor (Liu, Y.J. et al. 2007). Production of TSLP occurs primarily in keratinocytes in the skin and epithelial cells in the airways. Besides activating DCs, epithelial TSLP has been shown recently to activate cytokine secretion by mast cells (Allakhverdi et al. 2007). Elevated TSLP expression has been demonstrated in both atopic dermatitis and in asthma (Soumelis et al. 2002; Ying et al. 2005). A feature of its effects on DCs is that it activates costimulation processes without triggering DC generation of IL-12, a cytokine with potent Th1-skewing activity. Epithelial expression of TSLP is triggered by the TLR3 ligand dsRNA, by rhinovirus, and by Th2 cytokines and the process involves activation of NF-κB and IRF-3 in the case of dsRNA and STAT6 in the case of IL-4 (Kato et al. 2007). Other epithelial-derived factors that are likely to influence the Tcell-skewing characteristics of DCs are the type I interferons IFN-α and IFN-β (that skew toward Th1) and IFN-λ (IL-28/ IL-29) (skew toward Treg) (Rogge et al. 1998; Mennechet & Uze 2006). Epithelial cells can shape the tissue response during adaptive immune effector responses to conform to the nature of the T-cell response and the leukocytes required (neutrophils for Th1 and Th17 and eosinophils and basophils for Th2). For example, epithelial cells release Th1-related chemokines (CXCL10/IP-10 and CXCL9/MIG to attract Th1 cells and neutrophil chemoattractant chemokines) in response to IFN-γ produced by Th1 cells, and produce Th2-related chemokines (CCL22/MDC and CCL17/TARC to attract Th2 cells and eotaxins to attract eosinophils and basophils) in response to IL-4 and IL-13 produced by Th2 cells (Nickel et al. 1999). Epithelial cells produce CXCL8/IL-8 and CXCL1–3/GROα–γ in response to Th17 to recruit neutrophils (Laan et al. 1999; McAllister et al. 2005). The chemokines responsible for Th17 recruitment have not been evaluated, but it is reasonable to expect that they will be partly epithelial cell-derived. It is not clear whether epithelial cells also shape the T-cell response

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during initial sensitization, although it is tempting to speculate that epithelial cells may influence the phenotype of outgoing DCs to modify their T-cell differentiation tendencies in draining lymph nodes. Epithelial cells do express receptors for some factors that drive the T-cell differentiation process, such as IL-31, which may regulate both Th1 and Th2, and TGF-β, which skews toward Th17 (Chattopadhyay et al. 2007). It has not been tested whether epithelial cells have receptors for other such factors that influence the nature of the T-cell response, such as IL-25, that skews toward Th2, IL-28B/IL-29 that skew toward Treg, TSLP that skews to Th2 (see above), or IL-23 that skews to Th17 (see above). However, IL-22 appears to be a prominent Th17-produced cytokine and epithelial cells (keratinocytes) display a robust response to this cytokine (Zheng et al. 2007). Although epithelial cells express HLA-I, HLA-II and costimulatory molecules such as CD80, CD86 (Oei et al. 2004), B7-H1–B7-H4, B7-DC (Kurosawa et al. 2003; Kim et al. 2005; Tsuda et al. 2005), and CD40 (Propst et al. 2000), the roles of these molecules in antigen presentation to T cells and in modulation of T-cell responses are not completely understood. Studies in the 1970s demonstrated that IgA- and IgEexpressing B cells are found in the airways and that these cells produce IgE and IgA specific to known inhaled antigens (Huggins & Brostoff 1975; Nakajima et al. 1975). Several studies have shown that levels of aeroallergen-specific IgE are much higher in the airways than in the serum when normalized to total IgE or albumin. It is not uncommon for individuals with allergic rhinitis or CRS to manifest antigen-specific IgE in nasal secretions or nasal tissue with no apparent specific IgE in the serum for the same antigen, and documented cases exist in which nasal antigen challenge responses are elicited in individuals who lack skin-test sensitivity. A survey study found that 19% of patients with rhinitis and polyposis had specific IgE in the nose but not the serum (Shatkin et al. 1994). Recent studies that used timeresolved fluorescence immunosorbent assays to determine the relative proportions of total and specific immunoglobulins in the airways and circulation concluded that the majority of the total body aeroallergen-specific antibodies of the IgE and IgA isotypes are produced in the airways, and that systemic sensitization largely reflects spillover of immunoglobulins from the mucosal site of their production into the circulation (Yoshida et al. 2005). It has been suggested that IgE is tightly regulated in this way to avoid the danger of anaphylaxis that accompanies the presence of high concentrations of circulating IgE (Geha et al. 2003). It is now clear that antigen-specific B cells are activated and undergo class-switch recombination in the gut and airway submucosae. It thus becomes important to consider the role that local factors in the mucosae play in the recruitment, differentiation, activation, and survival of B cells. Epithelial cells, especially in the gut, have been shown to release chemokines that attract B cells in general, and IgA-secreting B cells

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and plasma cells in particular. These chemokines include CCL25/TECK, CCL28/MEC, CXCL13/BLC (B-lymphocyte chemoattractant), and CXCL12/stromal-cell derived factor (SDF)-1α. In many cases, studies have been restricted to mucosal epithelium of the intestine (Hieshima et al. 2004). In the airways, epithelial production of CCL28/MEC has been largely of interest since it attracts eosinophils and Th2 cells via CCR3 and CCR10 (John et al. 2005). However, this chemokine is also likely to be an important epithelial-derived chemokine that attracts B cells in both the gut and the airways (Lazarus et al. 2003; English et al. 2006). CXCL12/ SDF-1α is another known B cell-attracting chemokine that has nonetheless been of interest in airway disease for another reason; it has recently also been shown to play a role in recruitment of epithelial stem cells to injured trachea as part of the repair process mediated by keratinocyte growth factor (Gomperts et al. 2006, 2007). More information is needed to better clarify the role of epithelium in B-cell recruitment. Recent studies indicate that epithelial cells produce several factors that can modify the differentiation of B cells, much in the same way that has been described above for DCs and T cells. Epithelial cells have long been known to be a rich source of IL-6 and TGF-β, cytokines that have profound B cell-activating properties (see above). In addition, recent studies indicate that epithelial cells produce B-cell activating factor (BAFF)/BLyS (see Table 16.5) or TNFSF13B, a member of the TNF superfamily that is essential for B-cell development via the BAFF receptor and which mediates B-cell class-switch recombination through another receptor, TACI [transmembrane activator and CAML (cyclophilin ligand protein) interactor] (Mackay et al. 2003; Kato, A. et al. 2006). More studies are needed to determine the relative importance of epithelial-derived BAFF versus other sources of BAFF on local class-switch recombination and plasma cell differentiation of B cells in the airways. In the intestine, it has been concluded that epithelial BAFF is the major trigger for regulating immunoglobulin class switching and that this process is promoted further by epithelial-derived TSLP and regulated by the protease inhibitor SLPI (Xu et al. 2007). Epithelial cells perform a well-known role in the transport of IgA and IgM across the epithelium into mucosal secretions (Kaetzel 2005; Brandtzaeg et al. 2006). This process is likely to be of importance both in innate and adaptive immunity, as natural IgA antibodies, i.e., those not generated by somatic hypermutation, can be produced locally in mucosal tissues, in some cases without the participation of T cells. Mucosal B cells produce dimeric IgA or pentameric IgM with monomers joined by the J chain. These multimers bind to the polymeric immunoglobulin receptor (pIgR), which transports them across epithelial cells into the airway lumen. This process occurs to a significant extent in airway mucosal glands as well as in the lamina propria of intestine and conducting airways (Fagarasan & Honjo 2004). During the process of

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transport of IgA (or IgM) by pIgR, the transported antibodies are covalently linked to a portion of pIgR that becomes the secretory component (SC) to produce the secretory forms sIgA or sIgM. This process is quite important in mucosal immunity as well as in the neutralization of potential antigens in the gut and airways (immune exclusion). pIgR and SC have important immunologic roles beyond immunoglobulin transport. It has been established that epithelial pIgR expression and function is regulated by numerous cytokines, hormones and PAMPs (Kaetzel 2005). There are reports suggesting that defective epithelial transport of IgA may play a role in mucosal airway diseases such as COPD, CRS, and asthma (Buckley 1975; Daele 1997; Chee et al. 2001; Pilette et al. 2004). More information is needed to determine the role of local B-cell responses in both inflammatory disease and protective inmmunity in the airways. It should be clear from this section that awareness that epithelial cells drive adaptive immune responses in the airways at the level of DCs, T cells and B cells has clearly risen significantly.

Enzymes and enzyme inhibitors Several epithelial-derived enzymes and their inhibitors participate in airway inflammation and are briefly mentioned here. Protease-activated receptors (PARs) are seven-transmembrane G protein-coupled receptors that are activated by protease cleavage (Sokolova & Reiser 2007). The proteolytic cleavage of the extracellular N-terminal domain of PAR by proteases present in inflammation such as tryptase, cathepsin G, elastase, and allergen proteases (i.e., dust mite proteases) exposes a new N-terminus that is the PAR’s own ligand. This new N-terminal ligand folds onto the extracellular loops of the PAR, activating the receptor. G-protein signaling then induces diverse responses. Airway epithelium expresses all four members of the PAR family, particularly PAR-1 and PAR-3. PAR-1 and PAR-2 transactivate epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), resulting in cell proliferation or migration (wound repair). PAR-2 is increased in airways of asthmatic individuals and seems to mediate allergen-induced airway hyperresponsiveness and eosinophil influx, although this is controversial. PAR-2 activation by serine proteases Der p3 and Der p9 from house-dust mite induces epithelial secretion of IL-6, IL-8, and CCL17/TARC. Matrix metalloproteinases (MMPs) depend on zinc to degrade matrix proteins (Elliott et al. 2007). There are 24 MMPs that function as collagenases, gelatinases, or stromeolysins. In homeostatic conditions they are inhibited by tissue inhibitors of MMP (TIMP) that maintain the balance of formation and destruction of the extracellular matrix. In asthma, the elevated ratio between MMP-9 (gelatinase B) and TIMP-1 correlates with airflow obstruction, although these products may originate from other cell types other than epithelial cells. MMP-9 is increased in epithelium of nasal polyps (Chen et al. 2007) and is secreted by epithelial cells

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infected with RSV or exposed to eosinophil peroxidase and MBP (Pegorier et al. 2006). Epithelial cells also express surface-bound proteinases of the ADAM family (disintegrin and metalloproteinase domain), which are membrane proteins containing a signal peptide followed by proprotein, zinc metalloprotease, disintegrin, transmembrane, and cytoplasmic regions. Most noteworthy in airway epithelial cells is ADAM17, or TNF-α-converting enzyme, which cleaves membrane-bound TNF-α and TGF-α, which in turn become soluble and mediate inflammation and mucin production, respectively. Genotype variations of ADAM33 have been associated with asthma (Holgate et al. 2006) and its airway expression increases as disease severity worsens (Foley et al. 2007), although its function is not completely understood. Besides TIMP, epithelial cells produce another enzyme inhibitor, neutral endopeptidase, which degrades endothelins (Goldie & Henry 1999), SP, and other peptides (Widdicombe 1991). Chitin is the second most abundant biopolymer in nature after cellulose (Elias et al. 2005). It is not produced in humans but is a component of the walls of fungi; the exoskeleton of crabs, shrimp and insects; the microfilarial sheath of parasitic nematodes; and the lining of the digestive tracts of many insects. When mammalians are infected or infested with these organisms, they produce acidic mammalial chitinase (AMC). Neutralization of AMC in an asthma mouse model attenuated Th2 inflammation and airway hyperresponsiveness by inhibiting chemokine production and the IL-13 pathway (Zhu et al. 2004).

Conclusion We hope that this review illustrates the diverse and essential roles played by epithelium in homeostasis as well as in host responses to the environment and in disease. The older concepts of epithelium as a relatively simple hydrated barrier have been supplanted by the recognition that epithelial cells are centrally involved in immunity and inflammation, airway physiology, and repair. Inasmuch as numerous severe diseases of the airways are characterized by abnormal or excessive activation of one or more of these processes in the epithelium, future investigations to better understand the function of epithelium will certainly pay dividends in the form of improved understanding of disease and new therapeutic strategies to alleviate airways disease.

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Airway Vascularity in Asthma John W. Wilson

Summary

Role in asthmatic responses

The airway circulation has many potential roles in asthma. The vasculature has a major influence on upper airway patency in nasal infection and atopic responses. Given its responsiveness to provocative stimuli in asthma and vasoconstrictive and antiproliferative response to corticosteroids, the bronchial circulation is likely to be a future target for novel asthma therapies.

The classical early asthmatic response to allergen is associated with the release of numerous preformed and newly generated inflammatory mediators capable of causing vasodilatation and capillary leakage. Edema formation is an important component of this response, and may be life-threatening when critical upper airway structures are involved. The nasal obstruction seen in susceptible individuals after aeroallergen exposure is a manifestation of highly reactive airway vessels responding with dilation and leakage, though difficult to detect in the stable state between episodes (Braunstahl et al. 2003). In addition, the airway vascular response accompanying viral infection may be significantly upregulated by vascular endothelial growth factor (VEGF) released by fibroblasts (De Silva et al. 2006). The cellular infiltration that is characteristic of the late asthmatic response is a complex process that involves mobilization of cells from bone marrow and vessel margination sites, adhesion to vessels in the local inflammatory environment, cell rolling and transmigration between endothelial cells (von Andrian & Mackay 2000). The cell infiltrate includes eosinophils, mast cells and activated Th2 lymphocytes (Djukanovic et al. 1990; Wilson et al. 1992; Jeffery 1999). The central role of the eosinophil in the pathogenesis of asthma is well described (Kay 2005). Regulation of eosinophil numbers in the airway may be achieved through a number of therapeutic strategies including antagonism of the trophic factor interleukin (IL)-5 (Menzies-Gow et al. 2003), the use of corticosteroids acting through multiple pathways (Djukanovic et al. 1992), as well as via effective inhibition of adhesion glycoproteins in the bronchial circulation (Wegner et al. 1990; Sedgwick et al. 2005). Vessels in asthma are therefore capable of acting through a range of mechanisms to promote the inflammatory response and airway remodeling typical of asthma (Fig. 17.1).

Introduction Increased vascularity of the bronchial wall is now recognized as a key feature of the tissue remodeling that is characteristic of asthma, (Li & Wilson 1997b; Bousquet et al. 2000). Originally described in the era of Leonardo da Vinci, the bronchial circulation has until recently been considered to be primarily a source of oxygenation and tissue nutrients for the airway wall (Mitzner & Wagner 1992; Widdicombe 1993), as well as thermoregulation and humidification of inspired gas. It is also recognized as a primary pathway for elimination of drugs. The bronchial circulation has specific functions of relevance to asthma through regulation of fluid shift into the submucosa, by attracting and controlling the migration of inflammatory cells into the airway wall, as well as being a source of microenvironmental factors for the regulation of tissue remodeling. It is reactive to multiple stimuli and is able to increase blood flow dramatically to provide effective thermoregulation in response to heat stress. Its responsiveness to chemical mediators of inflammation, capacity to participate in immunologic responses and ability to remodel in response to trophic stimuli make the airway circulatory bed both an important determinant of airway wall thickness and the acute changes in airflow characteristic of asthma.

Descriptions of airway vessels Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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Modern descriptions of airway vessels have been qualitative comparisons of findings in postmortem specimens. In these

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Airway vessels

Fig. 17.1 Role of bronchial vessels in asthma.

Angiogenesis

studies, vessel congestion, edema and airway fluid were characteristic findings (Dunnill 1960; Dunnill et al. 1969). Using rubber injection-digestion preparations, the rich network of vessels deep to the surface mucosa appears as a superficial plexus, which is joined to a deeper system of capacitance vessels through connecting perforator vessels (Widdicombe 1992, 1993). This is exemplified by the typical findings seen in animal models of the bronchial circulation in proximal airways (Fig. 17.2). Flow in the bronchial circulation can increase dramatically in heat stress, with unit flow of over 7 mL/min (Baile et al. 1985), compared with 4 mL/min in cardiac muscle and kidney or 1 mL/min in resting skeletal muscle (Schindt & Thews 1987). The combination of constriction of efferent vessels and increased flow causes congestion of the submucosal plexus, evidenced by vasodilatation and vessel leakage. The net effect of these changes is thickening of the airway wall (Corfield et al. 1991a). Confined by remodeled components of the bronchial wall, including intramural scar-type collagen (Li & Wilson 1997a), swelling in the submucosa further contributes to luminal narrowing in bronchoconstriction and loss of distensibility during inspiration, typical of asthma (Wilson et al. 1993). Direct evidence for hypervascularity and angiogenesis in asthma has come from bronchial biopsy studies in volunteers using anticollagen IV to detect vessels (Li & Wilson 1997b). Earlier studies describing inflammation used nonspecific

Dilatation

Permeability

Cell infiltrate

Mediators

stains that were not vessel specific and found no increase in vessel numbers in asthma (Beasley et al. 1989). The use of factor VIII antigen to detect vessels in postmortem and resected lung specimens found that the vessel contribution to the thickness of the airway submucosa in sections was 3.3% in asthma and 0.6% in controls (Kuwano et al. 1993). This marginal increase was assessed as being unlikely to be a significant contributor to thickening of the airway wall. A later study in postmortem specimens from fatal asthma also using factor VIII Ag, in which vessel congestion was maximal, found dilatation of larger vessels and vascularity of 10.2% compared with 6.6% in controls (Carroll et al. 1997). This study also concluded that the increase in vascularity was not contributory to bronchial wall thickening and, indeed, no increase in vascularity was seen in nonfatal asthma. Possibly, vessel markers such as factor VIII Ag and CD31 are indicators of vessel activation or maturity (Tanaka et al. 2001; Kinouchi et al. 2003). The method of measurement in histological material appears to be crucial to discern differences in airway vascularity. It is now apparent that collagen IV is likely to be a component of almost all airway vessels and does not define any specific subset (Baluk et al. 2003). Virtually all bronchial vessels have collagen IV as a basement membrane (Baluk et al. 2003). In mild asthmatics, it has been established that 17% of the submucosal section is covered in vessels with a density of 740/mm2 (Li & Wilson 1997b). These are both significantly greater than the 10% of submucosal area and density of 555/mm2 seen in control subjects (Fig. 17.3).

Airway blood flow

Fig. 17.2 Bronchial capillary bed in the proximal sheep airway. A, afferent arteriole; C, capillary; V, venule. (See CD-ROM for color version.)

The total volume of blood flow through a tissue bed is determined by resistance (vessel caliber) and perfusing pressure (blood pressure). Variations in these determinants can result in marked changes in tissue blood flow. Physiologic examples include skeletal muscle during exercise and erectile tissue after stimulation. Flow in the tracheal capillaries in heatchallenged dogs may be as high as 7 mL/min per unit volume, compared with maximal flow in cardiac muscle of 4 mL/min or skeletal muscle of 1 mL/min (Baile et al. 1985, 1987). The increase in flow in specific capillary beds occurs because of diversion in blood flow from deep capacitance vessels into more superficial capillaries (Widdicombe 1992). It is likely that the combination of increased flow and vasodilatation in these vessels leads to mucosal thickening (Corfield

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Inflammatory Cells and Mediators 1994). Although bronchopulmonary anastamoses are well described (Jindal et al. 1985; Charan et al. 1986), there do not appear to be significant changes in bronchial flow after lung parenchymal injury (Behera et al. 1995); however, the addition of positive end-expiratory pressure (PEEP) does reduce flow (Behera et al. 1995). Bronchial flow is both highly tissuedependent and reactive to provocative stimuli. It provides both a mechanism to regulate humidification and heat exchange as well as acting as a means to increase the thickness of the bronchial submucosa. The homeostatic role of the bronchial circulation in maintaining thermoregulation was initially made evident from studies in animal models (Baile et al. 1985; Solway et al. 1986; Manohar 1990). Saturation with water vapor increases as inhaled gas is heated to body temperature and hence thermoregulation is associated with net water loss (Hanna & Scherer 1986). This has been the subject of controversy in the study of exercise-induced asthma, where both heat and water loss may occur (Gilbert et al. 1987; McFadden 1992). One view is that exercise-induced asthma may be almost solely a vascular phenomenon (McFadden 1990). Heat loss occurs in association with the hyperventilation of exercise and water loss may cause mucosal hyperosmolarity, leading to secondary mast cell and eosinophil activation with spasmogenic mediator release (McFadden 1990; Anderson & Daviskas 1992; McFadden & Hejal 1995; Moloney et al. 2003; Anderson 2006). Of importance is the finding that heat loss from the airway in asthma is increased (Paredi et al. 2002), although reduced in chronic obstructive pulmonary disease where vascularity is not increased (Paredi et al. 2003). It is likely that the vitally important role of the airway vasculature in maintaining thermoregulation in smaller animals is less significant in humans, but that during hyperventilation heat and water losses are substantial. Local mast cell activation produces inflammatory mediators that cause bronchoconstriction, but may be inhibited by mast cell stabilizing agents (Spooner et al. 2003). The role of the vasculature appears to be crucial in maintaining airway surface conditions, thereby avoiding destabilizing of mast cells and promoting the removal of released mediators from the local microenvironment.

(a)

(b)

Impact of increased airway vascularity on airway caliber (c) Fig. 17.3 Photomicrograph of vessels: (a) asthma. (b) control. (c) cystic fibrosis (× 400). (See CD-ROM for color version.)

et al. 1991a). In addition, blood flow in the human trachea at rest has been estimated to be 7.2 mL/min, falling to 3.3 mL/min during Valsalva maneuver and increasing to 17.1 mL/min with the Muller maneuver (Breitenbucher et al.

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Early studies in severe and fatal asthma showed evidence of tissue swelling and vasodilatation (De Burgh Daly 1935; Renault et al. 1943; Vallery-Radot et al. 1950; Dunnill 1960; Dunnill et al. 1969; Li & Wilson 1997b). The association of increased airway vascularity with asthma has led to speculation regarding a causal relationship (Baier et al. 1985; Lockhart et al. 1992; Li & Wilson 1997b). There is compelling evidence for a causal relationship based on detailed

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Poor distensibility of airway lumen on inspiration

Submucosal swelling internal to rigid zone with edema Remodeled airway Limited response to bronchodilator or antiinflammatory therapy


Sheep and dog models do, however, differ from the human airway in having significantly denser subepithelial capillary networks (Widdicombe 1996). Additionally, airway smooth muscle constriction may itself cause increased local tissue pressure, causing impaired bronchial venous drainage and vessel dilatation. The site of airway vascular engorgement is important in relation to any potential effect on airflow obstruction. Proximal airways are larger with deep and superficial plexuses of vessels (Widdicombe 1992). Maximal flow regulation appears to occur in the postcapillary venules, resulting in proximal congestion. Additional flow limitation may occur in the arterioles during acute events (Salvato 2001). Together, this evidence demonstrates the response of the airway to fluid loading and vascular expansion, as well as providing a view of how submucosal swelling may add to factors that reduce airflow in asthma.

Fig. 17.4 Potential limitations on airway function caused by submucosal swelling. (See CD-ROM for color version.)

Vasodilatation and angiogenesis morphologic analysis of bronchial biopsies in asthma of varying severity (Vrugt et al. 2000) and extent of inflammatory response (Salvato 2001). Theoretical models taking into account the site and degree of mucosal swelling have contributed significantly to the understanding of the role of minimal increases in submucosal edema (Hogg et al. 1987). Moderate submucosal swelling in conjunction with smooth muscle shortening of 20– 40% can lead to profound airway narrowing in this model (Wiggs et al. 1990, 1992). Mucosal vascular congestion and edema may: (i) reactively contribute to airway wall thickening, (ii) impinge on the airway lumen through swelling internal to submucosal, scar-type collagen (Wilson & Li 1997) and smooth muscle hyperplasia (Ebina et al. 1993), (iii) cause loss of distensibility of the airway wall (Wilson et al. 1993), (iv) potentially reduce responsiveness to bronchodilators (Li & Wilson 1997), and (v) reduce clearance of locally active inflammatory mediators (Cabanes et al. 1989; Lockhart et al. 1992) (Fig. 17.4). In a series of studies in sheep, it has been shown that fluid loading, resulting in elevated left atrial pressure, caused airway wall thickening and luminal narrowing (Wagner & Mitzner 1990; Blosser et al. 1994). The effect of fluid loading with saline appeared to cause greater wall thickening than loading with blood, possibly because of transudation associated with lower oncotic pressure. The important observation of increased reactivity to methacholine in left ventricular failure further supports the view that bronchial vascular engorgement will alter the mechanics of airway narrowing to stimuli. It also gives credence to the concept of cardiac asthma being potentially responsive to diuretic therapy through fluid unloading. Earlier use of the sheep model has shown a significant increase in bronchial blood flow after allergen challenge in animals sensitized to Ascaris suum antigen (Long et al. 1988).

The expansion of the bronchial vasculature may occur through either neovascularization or vasodilatation, or potentially both mechanisms. Vasodilatation may occur following exposure to a wide variety of inflammatory mediators and pharmacologic agents (Table 17.1). Much evidence for the potential role of these factors is circumstantial, as they have in many cases been shown to be active in animal models, as well as having been shown to be present or relevant to human asthma. Early studies in bronchial biopsies from mild asthma suggested that larger vessels over 300 mm in diameter were increased in number (Li & Wilson 1997). Whether this was

Table 17.1 Vasodilating factors acting on airway vessels. Angiogenin (Delvigne & Rozenberg 2002; Distler et al. 2003) bFGF (Tiefenbacher & Chilian 1997) Bradykinin (Laitinen et al. 1986) Histamine (Alving 1991) Heparin (Compton et al. 2002) Hypoxia (Wagner & Mitzner 1988) Hyperosmolar stimuli (Widdicombe 1996) LTD4 (Bisgaard 1987) Methacholine (Laitinen et al. 1986) Nitric oxide (NO) (Barnes 1996) Platelet-activating factor (PAF) (Corfield et al. 1991b) PGD2 (Alving et al. 1991) Salbutamol (Laitinen et al.1987b) Substance P (Laitinen et al. 1986) Transforming growth factor (TGF)-b (Armstead et al. 1993) Tumor necrosis factor (TNF)-a (Patel et al. 2002) Tryptase (Dumitrascu 1996) VEGF, via NO and PGI2 (He et al. 1999) Vasoactive intestinal polypeptide (VIP) (Laitinen et al. 1986)

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due to stretch vasodilatation or nonsprouting proliferation of endothelial cells was unclear. Intrinsically, bronchial endothelial cells have little replicative activity. Currently, evidence suggests that angioblasts and hemopoietic progenitors may be derived from a common lineage, sharing surface markers including CD31, CD34, and a receptor for vascular endothelial growth factor (VEGFR-2) (Shalaby et al. 1995). Endothelial progenitors may be either local in origin, or migrate to sites of injury from the circulation. This process is dependent on attraction to local sites (chemotactic) and specific adhesion to activated vessel walls (von Andrian & Mackay 2000). Factors likely to be responsible include VEGF, fibroblast growth factor (FGF)-2, angiopoietin, stromal cell-derived factor-1, and insulin-like growth factor (IGF) (Hattori et al. 2001; Moore et al. 2001). The active production of angiogenic factors by local endothelial cells and their precursors appears to be a potent stimulus for remodeling of airway vessels and accompanies intense vascular remodeling such as that seen in cystic fibrosis (Solic et al. 2005). In the latter study, the role of storage sites of IL-8 was emphasized, when bound to glycosaminoglycan in airway cells. The process of angiogenesis proceeds along a sequence of regulated steps (Conway et al. 2006). In sprouting angiogenesis, breakdown of the extracellular matrix (ECM) occurs initially, followed by elaboration of chemotactic factors and extracellular proliferation. Lumen formation is accompanied by collagen deposition and vessel maturation. Matrix metalloproteinases (MMPs) perform multiple roles in this environment including the degradation of matrix (Visse & Nagase 2003; Chung et al. 2004; Conant et al. 2004; Jozic et al. 2005; Nagase et al. 2006) and release of growth factors from known ECM storage sites (Visse & Nagase 2003; Solic et al. 2005; Nagase et al. 2006). The leakage of plasma proteins then contributes to the migration and differentiation of angioblasts in extravascular sites. This process is tightly regulated by local tissue inhibitors of metalloproteinases (TIMPs), that act to counterregulate ECM breakdown as well as performing multiple immunologic roles (Visse & Nagase 2003).

Table 17.2 Factors and states known to stimulate angiogenesis. aFGF (Rogala et al. 2001) avb3 integrin (Drake et al. 1995) Angiogenins (Hoshino et al. 2001b) Angiopoietin-1 (Koblizek et al. 1998) bFGF (Folkman et al. 1988) Calcitonin gene-related peptide (CGRP) (Alving 1991) ELR + CXC chemokines IL-8 (Koch et al. 1992; Norrby 1997) ENA-78 (Gillitzer et al. 1996) GCP-2 (Addison et al. 2000; Keane et al. 2002) ECM (Ingber et al. 1987) ENA-78 (Donninger et al. 2003) Ephrin-B1 (Sawai et al. 2003) Ephrin-B2 (Murohara et al. 1998) Estrogens (Johns et al. 1996) GRO-a (Shibata et al. 2003) Hepatocyte growth factor (HGF) (Bevan et al. 2004) Histamine (Sorbo et al. 1994; Norrby 1997) Hypoxia and HGF-1 (Bevan et al. 2004) IGF-1 (Warburton et al. 2000) IL-1 (Li et al. 1995) IL-4 (Fukushi et al. 2000) IL-13 (Fukushi et al. 2000) IL-6 (Huang et al. 2004) Lipopolysaccharide (Mattsby Baltzer et al. 1994) LTC4 (Kanayasu et al. 1989) MMP (Haas et al. 1998; Foda et al. 1999) Neurokinin (NK)A (Martling et al. 1990) NO (Morbidelli et al. 1996) PAF (Murohara et al. 1998; Russo et al. 2003) Prostaglandins (Ruegg et al. 2004) Pulmonary arterial occlusion (Mitzner et al. 2000) Stromal cell-derived factor-1 (Hoshino et al. 2003) Substance P (McDonald et al. 1996) TGF-a (Norrby 1997) TGF-b (Roberts et al. 1986) TNF-a (Norrby 1997) Vascular cell adhesion molecule (VCAM)-1 (Fukushi et al. 2000) VEGF (Leung et al. 1989; Jakeman et al. 1993; Hoshino et al. 2001a) VIP (Laitinen et al. 1987b)

Drivers of angiogenesis The phenotypic expression of angiogenic factors and their receptors is counterregulated to allow appropriate tissue homeostasis without hypervascularity (Hoshino et al. 2001b; Favre et al. 2003; Russo et al. 2003; Thurston et al. 2005). However, the genetic polymorphisms in angiogenic factors or their receptors may also contribute to the wide variation in response seen in human disease (Thurston et al. 1998). A range of growth factors and local conditions play defined roles in inflammation and angiogenesis through effects on local cell activity and the endothelium (Ribatti et al. 2004; Khurana et al. 2005) (Table 17.2). Neovascularization and the remodeling of existing vessels are likely to be induced by

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multiple growth factors, including VEGF (Hoshino et al. 2001a), angiopoietin 1 (McDonald 2001), and stromal cell-derived factor-1 (Hoshino et al. 2003). Many counterregulatory factors inhibit angiogenesis. Amongst these, corticosteroids, angiostatins (O’Reilly et al. 1997), and TIMPs (Johnson et al. 1994) are significant in asthma (Table 17.3). VEGF exists in a number of well characterized isoforms, including vascular permeability factor (VPF) (Tammela et al. 2005; Voelkel et al. 2006). Evidence of increased levels of VEGF has been found in the bronchial submucosa in asthma (Hoshino et al. 2001a), and sputum levels have been found to correlate with disease activity (Abdel-Rahman et al. 2006). Airway vascularity was found to be dependent on gene

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Table 17.3 Antiangiogenic factors relevant to asthma. Angiopoietin-2 (Maisonpierre et al. 1997; Holash et al. 1999; Kim et al. 2000) Angiostatin (O’Reilly et al. 1994) Anti-VCAM-1 (Fukushi et al. 2000) Anti-IL-8 antisera (Smith et al. 1994) Cancer therapy (Gourley & Williamson 2000) Corticosteroids (Smink et al. 2003) Endostatin (O’Reilly et al. 1997) IL-2 with histamine (Johansson et al. 2000) IL-12 (Voest et al. 1995; Yao et al. 2000) Immunization to CM101 (Fu et al. 2001) Monoclonal antibody blockade of VEGFR2 (Witte et al. 1998) MMP inhibition (marimistat) (Wada et al. 2003) Platelet factor 4 inhibits VEGF (Gengrinovitch et al. 1995) Prolactin (anti-VEGF) (D’Angelo et al. 1999) Suramin (Danesi et al. 1993) TIMP-1 (Johnson et al. 1994) TSP-1 (DiPietro et al. 1994) Thalidomide (Folkman 2001) Thrombospondin (Iruela-Arispe & Dvorak 1997; Tokunaga et al. 1999) Vasostatin (Yao et al. 2000) VEGFR-2 antibodies (Menrad et al. 1997) VEGFR2 blockade using VEGF-165 peptides (Siemeister et al. 1998; Piossek et al. 1999) TSP, thrombospondin.

expression of VEGF and its receptors flt-1 (VEGFR-1) and flk-1 (VEGFR-2) (Hoshino et al. 2001a). Given the potentially important role of VEGF in determining the extent of airway vascularity, identification of cellular origins becomes pivotal to understanding the regulation of production of this factor. A range of cells have been identified as sources in the airway, including smooth muscle (Alagappan et al. 2007), epithelium (Verhaeghe et al. 2007), mast cells (Chetta et al. 2005), macrophages, eosinophils, and CD34+ cells (Hoshino et al. 2001a) (Fig. 17.5).

Epithelium Mast cell

VEGF Poly

Eos

Vessel

EC Mf

Smooth muscle Fig. 17.5 Local sources of VEGF in asthma. EC, endothelial cell, Eos, eosinophil, Mf, macrophage; poly, polymorphonuclear leukocyte. (See CD-ROM for color version.)


VEGF, through the isoform VPF, increases vascular permeability and vessel fenestration (Roberts & Palade 1997); however, angiopoietin-1 has the opposite effect (McDonald 2001). Opposing functions of VEGF and angiopoietin-1 may signify complementary and coordinated roles in regulation of the bronchial vasculature. Fundamentally, regulation of vascular permeability by VEGF/VPF and angiopoietin-1 may be their most significant action in asthma, as nongenomic actions are more rapid than their more protracted effects on angiogenesis and vessel remodeling. The relative contributions of growth factors and inflammatory, vasoactive mediators remain undetermined. Identification of the inhibitory effect of angiopoietin and other factors on vascular leakiness raises the potential for new strategies for reducing airway obstruction in asthma and chronic bronchitis (McDonald 2001).

Bronchial vessels and the immune response Local microenvironmental factors are crucial in determining both susceptibility to vascular remodeling and the extent of angiogenesis. Major exogenous triggers of airway inflammation in asthma include viruses and inhaled aeroallergens, both of which are known to be associated with the production of angiogenic factors (Ribatti et al. 2004; Ghildyal et al. 2005). These stimuli elicit reciprocal immune responses, through elaboration of Th1 and Th2 cytokines. While there may be a genetic predisposition to bronchial hyperreactivity (Van Eerdewegh et al. 2002), the link to exaggerated Th2 responses characteristic of atopic asthma (Robinson et al. 1992) is currently less well defined. A link between airway inflammation and vessel remodeling may be understood by examining nonvascular actions of VEGF. Vascular endothelial growth factor levels are associated with increased severity of clinical asthma (Lee & Lee 2001), and may enhance Th2 lung inflammation murine asthma models (Lee et al. 2004). Together with the finding of VEGF production by mast cells (Ribatti et al. 2004), it is clear that there is a role in addition to its action on vessels in the effector arm of the early and late asthmatic responses. Regulation of the inflammatory response in asthma occurs through intermediary transcription factors such as NF-κB, that also signal gene transcription for angiogenic factors including IL-8, ENA-78, and GRO-α (Shibata et al. 2003). Indeed, endothelial cell cytokine production of angiogenic factors such as IL-8 is mediated by NF-κB (Tanner 2004). Treatment of asthma with inhaled corticosteroids may suppress NF-κB, and, coincidentally, local inflammation with angiogenesis (Orsida et al. 1999, 2001; Wilson et al. 2001). Cell infiltration, angiogenesis, and vessel leakage may also occur through the activation of structural cells, including fibroblasts, by viral infection that induces production of IL-8 and ENA-78 (Donninger et al. 2003; Ghildyal et al. 2005). Nasal obstruction during viral upper respiratory tract infections indicates the

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significance of this mechanism to cause luminal occlusion, independent of atopic status. With the stimuli of either infection or atopic inflammation, increased vascular surface area and endothelial fenestrations lead to increased plasma protein leakage. As well as elevated baseline leakage, remodeled vessels in the submucosa are abnormally sensitive to substance P, but not to plateletactivating factor or serotonin, implying that at least infection is associated with a selective upregulation of NK1 receptors on the vasculature (McDonald 2001). Inflammation and angiogenesis are codependent phenomena (McColley et al. 2000). The preparation of vessels with upregulated adhesion glycoprotein expression in an appropriate field, allows angiogenesis to arise from resident tissue progenitors, or alternatively from migrating angioblasts with endothelial lineage capacity.

Angioblasts and stem cells It has been known for some time that hemopoietic progenitors can be mobilized from bone marrow by inflammatory responses (Metcalf & Wilson 1976). Stem cells from an adult are generally classified as hemopoietic stem cells (HSCs) or mesenchymal stem cells based on their differentiation capacity. There are no specific markers for stem cells in the postnatal period. Nonetheless, HSCs used in bone marrow transplantation for hematologic malignancies and bone marrow failure are distinguishable by the presence of CD34 and CD45 surface markers (Reyes et al. 2002; Dao et al. 2003). These cells have been shown to include a subpopulation of progenitors that can differentiate from angioblasts into endothelial cells (Reyes et al. 2002; Young 2004), migrate to sites of ischemic injury in the heart, induce angiogenesis, and lead to adaptive tissue remodeling with improved cardiac function (Wollert et al. 2004). Mesenchymal stem cells may also act in a similar capacity (Vulliet et al. 2004); however, these CD34– cells may be preangioblasts capable of bidirectional CD34 expression (Dao et al. 2003). There are at least two lineages of cells with potential to contribute to angiogenesis, detectable in the peripheral circulation. Other components of airway remodeling have been shown to arise from mobilized, homing progenitors (Grove et al. 2004; Albera et al. 2005). Hemopoiesis and progenitor cell production in the bone marrow is stimulated by circulating factors elaborated as a byproduct of airway inflammation (Denburg 1998; Denburg et al. 2000). These mobilized cells may play an important role in determining chronic inflammatory responses in the airway. Typically this may be in infection, either as suppressive regulators of the immune response (Le Blanc et al. 2004), or as initiators of remodeling of different tissue components, including epithelium (Delplanque et al. 2000), collagen (Hashimoto et al. 2004), blood vessels (Kamihata et al. 2002), and smooth muscle (Yeh et al. 2003). It would seem likely

404

Angiogenesis Growth factors

CD34+ EC progenitor migration

EC proliferation

Homing to vessel

Vessel expansion/ sprouting

Angioblast proliferation

Angiogenesis

Angiogenesis

Fig. 17.6 Pathways to bronchial angiogenesis. (See CD-ROM for color version.)

that homing of angioblast progenitors to “prepared fields” is a mechanism of angiogenesis and a component of airway wall remodeling. Augmented mobilization of angioblasts through the use of granulocyte–macrophage colony-stimulating factor may improve graft function after pancreatic islet cell transplantation (Contreras et al. 2003), possibly because of the inflammatory microenvironment associated with the transplant, resulting in enhanced homing of angioblasts. In the absence of a “prepared field,” there is little possibility of homing and transmigration of circulating progenitors (Davies et al. 2002). Abrogation of homing through the use of integrin antagonists has been found to reduce airway hypervascularity and airway remodeling (Simper et al. 2002), and airway hyperresponsiveness in a primate model (Wegner et al. 1990). The relative contribution of migratory versus endogenous endothelial cell progenitors to angiogenesis is unknown at present (Fig. 17.6). The resolution of this issue may be key to novel strategies aimed at addressing remodeling associated with chronic inflammatory diseases.

Vessel leakage The morphologic description of edema in severe and fatal asthma is consistent with the role of the bronchial vasculature and the known actions of angiogenic factors, inflammatory mediators, and their counterregulators (Table 17.4). Observations in fatal asthma identified tissue expansion secondary to classical vasodilatation and plasma exudation (Dunnill 1960; Dunnill et al. 1969). Recognition of the importance of plasma extravasation has led to interpretations of the role of current asthma therapies in limiting airway edema based on animal studies (Barnes et al. 1990; Chung et al. 1990; Boschetto et al. 1991). Accurate quantification of plasma leakage in human asthma has been difficult to achieve; however, indirect assessment using plasma protein detection methods (Persson et al. 1986) has enabled quantitative assessments to be performed (Chu et al. 2001; Wilson & Wilson 2001). The action of corticosteroid therapy to limit extravasation has been inferred from levels of α2-macroglobulin in bronchoalveolar

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Table 17.4 Factors known to regulate microvascular leakage. Bradykinin (Laitinen et al. 1987b; Murohara et al. 1998) CGRP (Madeddu et al. 2001) Endotoxin (Grimminger et al. 1997) ET-1 (Eibl et al. 2002) Formoterol (Baluk & McDonald 1994) Histamine (Murohara et al. 1998) LTB4 (Bannenberg et al. 2004) LTC4 (Bureau et al. 1997) LTE4 (Joris et al. 1987) Mycoplasma (Chu et al. 2001) NKA (Baluk et al. 1999) NO (Murohara et al. 1998) O 2– (Dauber et al. 1991) PAF (Murohara et al. 1998) (Bjork & Smedegard 1983) PGI2 (Murohara et al. 1998) Salmeterol (Bolton et al. 1997) Substance P (Baluk et al. 1997; Van Rensen et al. 2002) Terbutaline (Svensjo et al. 1977) TNF-a (Koizumi et al. 2003) VEGF (Esser et al. 1998; Hippenstiel et al. 1998) VPF (Connolly et al. 1989)

lavage fluid from asthmatics (Nocker et al. 1999), and estimates have been made in noninvasive studies of induced sputum (Van Rensen et al. 2002; Kanazawa et al. 2007). Plasma leakage from vessels may be inhibited by βadrenergic agonists (Tokuyama et al. 1991; Bolton et al. 1997), corticosteroids (Bowden et al. 1994), and angiopoietins through regulation of the role of VEGF/VPF (Gamble et al. 2000; Koh et al. 2002; Makinde et al. 2006). Vessel dilatation cannot be easily separated from microvascular leakage, as they frequently increase under similar conditions. Leakage is an important component of the vascular response that enhances thickening of the airway wall.

Pharmacologic responsiveness Early information on the response of bronchial vessels to pharmacologic agents is available from canine models (Laitinen et al. 1986, 1987a). The β2-adrenergic agonists including salbutamol (Laitinen et al. 1987b) dilate vessels, while both corticosteroids (fluticasone propionate) and leukotriene receptor antagonists (monteleukast) are vasoconstrictors in the airway (Mendes et al. 2004). Using a soluble, inert gas uptake method, Wanner and colleagues have shown relative potencies of inhaled corticosteroids to cause bronchial vasoconstriction and found budesonide to have the greatest relative effect compared with beclomethasone dipropionate and fluticasone propionate in asthma (Mendes et al. 2003). The actions of known asthma treatments to modify airway blood flow


are relatively minor compared with their other known properties as antiinflammatory and bronchodilating agents.

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Fibroblasts and the Extracellular Matrix Lynne A. Murray, William G. Glass, Anuk M. Das and Geoffrey J. Laurent

Summary Chronic respiratory diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and asthma, collectively affecting more than 300 million people worldwide, place a huge burden on our health systems. A feature of these diseases, which currently remains poorly treated, is the destruction of normal airway and parenchymal structures and replacement with scar tissue, or fibrosis. We propose that the aforementioned changes not amenable to current therapies are integral to the pathophysiology of these diseases. The nature of this fibrotic response, characterized by proliferation of fibroblasts and myofibroblasts, is being challenged recently as it becomes clear that these cells may be derived from blood-borne precursors, or fibrocytes, as well as from transdifferentiation of resident cells, including epithelial cells. This chapter proposes that remodeling and fibrosis are central to normal host defense and are driven by multiple pathways including the coagulation and inflammation cascade. In disease settings, these pathways may proceed unabated, resulting in aberrant tissue function. For example, the pathways that give rise to key growth factors and cytokines including transforming growth factor (TGF)-β, thrombin and interleukin (IL)-13 are discussed. Furthermore, we describe the activation of resident cells including alveolar macrophages and epithelial cells as well as the processes that cause the cells to adopt a pathogenic phenotype. Finally, we review the opportunities arising out of research for better therapeutic intervention strategies that will either halt or potentially reverse fibrosis.

Introduction Fibrosis as a component of asthma Fibrosis with excessive accumulation of extracellular matrix (ECM) represents an element of airway remodeling and as such is part of the wider structural changes to lung archiAllergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

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tecture. The features of airway remodeling associated with asthma that contribute directly to an increase in airway wall thickness are subepithelial fibrosis, angiogenesis, and smooth muscle and mucous gland hypertrophy (reviewed by Cohn et al. 2004). The consequences of this airway wall remodeling include airway narrowing leading to lung function impairment (Paganin et al. 1996; Chetta et al. 1997; Bousquet et al. 2000; Cohn et al. 2004). Furthermore, inflammation and increased wall thickness is more pronounced in fatal asthma compared with mild/moderate asthma, suggesting an association between the degree of remodeling and disease severity (Carroll et al. 1993, 2002). Fibrosis is defined as the excessive accumulation of ECM proteins. Although the fibrosis observed in many pulmonary diseases may be an important part of host defense akin to collagen deposition during acute wound healing, we propose that the increased deposition of ECM proteins leads to pathophysiologic symptoms of chronic asthma, particularly those seen in chronic severe asthma. Significant differences in collagen type and deposition have been observed in the lung of asthma subjects. Types I and III are the predominant collagen types in the normal lung, with type I collagen the most abundant (Laurent 1986). The subepithelial tissues of asthmatics have been shown to contain significantly higher levels of collagen types I and III, contributing to reduced elasticity and compliance of the tissue (Wilson & Li 1997; Hoshino et al. 1998; Ward et al. 2001). Increased levels of collagen type V, fibronectin, and tenascin have also been reported to occur in the airways of chronic asthmatics (Laitinen et al. 1997; Hoshino et al. 1998; Cohn et al. 2002). In another study, although no changes in the extent of collagen I was observed, asthma subjects exhibited an abundance of collagens III and V and fibronectin in the thickened basement membrane compared with nonasthmatic subjects (Roche et al. 1989). Finally, although the total amount of elastic fiber appears to be unchanged in asthmatics (Godfrey et al. 1995), subepithelial elastin fibers of the airways are fragmented and fibers in the deeper layer are often patchy, tangled, and thickened indicating impaired ECM formation (Bousquet et al. 1996). These data suggest that the precise ratios of ECM components may not be identical

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between all asthma subgroups. Further, diversity is observed between patient groups depending on which area of the lung is evaluated.

Fibrosis and altered ECM turnover is seen in diverse lung disease pathologies Fibrosis associated with exuberant matrix turnover is also a feature in interstitial lung disease (ILD). The most common ILD is idiopathic pulmonary fibrosis (IPF), with an incidence of 3–29 per 100 000 (Coultas et al. 1994). IPF is a fibroproliferative disease characterized by excessive collagen accumulation in the lung, which ultimately leads to impaired gas exchange. Characteristic computed tomography (CT) findings of IPF include the presence of patchy heterogeneous lung fibrosis, which as the disease progresses often results in alveolar collapse, bronchiolectasis, and honeycombing (Dacic & Yousem 2003). As the underlying cause of IPF is unknown, many hypotheses have arisen as to the mechanisms of disease exacerbation and perpetuation. There is current debate as to whether IPF is due to repeated injury to the epithelium driving the fibrotic response. Another hypothesis suggests that chronic inflammation drives IPF. Both are likely important and further research is needed to dissect which of multiple pathways predominates in individual patients. Airway wall remodeling, characterized by emphysema (alveolar wall breakdown and airspace enlargement) and small airways fibrosis, is seen in the small airways of patients with chronic obstructive pulmonary disease (COPD). This raises an interesting phenomenon where excessive ECM breakdown and ECM deposition are occurring in adjacent sites in the same disease state. It is thought that the main pathologic alterations in the lungs of patients with COPD are due to impaired protease–antiprotease balance, resulting in dysregulated matrix degradation and generation (Shapiro 2003; Shapiro & Ingenito 2005). The extent of peribronchial fibrosis (which, like asthma, also occurs in the lamina reticularis of the basement membrane) increases with disease severity (Hogg et al. 2004; Chung 2005) and increased collagen and elastin deposition have also been observed in smokers with emphysema (Lang et al. 1994). COPD is hypothesized to be due to exposure to particulate matter such as occupational exposure or cigarette smoke. The increase in ECM found in the lungs of these patients may be due to pronounced activation of cells by the foreign particles, resulting in downstream inflammatory events mediating disease pathology. However, as has been hypothesized for IPF, the remodeling events may be an aberrant homeostatic process serving to protect the lung. This chapter explores the mechanisms involved in the generation and maintenance of the asthma-specific subepithelial fibrosis in the lamina reticularis of the basement membrane. The diversity in ECM components and how, when organized, their ultimate structure is central to tissue function

Fibroblasts and the Extracellular Matrix

is described. The main area of discussion with regard to the ECM focuses on the alteration and potential role the ECM may play during airway disease. The mechanisms by which the ECM signals to cells to regulate cellular function are also described. Further, the numerous cell types that directly and indirectly contribute to ECM generation, as well as how these cells often have altered phenotype and function during fibrotic disease states, is discussed. Other pulmonary diseases associated with fibrotic remodeling such as IPF and COPD are highlighted to better elucidate underlying mechanisms. Although the fibrosis associated with these diseases occur in distinct anatomic sites, we propose that common final pathways are likely in these apparently diverse diseases.

ECM and the basement membrane The ECM comprises a diverse group of proteins and glycoproteins that provide structural integrity and mechanical support for tissues and provides a meshwork for cell adhesion and motility. Cells are tethered to components of the ECM through cell–ECM adhesions mediated by a variety of cellsurface receptors such as integrins. The ECM can also bind growth factors and cytokines. This provides a readily available reservoir of mediators that may be released by either enzymatic activity or changes in the chemistry of the environment. Due to the diversity of the ECM components, this section initially focuses on each major component individually: collagen, elastin, proteoglycans (and glycosaminoglycans), laminin, and tenascin. It then describes how specific components of the ECM come together to mediate tissue integrity and function in the basement membrane.

Components of the ECM Collagen There are currently 28 types of collagen and they can be divided into fibrillar and nonfibrillar collagen subtypes. Fibrillar collagen, such as types I, II and III, forms banded fibrils and is the main constituent of connective tissue, cartilage, and bone. The basic unit of collagen is the triple helix, which consists of three polypeptide chains containing Gly-X-Y repeated amino acid sequences. Approximately every third X is proline and every third Y is hydroxyproline. Given the high concentration of hydroxyproline, this analyte is often used to quantitate the extent of collagen deposition in tissues. Collagen generation, with respect to amount and relative quantities of the different types, is site specific and is regulated at both the transcriptional and translational level, as well as being subject to posttranslational control. Ultimately, the physical properties of collagen depend on final structural assembly (Khoshnoodi et al. 2006). For example, tissues that are required to withstand high tensile strength such as tendons have a higher fibrillar to nonfibrillar collagen ratio.

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Collagen forms a scaffold for supporting cells and connects to cells directly through interactions with collagen receptors such as integrins α1β1 and α2β1 or the discoidin domain receptors, and indirectly via fibronectin–α5β1 and αVβ3 integrin bonds (Vogel et al. 1997; Vogel 1999). Other functions of collagen, besides providing tissue integrity, include activating platelets at sites of wound healing, thus triggering the formation of a hemostatic plug (Sixma & Wester 1977; Laurent 1986; Stassen et al. 2004). Furthermore, collagen can inhibit cell apoptosis in adherent cells thus promoting cell survival. This may be potentially either deleterious if the cell is contributing to aberrant pathogenesis such as fibrosis or be protective if the cell is maintaining homeostasis (Laurent 1986; Bijian et al. 2004; Sturm et al. 2004).

Elastin Elastic fibers are made up of a core of insoluble elastin and microfibrils predominantly consisting of fibrillin. Elastin fibers provide the required flexibility of lung tissue (Mithieux & Weiss 2005) and are essential for lung development, as elastin gene-deficient mice fail to develop adequately branched terminal bronchi and die 3 days postnatally (Dietz & Mecham 2000; Wendel et al. 2000). Fibrillin I-deficient mice also have severe airway malformation. In contrast, tight-skin mice, which have a duplication in the fibrillin I gene resulting in overexpression of fibrillin I, develop emphysema (Foster & Curtiss 1990). This suggests that both the elastin core and microfibril components of elastic fibers are required for normal lung development. Within the lung, elastin is produced by myofibroblasts and fibroblasts, endothelial cells, and smooth muscle cells (Foster & Curtiss 1990; Starcher 2000). Mechanical stress has been shown to upregulate elastin expression. Furthermore, mediators such as TGF-β1 and tumor necrosis factor (TNF)-α also increase elastin. Interestingly, although elastin expression declines after lung development, aberrant disorganized elastin production has been observed during IPF (Laurent & Tetley 1984).

Proteoglycans Proteoglycans are hydrophilic molecules consisting of a core protein with at least one of a diverse group of covalently attached glycosaminoglycan (GAG) chains that confer hydrophobicity by binding to water and cations (Hardingham & Fosang 1992). More than 20 genetically different species of core proteins have been identified (reviewed by Iozzo & Murdoch 1996). Proteoglycans are also found in cell membranes and thus may exert pleiotropic biological activities (Yanagishita & Hascall 1992; Iozzo & Murdoch 1996). The proteoglycans versican, biglycan, and decorin predominantly localize to the subepithelial layer, whereas perlecan is associated with the basal lamina (Huang et al. 1999; Johnson et al. 2004; de Medeiros Matsushita et al. 2005). Versican levels have been shown to be increased in a number of lung pathologies associated with fibrosis including

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IPF (Bensadoun et al. 1996). Interestingly, altered mass and composition of proteoglycans have been observed in the asthmatic lung (Huang et al. 1999; de Medeiros Matsushita et al. 2005). Patients with severe asthma also have increased versican and biglycan, which has been shown to correlate with disease severity (de Medeiros Matsushita et al. 2005). The GAGs most commonly found in the lung include heparan sulfate, hyaluronan (HA), chondroitin/dermatan sulfate, and heparin. GAGs can also act as docking stations for growth factors and cytokines, allowing retention of mediators locally and protection from proteolytic degradation and/or cleavage (Li et al. 2002). In disease models, alterations in GAG constituents have been observed, with the rank order of predominance being shown to shift from heparan sulfate to chondroitin/dermatan sulfate (Cantor et al. 1983). One of the GAGs widely distributed in the body is HA, which binds to various molecules including other matrix proteins such as versican (Laurent & Fraser 1986; LeBaron et al. 1992). Relatively low levels of HA are distributed ubiquitously in most tissues, although the amount of HA increases during wound healing responses (Hamann et al. 1995; Laurent et al. 1996). HA has been shown to promote cell migration and proliferation through binding to the cell-surface receptor CD44, which is expressed on many cell types including hematopoietic cells (Jalkanen 1987; Miyake & Kincade 1990; Hamann et al. 1995; Laurent et al. 1996; Teder et al. 2002). Increased levels of HA have been seen in the bronchoalveolar lavage (BAL) fluid of asthmatics and the level of HA correlates with disease severity (Bousquet et al. 1991).

Laminin Laminins are the predominant noncollagenous component of the basal lamina (Aumailley & Smyth 1998). They contribute to cell attachment, differentiation and cell motility, and have anti-apoptotic effects on cells through interactions with other ECM components such as collagen type IV (Wondimu et al. 2004). Furthermore, laminin expressed on epithelial cells has recently been shown to be essential in lung development and in epithelial cell differentiation, a process that may contribute to lung fibrosis and which is discussed further in the section on epithelial cells (Nguyen et al. 2005).

Tenascin Tenascin is another ECM glycoprotein which is upregulated during morphogenesis and tissue repair (Mackie et al. 1988; Sechler et al. 1998). Increased levels of tenascin have been observed in the bronchial lamina reticularis in various asthma subgroups such as patients with chronic asthma or seasonal asthma, in comparison with control patients (Laitinen et al. 1997). No correlation was reported between the number of eosinophils or lymphocytes and the level of tenascin expression, indicating that tenascin levels may correlate with the extent of tissue remodeling as opposed to inflammatory events or exacerbations (Laitinen et al. 1997). The potential

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contribution of eosinophils and lymphocytes to fibrosis is discussed later in this chapter (see section Indirect mediators of fibrosis).

The basement membrane During airway remodeling in asthma, it is collagen accumulation in the lamina reticularis layer of the basement membrane that leads to the characteristic subepithelial fibrosis associated with the disease (Roche et al. 1989; Brewster et al. 1990; Evans et al. 1993). The structure of the basement membrane is maintained through interactions with ECM components, for example homeotypic cross-linking between collagen types or proteoglycans. Specific cell–matrix interactions can regulate cell functions such as proliferation, differentiation, and survival and these are discussed further in this chapter. The true basement-membrane zone appears as three component layers: the lamina lucida, lamina densa, and lamina reticularis (Fig. 18.1). Together, the lamina lucida and lamina densa make up the basal lamina. The lamina lucida functions as the region of attachment between the epithelium and lamina densa and contains cell adhesion molecules and anchoring filaments of laminin. In contrast, the lamina densa is predominantly composed of collagen type IV, laminin, entactin, and heparan sulfate proteoglycans. On the ECM side of the lamina densa, anchoring fibrils of type VII collagen loop through strands of collagen in the lamina reticularis (Merker 1994). The lamina reticularis is variable in distribution, thickness, and composition. It forms a complex structure within the tracheal basement-membrane zone, under the respiratory epithelium (Evans et al. 2000). The lamina reticularis is especially pronounced under the respiratory epithelium of large conducting airways, where it may be several micrometers thick and becomes thicker as the airway increases in diameter. Immunohistochemical studies have shown that the collagen fibrils of the lamina reticularis consist of types I, III, V, VI, and VII collagen. Within and around these collagen fibrils are fibronectin, tenascin, and proteoglycans (Merker 1994).


ECM-producing cells Most cells in the body have the ability to produce matrix proteins (Fig. 18.2). The major ECM-producing cells are fibroblasts and myofibroblasts, although smooth muscle cells also generate large quantities of ECM (Dabbagh et al. 1998). The derivation of both fibroblasts and myofibroblasts is currently under examination and reports indicate that there may be multiple pathways through which fibroblasts and myofibroblasts are derived. For example, bone marrow-derived circulating collagen I-positive cells, or fibrocytes, have been shown to traffic to sites of active lung fibrosis (Quan et al. 2006). These cells have been hypothesized to differentiate into fibroblasts or myofibroblasts (Quan et al. 2004). In addition, studies in IPF patients have also highlighted the airway epithelium as being a source of fibroblast-like and myofibroblastlike cells during remodeling (Willis et al. 2005). The magnitude of these pathways during fibrosis is currently uncertain. These cell differentiation pathways are discussed below.

Fibroblasts Fibroblasts produce numerous matrix proteins including collagen, proteoglycans, and glycoproteins (Wahl et al. 1978; Hibbs et al. 1983; Derdak et al. 1992; Sheppard & Harrison 1992). In the lung they are associated with the ECM in the subepithelial layer, conducting airways, and also in the interstitial space of the lung parenchyma. They also interact with airway epithelial cells; this interaction may be important in disease settings and is discussed later in this chapter. Fibroblasts play a key role in ECM homeostasis. For example, they generate matrix metalloproteinases (MMPs), which break down collagen thereby maintaining homeostasis of the subepithelial basement membrane. However, the pleiotropic functions of fibroblasts, including the production of many cytokines and growth factors, may contribute to the perpetuation and maintenance of the aberrant fibrotic environment (Bergeron et al. 2003).

Lamina lucida

Basal lamina

Lamina densa

Basement membrane zone

Lamina reticularis Fig. 18.1 Schematic of the anatomic distribution within the basement membrane zone. The basement membrane consists of three layers: the lamina reticularis, lamina densa, and lamina lucida. Each layer contains multiple different extracellular matrix components that provide tissue architecture and strength, as well as influencing cellular function. (See CD-ROM for color version.)

ECM components of the lamina densa: Collagen type IV, heparan sulfate, laminin, entactin Collagen type VII ECM components of the lamina reticularis: Collagen types I, III, V, VI, VII; Fibronectin; Tenascin; Proteogyclans Epithelial layer

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Inflammatory Cells and Mediators Fibrocyte Recruitment and Accumulation Chemokines (e.g. MCP-1/CCL2, SDF1a/CXCL12)

Fibrocyte Differentiation Growth factors (e.g. TGF-b1) Fibroblast Activation Proliferation and Survival

Myofibroblast Activation and Survival Differentiation (e.g. TGF-b1, thrombin, IL-13)

Epithelial to Mesenchymal Transition Growth factors (e.g. TGF-b1)

Epithelial Cell Injury

Fibroblasts are activated by numerous signals: mechanical forces imposed during bronchoconstriction, matrix interactions, hypoxia, growth factors such as TGF-β1 and platelet-derived growth factor (PDGF), and cytokines including the type 2 cytokines IL-4 and IL-13 (Desmouliere et al. 1993; Doucet et al. 1998). Fibroblasts are capable of generating chemokines, including macrophage chemotactic factor (MCP)-1/CCL2, RANTES/CCL5, and eotaxin/CCL11 (Hogaboam et al. 1999; Teran et al. 1999; Wenzel et al. 2002; Chibana et al. 2003). Indeed, the Th2-associated cytokines IL-4 or IL-13 synergize with TGF-β to further enhance the secretion of proinflammatory mediators such as eotaxin/CCL11 from human airway fibroblasts in vitro (Doucet et al. 1998; Wenzel et al. 2002), demonstrating the interactions between the proinflammatory and profibrotic processes in the lung. Further, lung eosinophilia has been demonstrated to correlate with the extent of subepithelial fibrosis (Wenzel et al. 1999), again emphasizing the potential links between inflammation and fibrosis. The aberrant fibrotic response associated with diseases may be due to phenotypic alterations in fibroblasts (Scaffidi et al. 2002; Moodley et al. 2003). These studies have been performed with fibroblasts isolated from the lungs of patients with IPF. IPF-derived fibroblasts are more resistant to apoptosis, which is partly mediated through a TGF-β-independent pathway (Scaffidi et al. 2002). These cells also have low levels of cyclooxygenase (COX)-2 activity and an inability to upregulate COX-2 that may impact the proliferative capacity of fibroblasts (Moore et al. 2003). Further COX-2-deficient mice are more susceptible to bleomycin-induced lung fibrosis due to loss of the regulatory functions mediated by this component of the arachidonic acid cascade (Keerthisingam et al.

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Fig. 18.2 Derivation and potential differentiation of matrix-producing cells. Fibroblasts and myofibroblasts are the major sources of collagen within the lung. During fibrotic pathologies such as asthma and idiopathic pulmonary fibrosis, the extent of collagen production increases. This is due to heightened fibroblast and myofibroblast activation and number, as well as the recruitment of bone marrow-derived fibrocytes and differentiation of epithelial cells into a collagen-producing phenotype. See text for definition of abbreviations. (See CD-ROM for color version.)

2001; Hodges et al. 2004). Fibroblasts from a profibrotic environment exhibit altered responsiveness to growth factors, express enhanced receptor levels for chemokine receptors such as CCR7 and cytokines such as IL-4 and IL-13, and secrete a different array of mediators including MCP-3/CCL7 (Hogaboam et al. 1999; Choi et al. 2004, 2006; Jakubzick et al. 2004a,b; Renzoni et al. 2004). Another functional difference in fibroblasts derived from the lungs of IPF patients is the extent to which this cell type can modulate angiogenesis. Lung tissue sections from IPF patients express increased levels of the angiogenic chemokine IL-8/CXCL8 and decreased levels of the angiostatic chemokine IP10/CXCL10, thereby suggesting an imbalance in net angiogenesis in the lungs of IPF patients. Interestingly, IPF patientderived fibroblasts express increased levels of IL-8/CXCL8, suggesting that the fibroblast is the main effector cell causing the angiogenic imbalance (Keane et al. 1997). Reports dating back to 1952 have indicated that fatal asthma is associated with dilated, congested blood vessels (Walzer & Frost 1952; Dunnill 1960). Vascular changes often occur early in the disease process (Orsida et al. 1999) and studies have suggested that the extent of angiogenesis parallels the severity of asthma (Vrugt et al. 2000). Due to the other pathologies associated with severe asthma, such as thickening of the airways, an increase in vascularization is required to support the increased tissue mass. However, the extent and function of angiogenesis and whether angiogenesis is a major contributing factor to disease severity remains to be elucidated. Increased angiogenesis in the bronchial vasculature in asthma may contribute to the increased interstitial edema due to microvasculature leakage. Also, an increase in vessel number may

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increase leukocyte infiltration into the lung, thus perpetuating the inflammatory cascade in the lung. Interestingly, phenotypic differences have also been observed between fibroblasts isolated from mice with Th1-biased or Th2-biased experimentally induced granulomas (Hogaboam et al. 1999). Pulmonary fibroblasts isolated from the Th2 fibrotic environment expressed increased levels of MCP-1/ CCL2 and its receptor CCR2. These fibroblasts also expressed increased levels of procollagen mRNA when stimulated with MCP-1/CCL2, in comparison with Th1 environment-derived fibroblasts (Hogaboam et al. 1999). A better understanding of the phenotypes of disease-associated fibroblasts may highlight pathways specific to disease pathologies giving rise to targeted therapeutics.

Myofibroblasts Myofibroblasts were first described by Gabbiani and colleagues as cells central to wound healing (Gabbiani & Badonnel 1976). As has already been alluded to, the presence of myofibroblasts in the lung is characteristic of aberrant fibrotic remodeling and IPF (Desmouliere & Gabbiani 1995; Phan 2002). They are “smooth muscle-like” cells as myofibroblasts are morphologically similar to fibroblasts; however, they also express α-smooth muscle actin (α-SMA) fibers (Ohta et al. 1995). The actin filaments result in myofibroblasts having a contractile phenotype, which at sites of wound healing serves to close the wound. Myofibroblasts are rarely seen in the normal lung, although the presence of myofibroblasts is characteristic of fibrotic remodeling and IPF (Adler et al. 1989; Mitchell et al. 1989; Kuhn & McDonald 1991; Pache et al. 1998). However, the presence of contractile myofibroblasts in the interstitium of the lung may cause either retraction of parenchymal tissue, resulting in alveolar collapse and creating the characteristic honeycombing observed in the lungs of IPF patients, or add to the increase in alveolar size, which is characteristic of COPD (Dacic & Yousem 2003). There is also a significant increase in myofibroblast numbers in the lungs of asthmatic patients following allergen challenge (Gizycki et al. 1997). An increase in the number of myofibroblasts in the mucosa of asthmatics has been shown to correlate with subepithelial collagen deposition (Brewster et al. 1990). Indeed, during fibrotic pathologies, myofibroblasts have been shown to be the predominant contributors to ECM deposition and structural remodeling (Phan 2002). For example, colocalization of myofibroblast α-SMA and procollagen I at the mRNA level has been reported by in situ hybridization, indicating that these cell types are a significant source of collagen during pulmonary fibrosis (Zhang et al. 1994). Additionally, myofibroblasts also produce elastin, fibronectin, and laminin and they are significant sources of chemokines and growth factors (Baur & Parks 1983; Singer et al. 1984; Zhang et al. 1994; Weill et al. 1997; Thannickal et al. 2003). Given the importance of myofibroblasts in the fibrotic state, defining both the origin and mechanisms leading to the


clearance of these cells will greatly add to our understanding of the role of this cell in driving fibrosis. Myofibroblasts express a panel of markers and these markers have been correlated with site of derivation. For example, myofibroblasts found in the peripheral and subpleural regions of fibrosis express α-SMA, vimentin, and desmin, whereas cells found in other regions of the lung do not express desmin (Zhang et al. 1994). This suggests that there may be differential sources of myofibroblasts and indeed multiple potential processes have been described. Fibroblast-to-myofibroblast transdifferentiation can be induced in vitro by TGF-β1 stimulation of fibroblasts and it has been hypothesized that TGF-β1 found locally at sites of fibrosis will differentiate resident fibroblasts into myofibroblasts (Desmouliere et al. 1993; Chambers et al. 2003). The environment at sites of fibrotic remodeling also contains a variety of other soluble factors, such as type 2 cytokines which have also been shown to potentiate fibroblast differentiation (Mattey et al. 1997). At sites of normal wound healing, once sufficient matrix has been deposited, fibroblasts and myofibroblasts undergo apoptosis (Darby et al. 1990; Desmouliere et al. 1995). This serves to limit the excessive deposition of ECM and also dampen the proinflammatory and profibrotic milieu. However, myofibroblasts persist in the IPF lung and this may be due to the profibrotic environment as TGF-β1 promotes IPF lung-derived myofibroblast survival (Kuhn & McDonald 1991; Zhang & Phan 1999). These cells generate mediators such as TNF-α and IL-1α that may drive epithelial cell apoptosis yet prevent eosinophil apoptosis (Zhang et al. 1996). Thus, myofibroblasts appear to have an integral role in perpetuating the inflammatory processes involved in asthma and fibrosis. Another possible source of myofibroblasts in the fibrotic lung is the epithelium, through a process of epithelial-tomesenchymal transition (EMT) (Kalluri & Neilson 2003). Interestingly, this process was also alluded to in 1976, with epithelial cells being described as differentiating into spindlelike myofibroblasts during reepithelialization (Gabbiani & Badonnel 1976). Overall, EMT describes a process where resident epithelial cells can be induced, through stimulation with TGF-β1, to transition into a mesenchymal phenotype, thus changing their properties such as the extent of ECM generation and enhanced motility (Okada et al. 1997). The hypothesis that EMT may be driving fibrosis was first described in vivo at sites of renal fibrosis and is discussed later in this chapter (Okada et al. 1997). Another mechanism by which myofibroblasts may arise is due to differentiation of recruited bone marrow-derived collagen I-positive cells, commonly referred to as “fibrocytes” (Epperly et al. 2003; Direkze et al. 2004; Forbes et al. 2004; Hashimoto et al. 2004; Deb et al. 2005; Yamaguchi et al. 2005; Quan et al. 2006).

Fibrocytes Circulating type I collagen-positive (collagen I+) cells have been identified as having a role in collagen deposition during

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remodeling pathologies (Quan et al. 2006). A variety of extracellular and intracellular markers have been used to identify fibrocytes. Fibrocytes express CD45, indicating hematopoietic origin. They also express stem cell markers, for example CD34 or CD13, as well as intracellular procollagen I. Furthermore, fibrocytes have been shown to express multiple chemokine receptors including CCR2, CCR7, and CXCR4 (Hashimoto et al. 2004; Phillips et al. 2004; Moore et al. 2006). Inhibition of the respective chemokine ligands in mouse models of fibrosis inhibits fibrocyte recruitment and ultimately reduces collagen deposition (Hashimoto et al. 2004; Phillips et al. 2004; Moore et al. 2005, 2006). Interestingly, fibrocytes also express major histocompatibility complex (MHC) class II molecules and have been shown to be able to present antigen to naive T cells, indicating that these cells may function as antigen-presenting cells at sites of fibrosis (Chesney et al. 1997, 1998). Fibrocytes have also been hypothesized to be a circulating progenitor cell in that they have pleuripotent potential to differentiate into other cell lineages, as has been demonstrated with fibrocyte-derived adipocytes (Hong et al. 2005). These circulating collagen I+ cells, or “fibrocytes,” were initially described at sites of wound repair (Bucala et al. 1994). However, fibrocytes have been demonstrated to generate a variety of ECM components and cytokines both in vitro and at sites of tissue remodeling (Chesney et al. 1998; Yoshida et al. 1999; Abe et al. 2001; Phillips et al. 2004; Hong et al. 2005; Moore et al. 2006). In particular, these cells have been demonstrated to secrete other matrix proteins, including type I and type III collagen as well as fibronectin (Bucala et al. 1994; Quan et al. 2006; Varcoe et al. 2006). Fibrocytes may also play a role in tissue remodeling through the production of MMPs (Hartlapp et al. 2001). Moreover, it has recently been postulated that an overexuberant recruitment of these cells to sites of pulmonary injury contributes to the aberrant deposition of collagen, which ultimately induces pathologic fibrosis (Bucala et al. 1994; Chesney et al. 1998; Chesney & Bucala 2000; Abe et al. 2001; Schmidt et al. 2003). It has been suggested that fibrocytes have the ability to differentiate into myofibroblasts, although this is not consistently reproducible (Jester et al 1987; Delanian et al. 1998; Schmidt et al. 2003). In vitro stimulation of fibrocytes with TGF-β1 results in the cells transitioning into a myofibroblast phenotype and producing fibronectin and type III collagen (Schmidt et al. 2003). Using an adoptive transfer model of bone marrow cells from green fluorescent protein (GFP) transgenic mice into mice challenged with intratracheal bleomycin (to initiate lung injury), it was demonstrated that recruited GFPpositive fibrocytes differentiated into fibroblasts and resident lung fibroblasts differentiated into myofibroblasts (Hashimoto et al. 2004). Further studies are necessary to delineate the fate of fibrocytes and the total impact of this pathway to fibrogenesis. There is increasing evidence that fibrocytes may also have a pathogenic role in asthma (Schmidt et al. 2003; Phillips et al.

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2004). A correlation in the number of fibrocytes in the basement membrane of asthmatics and the extent of subepithelial fibrosis has been described (Nihlberg et al. 2006). Increased numbers of bone marrow-derived fibrocytes have been reported in the airways of asthmatics, and allergen challenge further induced CD34+ cell recruitment to the subepithelial region (Schmidt et al. 2003). Interestingly, CD34 colocalized with α-SMA, suggesting that CD34+ fibrocytes traffic to sites of active remodeling and differentiate into myofibroblast-like cells (Schmidt et al. 2003). The mobilization of fibrocytes in repair during other diseases has also been demonstrated with burn patients (Yang et al. 2002). Adherent cells isolated from the peripheral blood of patients with severe burns could be rapidly induced to a fibrocyte phenotype in vitro and the peak number of these potential “pre-fibrocyte” cells occurred 3 weeks after injury, which was also when peak circulating TGF-β1 levels were detected (Yang et al. 2002).

Epithelial cells Alterations in epithelial cell phenotype to a more mesenchymal phenotype, i.e., the process of EMT, is a concept that is emerging for IPF and pulmonary disorders associated with fibrotic remodeling. One of the causative factors hypothesized to be driving IPF is repeated injury to the epithelium, which cannot regenerate adequately to cover denuded surfaces and leads to epithelial cell hyperplasia (Hyde et al. 1992; Gross & Hunninghake 2001). IPF epithelial cells have altered synthetic capacity and produce increased levels of profibrotic factors such as TGF-β (Khalil et al. 1996). This finding has also been correlated with disease progression in that advanced IPF lesions have increased TGF-β1 expression compared with epithelial cells from early lesions (Khalil et al. 1996). EMT describes a process by which epithelial cells undergo phenotypic transition to fully differentiated mesenchymal cells, such as fibroblasts and myofibroblasts (Zavadil et al. 2004; Zavadil & Bottinger 2005). The differentiation of airway epithelial cells of one type to another, for example type I pneumocytes transitioning into goblet cells, has been previously described (Danto et al. 1995; Borok et al. 1998; Torday et al. 2003; Kim et al. 2006). However, the switching of an epithelial cell into a phenotype that moves beyond the original cell’s embryonic lineage has only recently been hypothesized as a driving factor in fibrosis (Kalluri & Neilson 2003; Selgas et al. 2004; Valcourt et al. 2005). EMT is a dynamic process in which the epithelial cells must lose polarity, downregulate adhesion molecule expression, and upregulate the machinery necessary for motility (Zavadil et al. 2004; Zavadil & Bottinger 2005). It is a wellestablished phenomenon in tumor metastases, in that primary tumor cells change phenotype to become more motile and mesenchymal in phenotype and then engraft at sites distal to the initial primary tumor (Kang & Massague 2004). Recent publications indicate that epithelial cells at sites of

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fibrosis also transition into a mesenchymal phenotype following injury and this can facilitate the fibrotic state in various organs (Strutz et al. 1995; Ng et al. 1998; Iwano et al. 2002). This has been shown using cells in vitro and also immunohistochemical colocalization of epithelial and mesenchymal cell markers on the same cells (Willis et al. 2005). Epithelial cells that undergo EMT downregulate epithelialspecific markers such as E-cadherin and concomitantly acquire some of the fibroblast and/or myofibroblast-associated markers such as expression of fibroblast-specific protein (FSP)-1 or α-SMA (Okada et al. 1997; Jechlinger et al. 2002; Grunert et al. 2003). The idea of EMT promoting the fibrosis observed in asthma and IPF is rapidly evolving. Induction of EMT requires the presence of profibrotic growth factors such as TGF-β1 (Citterio & Gaillard 1994; Pagan et al. 1999; Grande et al. 2002; Xu et al. 2003; Zeisberg et al. 2003; Liu 2004; Strizzi et al. 2004; Aguilera et al. 2005; Demir et al. 2005; Han et al. 2005; Zavadil & Bottinger 2005; Ahmed et al. 2006; Gotzmann et al. 2006; Lee & Kay 2006; Sam et al. 2006; Yang et al. 2006). Epithelial cells exposed to TGF-β1, alone or in combination with other growth factors such as epidermal growth factor, begin the process of EMT through a loss of polarity and the increased expression of MMPs, which enable basement membrane degradation. The cells also undergo cytoskeletal changes as well as altered expression of surface molecules. For example, downregulation of E-cadherin and zona occludens (ZO)-1 with a concomitant upregulation of vimentin is needed for migration and transition to a mesenchymal phenotype (Iwano et al. 2002; Grunert et al. 2003). The majority of the work evaluating EMT has been performed in vitro, and the full extent of this pathogenic pathway in vivo is currently being evaluated. It is still not known whether EMT contributes to the excess ECM deposition. Future work correlating the time-course of EMT induction with disease staging will also be very insightful. This pathogenic process may provide novel therapeutic targets such that inhibiting or reversing EMT may provide clinical benefit to patients with lung fibrosis-associated diseases. In asthma there is also evidence of altered epithelial cell phenotype and function (Fahy 2001). Not only is there an increased goblet cell number in asthma (Fahy 2002), which may occur through differentiation of type I cells into mucusproducing type II cells (Hogg 1993), but epithelial cell shedding (sloughing) is also a characteristic feature of asthma (Chanez et al. 1999). This phenomenon may be due to shearing of the epithelium following bronchoconstriction or airway hyperresponsiveness, or may be due to a rapid proliferative rate of the epithelium in asthmatics (Montefort et al. 1993). Interestingly, the epithelium has been demonstrated to regulate fibroblast proliferation through the generation of mediators including prostaglandins and chemokines (Moore et al. 2003). Therefore, any alteration in epithelial phenotype may deleteriously affect fibroblast function.


There is emerging data indicating that bone marrow-derived progenitor epithelial cells may play a role in re-modeling. Resident progenitor epithelial cells in the lung have been hypothesized to maintain a homeostatic epithelial layer. In contrast, circulating progenitor epithelial cells are recruited to promote repair following excessive injury. Evidence for this has come from sex-mismatched human allograft transplants where recipient epithelial cells contribute toward the regeneration of donor lung epithelium (Kleeberger et al. 2003; Spencer et al. 2005). Further, using an animal model of tracheal transplantation, recipient bone marrow-derived epithelial cells repopulated the transplanted tracheas (Gomperts et al. 2006). Once recruited, the progenitor epithelial cells localized to the basement membrane and then migrated through the basement membrane to the apical epithelial layer where the cells further differentiated (Gomperts et al. 2006). Therefore, as with the literature surrounding fibrocytes, this body of work demonstrates the plasticity of bone marrowderived cells in reconstituting damaged ECM. Although this field is still emerging, this work does highlight a role for the epithelium in remodeling through epithelial–fibroblast communication as well as transition into a fibroblast-like phenotype via EMT.

Indirect mediators of fibrosis The lung contains numerous other structural and inflammatory cells that have been shown to be involved in the fibrotic response. These cell types are not responsible for directly generating collagen, although they have been shown to be indirectly contributing to fibrosis through the generation of other mediators such as growth factors and cytokines. This section outlines some of the cell types involved indirectly in fibrosis and the current understanding of how these cells drive fibrosis.

Airway smooth muscle Airway smooth muscle (ASM) cells have the ability to secrete large amounts of cytokines and chemokines such as RANTES/CCL5, MCP-1/CCL2, eotaxin/CLL11, and MCP-3/ CCL7 (Baraldo et al. 2003; Wuyts et al. 2003a,b; John et al. 2004; Peng et al. 2004). These chemokines and cytokines likely play a strong role in both the initiation and maintenance of chronic airway inflammation by recruitment and activation of immune cells (Murray et al. 2006). ASM cells have been shown to produce MMPs which, as discussed later in the chapter, can also act on the local ECM environment (Xie et al. 2005). Increased procollagen I gene expression has been demonstrated in ASM cells, although their ability to produce other ECM components is currently undefined (Dabbagh et al. 1998). There is also evidence indicating that ECM components themselves induce chemokine production by ASM cells and that this synthesis is increased in asthma (Johnson 2001; Black et al. 2003; Elshaw et al. 2004).

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Alveolar macrophages Alveolar macrophages have been widely examined in the pathogenesis of asthma. Several studies have demonstrated a significant positive correlation between macrophage activation and the severity of asthma (Duddridge et al. 1993; Woolley et al. 1995; Virchow et al. 1996). Further, allergen challenge has been demonstrated to induce the activation of these cells (Carroll et al. 1985; Viksman et al. 2002; Ferreira 2003; Lensmar et al. 2006). It is long established that alveolar macrophages have a role in lung fibrosis. One role for macrophages in tissue remodeling may be through the production of MMPs, other proteolytic enzymes and mediators that degrade local tissue but which also exacerbate inflammation and enhance mucus production (Page & Morley 1986; Page & Coyle 1989; Henderson et al. 2000; Maisi et al. 2002). Crucially, several reports have shown that macrophages are sources of profibrotic growth factors such as PDGF, connective tissue growth factor (CTGF), and TGF-β1 (Chanez et al. 1995; Lee & Lee 2001; Burgess 2005; Chetta et al. 2005; Howell & McAnulty 2006). Further, the extent of growth factor production is enhanced in alveolar macrophages isolated form the lungs of patients with ILD, as is the levels of chemokine production (Bitterman et al. 1982; Nagaoka et al. 1990; Zhang & Phan 1996; Hasegawa et al. 1999). Alveolar macrophages from asthmatic patients also secrete significantly more fibronectin and TGF-β compared with macrophages from nonasthmatic patients (Vignola et al. 1996). Interestingly, stimulating macrophages with the type 2 cytokines IL-13 or IL-4, which are commonly associated with atopic diseases, results in an “alternatively activated macrophage” (aaAM) phenotype that may be additive to the fibrotic environment (Fig. 18.3) (Gordon 2003). These

Classically Activated Alveolar Macrophage

Predominant Response

Nitric oxide, IL-6, TNF-a

TGF-b1, PDGF, TARC, IL-10, arginase, fibronectin Alternatively Activated Fig. 18.3 Predominant responses of classically activated and alternatively activated alveolar macrophages. Classical activation of macrophages by, for example, interferon (IFN)-g or microbial pathogens induces microbicidal activity such as increased generation of nitric oxide and TNF-a. In contrast, stimulation of alveolar macrophages with type 2 cytokines such as IL-4 or IL-13 results in an alternatively activated phenotype. This results in the production of profibrotic growth factors and extracellular matrix components. See text for definition of abbreviations. (See CD-ROM for color version.)

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aaAMs have been demonstrated to generate fibronectin, promote proliferation, and augment ECM generation by fibroblasts in vitro (Song et al. 2000). Several reports have indicated altered cytokine and growth factor production by aaAMs, including increased TGF-β1, Th2-associated chemokines TARC/CCL17 and MDC/CCL22 (Andrew et al. 1998; Bonecchi et al. 1998; Imai et al. 1999; Song et al. 2000; Lee, C.G. et al. 2001; Fichtner-Feigl et al. 2006). Another chemokine associated with aaAMs is PARC/CCL18 and interestingly the majority of alveolar macrophages isolated from the BAL fluid of IPF patients spontaneously release PARC/CCL18 (Prasse et al. 2006). Moreover, aaAM-derived PARC/CCL18 has been shown to feed back to fibroblasts to promote collagen generation (Prasse et al. 2006). Currently the receptor for PARC/CCL18 is unknown. Another characteristic marker for the aaAM phenotype is the presence of arginase. Arginase inhibits nitric oxide production in an autocrine manner, potentially inhibiting the nitric oxide host defense role of these cells and has also been reported to further promote ECM generation (Hesse et al. 2001). This early work suggests that alveolar macrophages may be central to the pathogenesis of IPF and fibrosis in general. Emerging evidence suggests a role for aaAM in fibrotic disease pathologies. Further studies establishing the role of aaAM in diseases associated with fibrosis are warranted to identify novel pathways amenable to therapeutic intervention.

T cells T cells have the ability to orchestrate a number of features of remodeling (reviewed in Kay 1997). The airways of patients with moderate, severe, and fatal asthma have increased numbers of T lymphocytes including both CD4+ and CD8+ T cells (Kline & Hunninghake 1994; Mazzarella et al. 2000; Redington et al. 2000; Tsoumakidou et al. 2004; van Oosterhout et al. 2004). T cell-derived cytokines, in particular IL-4 and IL-13, can mediate various pathologies associated with the initiation, stabilization, and progression of fibrosis (Fig. 18.4). The animal models of asthma and fibrosis show a strong presence of CD4+ cells in the lung and these cells are thought to participate in the fibrotic process through the secretion of Th2 cytokines such as IL-4, IL-5, and IL-13 (Wise et al. 1999; van Rijt et al. 2005). CD4+ T cells are required for eosinophil recruitment since CD4 gene-deficient mice did not exhibit pulmonary eosinophilia (Gonzalo et al. 1996). Although the extent of eosinophilia does not correlate with disease severity in this model, it is interesting to note that the eosinophilia observed in asthmatic patients correlates with certain features of airway remodeling such as the extent of ECM deposition and subepithelial membrane thickness (Wenzel et al. 1999).

Eosinophils The role of eosinophils in fibrosis may be twofold: through the release of soluble mediators and through cell–cell

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Monocyte

IL-13 Fig. 18.4 Effects of IL-4 and IL-13 on key features of fibrotic remodeling in the lung. IL-4 and IL-13, generated by Th2 cells, exert pleiotropic functions on a variety of cells within the lung. Reports indicate that these two cytokines have overlapping roles yet each exert the pathologic changes to different extents and this is represented with the shaded gradients. For example, IL-13 is more efficient at promoting the generation of alternatively activated macrophages, whereas IL-4 is more efficient at inducing isotype switching in B cells. These cellular responses are hallmarks of asthma and idiopathic pulmonary fibrosis and have been associated with disease progression and severity. AHR, airway hyperresponsiveness; EMT, epithelial to mesenchymal transition. See text for definition of other abbreviations. (See CD-ROM for color version.)


Alternatively Activated Macrophage TGF-b production

Fibroblasts

ECM generation Proliferation Differentiation in myofibroblasts

Smooth Muscle Th2 Cell

Hyperreactivity Hypertrophy

Eosinophil Activation Survival Lung infiltration Epithelium

Mucos production Goblet cell hyperplasia EMT

B Cell

Mast Cell IgE

IL-4

interactions. For example, the eosinophil granule proteins major basic protein (MBP) and eosinophil peroxidase (EPO) can directly damage host tissue, and eosinophil cationic protein (ECP) directly induces fibroblast ECM production (Heyneman et al. 1975; Dechatelet et al. 1978; Bozeman et al. 1990; Hernnas et al. 1992; Romano et al. 2000). Further, eosinophils are sources of TGF-β1 during fibrosis (Wong et al. 1993; Elovic et al. 1994; Minshall et al. 1997). In fact, increased numbers of eosinophils as well as their products are often found in patients with IPF (Reimert et al. 1994; Fujimoto et al. 1995; Kroegel et al. 1998) and this cell type has been demonstrated to directly activate fibroblasts and promote fibroblast proliferation through cell–cell interactions (Shock et al. 1991). Further, fibroblasts generate granulocyte–macrophage colony-stimulating factor (GM-CSF), which has been shown to promote eosinophil survival (Vancheri et al. 1989). IL-5, another Th2-associated cytokine, has been shown to be central to eosinophil recruitment, activation, maturation, and survival (Yamaguchi et al. 1988; Clutterbuck et al. 1989). Treatment of asthmatics with a neutralizing monoclonal antibody to IL-5 reduced the number of eosinophils in the airways and also impacted subepithelial fibrosis (Leckie et al. 2000). Critically, however, lung function in asthmatic patients was not improved following a single dose of the anti-IL-5 antibody (Leckie et al. 2000; Flood-Page et al. 2003; Menzies-Gow et al. 2003). However it is important to note that the investigators in the anti-IL-5 study reported that there was not complete inhibition of eosinophil numbers in the lungs (Flood-Page et al. 2003). Overall, the role of the eosinophil in fibrosis requires further evaluation (Kay et al. 2004).

Degranulation Histamine release AHR

Mast cells Mast cells are strongly implicated in the pathogenesis of asthma, because these cells are found activated in the lungs of asthmatics (Synek et al. 1996; Boyce 2003). Although the major role of the mast cell appears to be limited to degranulation following engagement of cell-surface IgE receptors, evidence suggests that the mast cell plays a larger role in asthma and fibrosis. For example, mast cells produce the proteolytic enzymes tryptase and chymase, which may promote aberrant tissue degradation (Boyce 2003). Mast cell-derived chymase can have numerous downstream activities including the cleavage of latent TGF-β1 into active TGF-β1 (Rothe & Kerdel 1991; Howard et al. 2004). Chymase has also been demonstrated to generate type I collagen peptides which, by the nature of these peptides, may exacerbate the proinflammatory milieu (Kofford et al. 1997; Wang et al. 2001). Use of mast cell chymase inhibitors has shown efficacy in animal models of lung fibrosis, thus highlighting the potential deleterious role of these cells in fibrosis (Tomimori et al. 2003).

Neutrophils Neutrophils appear to be an indicator of the severity of asthma as they are found in the airways of patients with severe or chronic asthma (Martin et al. 1991; Sur et al. 1993; Carroll et al. 1996). One potential role of neutrophils in driving fibrotic disease is through the degradation of ECM by proteolytic neutrophil-derived mediators. Neutrophil elastasedeficient mice are protected from bleomycin-induced lung injury that is partly mediated through decreased TGF-β generation (Dunsmore et al. 2001). Further, during disease exacerbations in asthma, IPF and COPD, the pulmonary localization

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of neutrophils may excessively cleave ECM components into profibrotic and proinflammatory peptides. Increased levels of neutrophil-recruiting CXCR1- and CXCR2-binding chemokines such as ENA78/CXCL5 and IL-8/CXCL8 have been described in the IPF lung (Keane et al. 1997, 2001). However, the consequences of elevated levels of these chemokines has been correlated with an increase in angiogenesis, which may worsen fibrotic disease manifestations as described earlier (Folkman 1995). The potential consequences of matrix degradation are discussed subsequently in the section Matrix metalloproteinases and matrix degradation.

Mediators of fibrosis There are a number of mediators of fibrosis that have been shown to activate fibroblasts, including growth factors and cytokines. One of the most potent and well-described growth factors is TGF-β. This cytokine is found upregulated in numerous fibrotic diseases and not only contributes directly to fibrosis by acting on fibroblasts but also induces the secretion of other profibrotic mediators such as PDGF and CTGF. Of the classic Th2 cytokines, both IL-4 and, to a greater extent, IL-13 have been shown to be profibrotic. IL-4 is profibrotic predominantly via an indirect mechanism, whereas IL-13 acts directly on fibroblasts and virtually every other cell type involved in asthma and fibrosis. We also discuss thrombin as a soluble mediator of fibrosis that mediates its fibrotic activities via two distinct mechanisms: initiation of coagulation and activation of its cell-surface protease-activated receptors. Finally, we highlight the importance of ECM homeostasis, which is mediated by various proteases and protease inhibitors.

TGF-b Three TGF-β isoforms, TGF-β1, TGF-β2 and TGF-β3, have been described (Coker et al. 1997; Chakravarthy et al. 1999). TGF-β1 appears to be the predominant isoform found in fibrotic lung tissue. The extent and expression pattern of TGF-β2 and TGF-β3 are comparable between nonfibrotic and fibrotic tissue (Khalil et al. 1996). TGF-β1 is upregulated in bronchial epithelial cells and alveolar macrophages in the IPF lung (Khalil et al. 1991, 1996; Coker et al. 1996). Numerous cell types generate and secrete TGF-β1 including lymphocytes, macrophages, eosinophils, epithelial cells, fibroblasts, and endothelial cells (Yoshida & Gage 1992; Okumura et al. 1997). While not thought to be the predominant source of TGF-β1 in the local tissue environment, platelets store and can secrete large amounts of this potent growth factor (LangRollin et al. 2001). TGF-β1 regulates numerous biological activities such as proliferation, apoptosis, and differentiation (Pinkas & Teicher 2006; Sanders et al. 2006). This protein also acts on cells to induce secretion of the profibrotic growth factors such as CTGF and PDGF (Kothapalli et al. 1998).

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Interestingly, TGF-β1 is a bimodal protein: it induces the proliferation of smooth muscle cells and fibroblasts at low concentrations but inhibits cell growth at higher concentrations through the induction of PGE2 (Battegay et al. 1990; McAnulty et al. 1997). TGF-β1 expression is increased in the lungs of asthma patients (Vignola et al. 1997; reviewed in Howell & McAnulty 2006). It has been further demonstrated that TGF-β1 is nearly absent in bronchial epithelial cells but is highly expressed in inflammatory cells beneath the basement membrane where subepithelial fibrosis predominates (Magnan et al. 1997). Eosinophils in the lungs of asthmatic patients have also been shown to have increased expression of TGF-β1 compared with control patients (Nomura et al. 2002; Kay et al. 2004). Moreover, TGF-β1 expression in the patients correlated with the observed increase in fibroblast number, as well as with thickness of the basement membrane (Minshall et al. 1997; Kay et al. 2004; Balzar et al. 2005). Transient overexpression of TGF-β1 or pulmonary delivery of this cytokine to mouse lungs induces a pronounced interstitial fibrosis mediated by aberrant ECM generation and deposition, as well as the presence of myofibroblasts (Sime et al. 1997). The in vitro profibrotic functionality of TGF-β1 and the association of TGF-β1 with fibrotic pathologies suggest that TGF-β1 is a central mediator of fibrosis (Bergeron et al. 2003). A polymorphism in TGF-β1 conferring greater production has been associated with disease progression but not with predisposition to developing IPF (Xaubet et al. 2003). Interestingly, this same polymorphism has also been associated with COPD (Wu et al. 2004). The polymorphism occurs at a lower frequency in COPD patients, suggesting that this particular genetic alteration may be protective (Wu et al. 2004). The emergence of therapeutics directed against the TGF-β pathway will shed light on the role of this growth factor in disease (see Table 18.1).

IL-13 and IL-4 IL-13 and IL-4 are two pleiotropic Th2-associated cytokines, with numerous distinct and overlapping functions (see Fig. 18.4). IL-13 is elevated in the lungs of IPF patients and is associated with fibrotic pathologies and aberrant remodeling at various tissue sites (Hancock et al. 1998; Wynn 2004). IL-13 is produced predominantly by T cells and exerts the majority of its biological effects via a heterodimeric receptor composed of IL-4Rα and IL-13Rα1 (Schnyder et al. 1996; Akbari et al. 2003; Wynn 2003). The profibrotic role for IL-13 in asthma and pulmonary fibrosis has been reviewed (Finkelman et al. 2004; Wills-Karp 2004). IL-4 is a potent cytokine that shares many functions with IL-13, which is not surprising given that IL-4 and IL-13 share the common IL-4Rα receptor subunit (Wynn 2003; Finkelman et al. 2004; Wills-Karp 2004). IL-4 is also secreted predominantly by Th2

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cells and is a characteristic feature defining CD4+ T cells as Th2 cells (Mosmann & Coffman 1989). There are differences in the downstream events following IL-4 or IL-13 signaling as these two cytokines have overlapping but not redundant function. For example, IL-4 more potently induces B-cell proliferation as well as immunoglobulin production and class switching to expression of IgE, which in turn activates mast cells by cross-linking the Fcε receptor (Cocks et al. 1993). In contrast, IL-13 activates epithelial cells and goblet cells, causing mucus production, goblet cell hyperplasia, and EMT (Atherton et al. 2003; Yasuo et al. 2006). Moreover, IL-13, to a greater extent than IL-4, stimulates epithelial cells to generate and secrete eotaxin/ CCL11 and MCP-1/CCL2, chemoattractants for eosinophils and monocytes respectively (Pope et al. 2001; Moore et al. 2002; Ip et al. 2006). IL-13 also induces eosinophil activation and promotes survival, a function not yet attributed to IL-4 (Luttmann et al. 1996). Various animal models of fibrosis have indicated a more profibrotic role for IL-13 than IL-4. One study using pulmonary-specific IL-13 transgenic overexpression demonstrated many hallmarks of asthma and IPF (Zhu et al. 1999). There was an increase in subepithelial fibrosis, excess mucus production, and pronounced pulmonary inflammation (Zhu et al. 1999). IL-13 has also been shown to be involved in the maintenance of lung fibrosis. Selective depletion of IL-13Rα1-positive cells following establishment of experimental fibrosis improved disease pathology (Jakubzick et al. 2003). In the fluorescein isothiocyanate (FITC) mouse model of fibrosis, IL-4 gene-deleted mice were not protected from fibrosis while both IL-13 gene-deleted and IL-4/IL-13 dual gene-deleted mice were both significantly protected. This protection correlated with reduced eosinophilia but appeared to be independent of the TGF-β pathway (Kolodsick et al. 2004). Similarly, in the bleomycin-induced mouse model of pulmonary fibrosis, neutralization of IL-13, but not IL-4, attenuated the pulmonary fibrosis as assessed by collagen deposition (Belperio et al. 2002). Further, IL-13 has been demonstrated to play a significant role in maintenance of the subepithelial fibrosis seen in the ovalbumin-induced mouse model of asthma, as anti-IL-13 monoclonal antibody treatment halted development of subepithelial fibrosis and reversed mucous hyperplasia (Yang et al. 2005). Both IL-13 and IL-4 induce profibrotic responses in fibroblasts, responses comparable to those induced by TGF-β1 (Oriente et al. 2000; Hashimoto et al. 2001). Both IL-4 and IL-13 induce fibroblast proliferation (Saito et al. 2003). Interestingly, IL-4 and IL-13 induced greater proliferation of fibroblasts derived from patients with mild asthma compared with responses elicited on fibroblasts derived from subjects with severe asthma (Kraft et al. 2001). This suggests an altered fibroblast phenotype that is dependent on disease staging. IL-13 and IL-4 have also been demonstrated to play


profibrotic roles on airway epithelial cells by promoting epithelial cell proliferation and also inducing mitogenic TGF-β2 production from these cells (Richter et al. 2001). Interestingly, IL-13 but not IL-4 promotes the differentiation of fibroblasts to myofibroblasts (Kraft et al. 2001; Saito et al. 2003). The other mechanism by which IL-13 can mediate fibrosis is via the second IL-13 receptor, IL-13Rα2 (Fichtner-Feigl et al. 2006). This receptor was, until very recently, thought to be a decoy receptor as it has a very short cytoplasmic tail and is frequently shed from the surface of cells (Gauchat et al. 1997; Orchansky et al. 1997). However IL-13 binds to IL13Rα2 with a much higher affinity than IL-13Rα1 (Rahaman et al. 2002; Wu & Low 2003). Recent data suggest that IL13Rα2 has signaling capabilities resulting in TGF-β1 secretion from macrophages (Fichtner-Feigl et al. 2006). Overall the vast majority of literature suggests that IL-4 plays a role in asthma, likely at the acute stage. In contrast, IL-13 appears to play a more central role through the maintenance and progression of fibrosis. This is further highlighted by the recent evidence demonstrating a functional role for the IL-13-specific receptor IL-13Rα2.

Thrombin and coagulation pathway-associated proteases Thrombin is the final serine protease generated during blood coagulation and fibrin formation (Overduin & de Beer 2000). The extrinsic coagulation cascade is initiated following tissue injury, with tissue factor (TF) interacting with factor VII. This then initiates the conversion of factor VII to VIIa. Factor VIIa converts factor X to Xa, which acts in concert with factor V to convert prothrombin to thrombin (Chambers & Laurent 2002). Thrombin catalyzes the conversion of soluble fibrinogen to insoluble fibrin monomers. Thrombin can generate a positive feedback loop to achieve sustained coagulation by activating the intrinsic pathway, starting with the conversion of factor XI to XIa. Factor XIa converts factor IX to IXa. Factor IXa converts factor X to Xa, which, as with the extrinsic pathway, converts prothrombin to thrombin (Schenone et al. 2004). Thrombin has been implicated in pulmonary fibrotic diseases such as acute lung injury (Schmidt et al. 1996; Kipnis et al. 2004), acute respiratory distress syndrome (ARDS) (Burchardi et al. 1984), ILD (Fujimoto et al. 2003; Kimura et al. 2005), and IPF (Hernandez-Rodriguez et al. 1995; Howell et al. 2002; Ludwicka-Bradley et al. 2004). Indeed, BAL fluid from ARDS patients was shown to have complexes of TF– factor VII–VIIa capable of triggering the extrinsic coagulation cascade. Active thrombin has also been found elevated in the BAL fluid of scleroderma patients (Ohba et al. 1994). Active thrombin is also found at increased levels in patients with IPF and eosinophilic pneumonia (Kimura et al. 2005). Further, thrombin also represents a large proportion of the fibroblast proliferation capacity of BAL fluid (Hernandez-Rodriguez et al. 1995). Animal models of fibrosis have strengthened the

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connection between thrombin and fibrosis. Increased thrombin is found in the lungs of mice challenged with bleomycin and pharmacologic inhibition of thrombin significantly reduced collagen deposition (Howell et al. 2002). At the cellular level, thrombin has numerous biological effects that are divergent from its role as a coagulation pathway proteinase. It has been demonstrated to be a chemoattractant for leukocytes and can initiate the release of cytokines and chemokines such as MCP-1/CCL2, IL-6, and IL-8/CXCL8 from numerous cells (Sower et al. 1995; Gordon et al. 2000; Brandes et al. 2001; Liu et al. 2006). Specific to fibrosis, thrombin can act directly on smooth muscle cells and fibroblasts to induce matrix production by promoting the release of PDGF and TGF-β1 (Ohba et al. 1994; Chambers et al. 2000; Howell et al. 2002; Vesey et al. 2005). The cell-based activities of thrombin are mediated through a family of receptors termed proteinase-activated receptors (PARs). PARs are G protein-coupled receptors (GPCRs) that, unlike other GPCRs, are activated by proteolytic unmasking of a ligand attached to the receptor. Thrombin, trypsin, and tryptases are the most well-characterized proteinases capable of this unmasking (Molino et al. 1997; Fan et al. 2005). Four PARs have been identified in humans with differing functions and tissue expression patterns. Thrombin activates PAR-1, PAR-3, and PAR-4, whereas PAR-2 is activated by trypsin, mast cell tryptase, and coagulation factors VIIa and Xa (Molino et al. 1997; Camerer et al. 2000; Kawabata 2002; Ossovskaya & Bunnett 2004). As with thrombin, a profibrotic role for PARs has also been described. PAR-1, which is activated by thrombin, is expressed on T cells, monocytes, and dendritic cells, although the functional significance of this interaction is not completely understood (Steinhoff et al. 1999; Fields et al. 2003). PAR-2 is expressed in the skin, specifically keratinocytes, endothelial cells, and dermal dendritic cells (Steinhoff et al. 1999). However, PAR-2 expression has also been shown on vascular smooth muscle cells as well as on fibroblasts (Molino et al. 1998; Gruber et al. 2004). PAR-3, which is not as well characterized as PAR-1 or PAR-2, has been found on endothelial cells and platelets, although the function of this PAR is still being determined (Schmidt et al. 1998). PAR-4, which is cleaved by thrombin, is also found predominantly on platelets (Henriksen & Hanks 2002). Given the tissue localization and activation of PARs, it is interesting to note that both PAR-1 and PAR-2 have been implicated in fibrosis. Indeed, PAR-1-deficient mice are protected from bleomycininduced lung fibrosis (Howell et al. 2005). Further, PAR-2deficient mice had decreased eotaxin/CCL11 and reduced eosinophilia in the lungs following antigen challenge in an allergen sensitization and challenge model of asthma (Schmidlin et al. 2002; Takizawa et al. 2005).

Matrix metalloproteinases and matrix degradation The ECM is a dynamic structure that is continuously turning over and it is the fine balance between matrix generation and

424

breakdown that maintains the functional tissue environment (Laurent 1987; McAnulty & Laurent 1987; Mosher et al. 1992). Disruption of this homeostatic environment leads to excessive degradation or aberrant accumulation of the ECM. It has been hypothesized that breakdown of the matrix and its degradation products significantly contributes to the fibrotic process, potentially through the generation of proinflammatory and profibrotic ECM peptides (Laurent & Tetley 1984; Crouch 1990). Major elements associated with matrix degradation are proteases and protease inhibitors, which act on ECM components and also further enhance their own activity through activation of local protease cascades (Matrisian 1992; O’Connor & FitzGerald 1994). MMPs are central to ECM formation (Demedts et al. 2005). As well as directly altering ECM components, MMPs have been shown to cleave mediators such as chemokines from GAGs (Li et al. 2002). This cleavage increases the local concentration of these factors and may potentially increase their biological activity (Van den Steen et al. 2000). Tissue inhibitors of metalloproteinases (TIMPs) inhibit MMPs by binding to the catalytic site on these proteinases (Visse & Nagase 2003). A role for MMPs in disease activity has been reported for asthma and COPD (reviewed in Demedts et al. 2005). In asthma, it has been suggested that MMP-9 is central to disease activity and severity, with eosinophils and macrophages being the major source of this protease (Mautino et al. 1997; Ohno et al. 1997). Increased MMP-2 and TIMP-1 have also been detected in the lungs of asthmatics (Cataldo et al. 2000). However, only MMP-9 has been reportedly elevated in both BAL fluid and plasma of asthmatics during disease exacerbations, while MMP-2 and TIMP-1 levels were unaltered (Lee, Y.C. et al. 2001; Mattos et al. 2002; Cundall et al. 2003; Oshita et al. 2003). In contrast, increased MMP-8, MMP-9, and MMP-12 have been found to be elevated in the lungs of COPD patients (Finlay et al. 1997). In IPF, MMP-1, MMP-2, and MMP-9 were colocalized to the epithelium surrounding fibrotic lesions (Fukuda et al. 1998). There was also increased TIMP-2 in the myofibroblasts, which suggests that MMP activity may be inhibited and that the fibrotic region is not degraded (Fukuda et al. 1998). Another protease known to degrade matrix is the serine protease neutrophil elastase (NE). NE is released from azurophil granules in neutrophils and has microbicidal properties but it can also damage local tissue when expression is prolonged and thus promote aberrant remodeling (Travis 1988; Chua & Laurent 2006). Furthermore, as with MMPs, NE can cleave other proteolytic enzymes, rendering them more biologically active (Ferry et al. 1997). In disease, elevated levels of elastase have been detected in the sputum of asthmatic patients in comparison with control subjects, and elastase concentrations highly correlated with lung function as measured by FEV1 (Vignola et al. 1998). In addition, mice deficient in NE are resistant to bleomycin-induced pulmonary

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Table 18.1 Current therapeutics in development for disease indications where targeting these pathways may have profibrotic potential and thus may be suitable therapeutic candidates for diseases associated with pulmonary remodeling and fibrosis. Target

Compound

Company

Therapeutic strategy

Current reported indications

Status

CTGF

FG-3019

Fibrogen

Anti-CTGF mAb

Diabetic nephropathy, idiopathic pulmonary fibrosis, focal segmental glomerulosclerosis

Phase I

PDGF

ARC-127 CR-002

Archemix Curagen

Anti-PDGF-B aptamer Anti-PDGF-D mAb

Age-related macular degeneration Diabetic nephropathy, IgA nephropathy, lupus nephritis

Preclinical Phase I

TGF-b

GC-1008

Cambridge Antibody Technology/Genzyme Archemix

Anti-TGF-b mAb

Idiopathic pulmonary fibrosis, renal cell carcinoma, malignant melanoma

Phase I

ABX10-0122 DOM 1000P IMA-638 TNX-650

Abgenix/Amgen Domantis Millenium/Wyeth Tanox

Anti-IL-13 mAb Anti-IL-13 dAB Anti-IL-13 mAb Anti-IL-13 mAb

CAT-354

Cambridge Antibody Technology

DOM-0910 AMG-317

Domantis Amgen

IL-13

IL-13/IL-4

TGF-b2 aptamer

Preclinical Preclinical Preclinical Phase I Phase I

Anti-IL-13 mAb

Asthma Asthma Asthma Hodgkin’s lymphoma, moderate to severe asthma Severe asthma

Anti-IL-4/IL-13 dAb Anti-IL-4 receptor mAb

Asthma Asthma

Preclinical Phase I

Phase I/II

mAb, monoclonal antibody; dAb, domain antibody.

fibrosis possibly because NE plays a role in TGF-β1 activation (Dunsmore et al. 2001). Thus it might exert important profibrotic effects.

Clinical targeting of fibrosis in lung pathologies Until recently, analysis of the fibrotic component of pulmonary disease processes required the examination of resected tissue. However, advances in CT and other imaging modalities now permit more accurate and insightful analysis of the structure of the lung parenchyma and airway wall in a noninvasive manner (Coxson & Rogers 2005). These techniques will prove to be extremely important in the evaluation of disease staging and progression. For example, in COPD, accurate diagnosis of patients regarding emphysematous disease or a disease with more airway wall remodeling will allow patient stratification for therapeutic intervention, which will depend on the specific pathologic manifestation of the disease. Currently there are no therapeutic molecules in use in clinical respiratory medicine that specifically attenuate fibrotic remodeling in the lung. However, Table 18.1 shows examples of some of the approaches currently being applied within the pharmaceutical industry that may modulate some of the key pathways associated with fibrotic remodeling

described in this chapter. The table focuses on agents in development that are specific inhibitors of numerous molecular targets. The table also highlights the large number of ongoing trials with these potential therapeutics. Data from the clinical trials will provide some very important insights in the next few years into the role of these molecular targets in different diseases. Some of the therapeutics in development are targeting diseases associated with significant fibrotic pathologies. Outcomes from these studies will provide invaluable information regarding the importance of the molecular target in ECM accumulation. Further, information from ongoing trials in IPF may also give us some important insights into the potential contribution of aberrant ECM deposition to asthma physiology.

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Immune Complexes and Complement: Their Role in Host Defense and in Disease Michael M. Frank and C. Garren Hester

Summary The formation of circulating immune complexes is thought to play an etiologic role in many autoimmune and infectious diseases. These complexes activate a variety of effector systems that contribute to tissue pathology. One important, highly complex, effector system is comprised of the proteins of the complement system. Our current understanding of immune complexes, their formation, and their contribution to disease manifestations is reviewed. Similarly, we provide a brief review highlighting the chemistry, mechanisms of action, and function of the complement system proteins.

Overview During the course of ordinary life, normal individuals are exposed to microbes that have invaded the tissues or intravascular space as well as to circulating foreign proteins absorbed from the gut or transported across the respiratory mucosa. To deal with these kinds of threats even primitive animals and plants have evolved a variety of defense mechanisms that together are termed “innate immunity.” Innate immune mechanisms are “hardwired” in the genome. Animals more advanced than sharks have evolved adaptive immune responses as well. These responses allow for a greater degree of flexibility in that the profile of responding cells and proteins undergoes alteration to more precisely fit the antigenic target. The innate immune system has many overlapping systems for dealing with invaders. Toll-like receptors on a variety of cells, including all phagocytes, recognize repeating structures on microbes, as exist in the polysaccharide capsule of bacteria or microbial DNA and RNA, and activate the phagocytes to destroy the microbes (Hoebe et al. 2006). There are receptors on Kupffer cells that recognize the terminal


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mannose on the sugar moiety of partially degraded glycoproteins and there are scavenger receptors on phagocytic cells that also bind and phagocytose these materials. Defensins and granzymes, microbicidal peptides, are present in a variety of cells and are released to aid in host defense. The complement proteins represent yet another sophisticated innate immune system designed to deal with invaders or foreign materials both in circulation and in tissues. The adaptive immune response developed to provide more precise control and more rapid elimination of microbes from the circulation or tissues, since infection is such an important factor disrupting normal homeostasis. All these systems are designed to clear abnormal cells, foreign proteins, and microbes from the body, but, like many sophisticated systems, there are problems that may arise from their presence as well as the benefits usually obtained. Adaptive immune clearance mechanisms often involve the interaction of specific antibody with the antigenic or microbial target, leading to the formation of immune complexes in the circulation or in tissues. This interaction in turn often activates the complement system leading to yet another series of downstream consequences. This chapter reviews aspects of immune complex formation and pathophysiology, and the role of complement in the immunologic and inflammatory response. Investigators over a century ago recognized that the immune system responds to a foreign protein with the formation of antibody. Shortly thereafter, horse anti-diphtheria toxin antiserum was found to be successful in treatment of children with diphtheria; tens of thousands of such children were treated and a new illness was noted that had not been recognized earlier. This illness was systemically studied by von Pirquet and Schick (1905). They noted that the administration of horse serum did not produce immediate toxicity. However, 5–8 days later, the children frequently developed urticaria, particularly at the site of subcutaneous injection of the horse serum, fever, lymphoadenopathy of the draining lymph nodes in the region of the injection, leukopenia, edema, and proteinuria. This toxic effect of the horse serum disappeared over the next 4 or 5 days. In their careful clinical investigation of these phenomena, they came to realize that the phenomena were due to formation of antibody in the recipient to the infused horse protein, the formation of

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antigen–antibody complexes in the circulation, and toxicity derived from these antigen–antibody complexes. They further appreciated the fact that if a child was reexposed to horse serum at a later time the symptom complex reappeared, often in more severe form and much more rapidly. This was the first demonstration in humans that products of the immune response could be damaging as well as beneficial. They termed this phenomena “serum sickness” as it was brought on by the administration of horse antiserum. Further understanding of serum sickness was limited until the 1950s when radiolabeled proteins became available for study. In a classic series of studies, radiolabeled bovine serum albumin (BSA) was given to rabbits intravenously and the effects of the infusion followed (Dixon 1963). It was noted that there was a period of equilibration of the BSA in the circulation, followed by the normal degradation of the protein with its slow disappearance from the circulation. In the rabbit, as in humans, at 5– 8 days there was a sudden acceleration of clearance of the injected protein, as the animal made antibody, and a depression in serum complement associated with the appearance of circulating immune complexes in the rabbit’s blood. When the immune complexes were cleared some days later, free antibody appeared in the rabbit’s circulation. These animals developed marked organ pathology during the period of circulating immune complexes that resolved after the complexes were cleared from the circulation. An important feature of this disease in the rabbit was that it was much more severe than the disease usually associated with injection of horse serum in children with diphtheria. In rabbits, serum sickness included arthritis, glomerulonephritis, and vasculitis. The glomerulonephritis was characterized by proteinuria and swelling of endothelial cells, but there was little hematuria and few red cell casts. Neutrophils did not seem to be very important in the development of this acute glomerulonephritis, and, in fact, it was not clear that complement was important either, although C3 was deposited along the glomerular basement membrane in a typical granular pattern. Arteritis in this acute serum sickness in rabbit was noted at branching points of the aorta. In this case, both neutrophils and complement appeared to be required for the development of the lesions. A chronic rabbit model of serum sickness was also developed in which antigen was given repeatedly over a period of weeks. It was found that to develop chronic glomerulonephritis in these animals, it was important the animals made an immune response with circulating antibody, and the amount of the antigen given had to be titrated such that it overwhelmed the antibody at the time of each injection. If insufficient antigen was given, it was removed from the circulation rapidly by the large amounts of circulating antibody, and glomerulonephritis was not seen. If antigen excess was achieved, proteinuria became so severe that it caused a nephrotic syndrome. The striking thing about this model was

that it mimicked many of the features seen in patients with systemic lupus erythematosus (SLE). Studies showed that patients with SLE have circulating immune complexes to a wide variety of antigens, particularly antinuclear antibodies and antibodies to double-stranded DNA, and may develop vasculitis and glomerulonephritis, similar to the rabbits with circulating immune complexes. Thus, it was concluded that lupus erythematosus is the prototype of an immune complex disease in humans and that the rabbit model was an excellent model for defining the pathology of lupus. As work proceeded and many studies were moved into murine models, a variety of immune complex-mediated lupus-like models were also developed in mice. Studies in mice were particularly useful as, with the advent of genetic homologous recombination technology, it was possible to explore systematically the factors that lead to immune complex formation and tissue destruction in animals missing various proteins important in disease. Nevertheless, it is fair to say that there are still many features of immune complex pathology that are not fully understood, and there is a considerable way to go before we have detailed insight into these immune complex models. With the advent of a convenient model in rabbits of immune complex pathology, many studies were performed to determine the mechanisms by which immune complexes cause disease. It was clear that when immune complexes are formed in the circulation, they are rapidly removed by the cells of the mononuclear phagocyte system or, as it was originally termed, the reticuloendothelial system (Aschoff 1924). The majority of these cells are in the liver, but the spleen makes an important contribution to the clearance of immune complexes from the bloodstream and even the lung has some contribution to make. It was shown that when one forms immune complexes in the test tube and injects them into rabbits, high-molecular-weight complexes (greater than 11S in sedimentation characteristics) are rapidly removed from the circulation and that these appear to be responsible for the development of disease (Mannik et al. 1971). However, studies showed that experimentally it was extremely difficult to cause preformed immune complexes to be deposited in tissues like the kidney and, in fact, in most situations, only very tiny amounts of such preformed immune complexes escape the circulation to be deposited in tissues (Mannik & Arend 1971). The role of complement in immune complex clearance was studied by depleting animals of C3, alternative pathway proteins, and the late-acting complement components, by the injection of cobra venom factor (Mannik & Arend 1971). This protein, known for a century and isolated from the venom of cobras, is a C3b analog that causes massive alternative complement pathway activation and, therefore, depletes the animal of complement. The nature of the C3b fragment and the mechanism of its action are discussed later in the chapter.

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The use of cobra venom factor in studies of clearance of immune complexes from the circulation suggested that complement plays no role in their clearance: the rates of clearance of immune complexes were exactly the same in cobra venom and treated and control animals. Injected complexes were removed rapidly from the circulation and it was found that to have complexes deposited in tissue sites, they had to circulate for a prolonged period. One way this could be accomplished was by reduction and alkylation of disulfide bonds in the antibody used to form the complexes (Haakenstad & Mannik 1976). This chemically altered antibody interacted poorly with complement and poorly with cellular receptors for complement and antibody. The complexes continued to circulate for a longer time and were more likely to be deposited in tissues (Haakenstad et al. 1976). The characteristics of antigens associated with immune complex disease pathology were examined systematically. It was found that charged antigens, such as cationically charged proteins, were more likely to cause glomerulonephritis than neutral or negatively charged proteins (Gauthier et al. 1982). Presumably, the negative charges on the basement membrane of the glomerulus bound these positively charged proteins and facilitated their deposition in the kidney. Other factors that were determined to be important were the size of the antigenic protein, the degree of “foreignness,” hydrophobicity, and the ability to form a lattice with antibody (Berzofsky & Berkower 1993). IgG, IgE, and monomer IgA are divalent. IgM has five effective binding sites. IgG deposition in tissues is characteristic of immune complex diseases and the interaction of IgG with antigens was studied in detail. In the presence of antigen excess, that is, far more antigen than specific antibody in a mixture, antigen molecules will bind to each of the IgG antibody binding sites. Theoretically two antigen molecules can bind to each divalent antibody molecule. The resulting product is soluble. Similarly, in vast antibody excess the resulting product is usually soluble. Here, the number of antibody molecules that bind to each antigen is determined by the size of the antigen, and the number of antigenic groupings on the antigen surface. Thus, if the antigen has ten antigenic groupings, ten molecules of antibody theoretically can combine with each antigen, each by one of its two binding sites. In general, this combination still leads to soluble products. In the zone of equivalence, where neither the antigen nor antibody is in excess, there tends to form a lattice in which antibody molecules crosslink antigens and in the test tube form a precipitate. It was in slight antigen excess or equivalence that maximum pathology was observed in the animal models. The characteristics of antibody also were found to be important in determining the nature of the immunopathology observed. In most of the diseases studied, IgG antibody appeared to be more responsible for clinical symptomatology than IgM, although clearly there are situations in which IgM immune complexes are present. For example, in some cases

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of cryoglobulinemia, IgM antibody complexed to hepatitis C virus antigen on the virus surface may be responsible for the cold precipitins found in serum (Gertz 2005). Nevertheless, in most diseases, it appeared that IgG deposition was responsible for pathology. It was recently noted that the degree of sialation of IgG increases during immunization and the degree of sialation can be correlated with biological effect (Kaneko et al. 2006). IgA complexes were found present in such diseases as Henoch–Schönlein purpura, but these situations appeared to be in the minority (Coppo et al. 2006). The ability of the antibody to activate complement proved to be important (Cochrane & Koffler 1973). Although this did not appear to be true for clearance of the complexes, it did seem to be required for maximum damage, and often complement proteins were found deposited in the tissues at sites of inflammation. It was found that certain IgG antibodies, as well as IgM, activate the classical complement pathway. This is discussed in greater detail later in the chapter. The proteins or components of the classical pathway are activated by antibody binding to its antigen and in turn binding C1, C4, C2, and C3 of the classical pathway. A second pathway of complement activation, the alternative pathway, was later discovered, and it was found that antigen–antibody complexes of many classes and isotypes can activate this pathway and bind C3 (Thurman & Holers 2006). In early investigations, there was a particular focus on the ability of IgA to activate the alternative pathway, as it was found that IgA could not activate the classical pathway and C3 was often found in the IgA containing lesions in Henoch–Schönlein purpura. The lectin pathway, found more recently, has a reaction mechanism similar to that of the classical pathway (Jensenius 2005). It differs in that the recognition element is not antibody and C1, but mannose-binding lectin (MBL), a serum protein that recognizes the repeating sugar structures on many pathogens. The classical pathway is activated by IgM and IgG subclasses 1, 2, and 3. Subclass 4, like the other subclasses, activates the alternative pathway, but it does not activate the classical pathway to induce complement-mediated inflammation (Walport 2001a,b). The affinity of the antibody for antigen also proved to be important; in general, high-affinity interactions activate more complement and cause more damage than low-affinity interactions (Fauci et al. 1970). Because it proved difficult to develop models in which preformed immune complexes leave the circulation to deposit in tissues, there was interest in the possible formation of the immune complexes within the tissue that was damaged. It was shown that in some cases an antigen could be planted or deposited in the kidney and that antibody diffusing through tissue could meet that antigen, form immune complex in situ, and cause the immunologic damage observed (Nangaku & Couser 2005). Although in situ complex formation clearly occurs, and it has been shown that a renal metalloproteinase is the antigen in one rare type of membranoproliferative glomerulonephritis, it is not clear exactly how important this phenomenon

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is, and people have turned their attention back to deposition of circulating immune complexes as a major cause of disease. Our group was able to perform the only reported prospective study of serum sickness and immune complex disease in humans and the results are quite instructive (Lawley et al. 1984). At the time of our study, there was great interest in the use of horse anti-thymic globulin for treating patients with aplastic anemia. It had been found that this treatment leads to remission of disease in about one-third of patients. The treatment had been instituted to prevent graft-versus-host disease in patients who were being prepared for bone marrow transplantation, but in this one-third of patients the bone marrow transplantation was not needed. All patients receiving horse antithymic globulin were treated with high-dose steroids but, even so, they developed serum sickness. As in the original studies of von Pirquet and Schick, the serum sickness started 5– 8 days after the injection of horse antithymic globulin. Unlike the patients studied by von Pirquet and Schick, these patients, who presumably had immunologic abnormalities leading to aplastic anemia to begin with, and who tended to be adults rather than children, became far more ill. It was shown that they had circulating immune complexes at the time that they became ill and a striking fall in serum complement. At the time that immune complexes were in the circulation, they developed urticaria, but also a variety of other skin lesions. They developed mild albuminuria, edema, hematuria and decreased creatinine clearance, severe arthralgias, which in some patients progressed to arthritis, leukopenia, an increase in blood detected in the stool, an increase in hepatic transaminases, and many of the features that we consider part of the lupus syndrome. All these symptoms cleared when the immune complexes disappeared from the patients’ circulation. Thus, it was possible in this prospective study to show that many of the symptoms that we associate with immune complex diseases like SLE are associated with circulating immune complexes patients with a propensity to develop autoimmune disease. More recently, attention has turned to the molecular factors that promote the pathologic effects of immune complexes. As mentioned, it is known that the removal of immune complexes from the circulation is the responsibility of phagocytes predominantly of the liver and spleen. These cells have a wide variety of receptors including complement receptors and receptors for the Fc fragment of IgG (FcγR), as well as receptors for IgE, etc. It is known that in the mouse there are four groups of IgG Fc receptors. It is assumed that there are four groups of FcγR in humans as well, but only three groups have been identified and studied thus far (Ravetch & Bolland 2001; Salmon & Pricop 2001). These groups of receptors have extracellular domains that recognize the Fc fragment of IgG. We focus on IgG receptors in this chapter as receptors for IgE are considered in detail elsewhere in this book. In general, these receptors are associated with transmembrane proteins

and, usually, these receptors are associated with signaling functions within cells. FcγR have been divided into “activating” and “inhibiting” groups of receptors. Activating receptors include FcγRI, FcγRIII, and FcγRIV in the mouse, and FcγRI, FcγRIIA, and FcγRIII in humans. These receptors do not signal directly, but in most cases have an associated membrane protein, the γ chain, with a large intracytoplasmic tail that transduces the activation signal. Activation of the receptor is associated with phosphorylation of the γ chain. The γ chain resides in the membrane as a dimer or is associated with another chain, the ζ chain. All the receptors are transmembrane proteins except FcγRIII which exists in two forms, FcγRIIIa and FcγRIIIb. FcγRIIIa is a typical transmembrane protein while FcγRIIIb is linked to the membrane by a phosphatidylinositol linkage. Receptor activation leads in turn to phosphorylation of a series of intracellular signaling molecules including src and syk protein kinases, ultimately leading to cellular activation. FcγRIIB is the only FcγRII receptor in the mouse. It and FcγRIIB in humans are inhibitory receptors. Inhibitory receptors differ from the activating receptors in that they do not transmit their information via phosphorylation of the γ chain, but via an ITIM (immunoreceptor tyrosine-based inhibition motif) or inhibition motif on the intracellular portion of the Fc receptor. This is a 13-amino acid sequence in the cytoplasmic domain that becomes tyrosine phosphorylated upon activation, creating a binding site for an inhibitory signal protein, SHIP (SH2-containing inositol phosphatase), that hydrolyzes activated membrane inositol phosphate, thereby disrupting immunoreceptor tyrosine-based activation motifs and B-cell receptor-mediated calcium influx. Also, other cellular activation pathways are inhibited by ligation of the ITIM-bearing protein. Many studies have assessed the function of FcγR in the development of autoimmunity and in the development of the immune response. FcγRI in both human and mouse is a high-affinity receptor (KA ∼ 1 × 108 − 1 × 109) (Nimmerjahn & Ravetch 2005). Under usual circumstances, it is expected to be complexed with serum IgG and, presumably, crossligation of receptors is important in its function. Its function is the most poorly understood of the receptor group. FcγRII and FcγRIII are relatively low-affinity receptors (KA ∼ 1 × 107 and 3 × 107, respectively), and when cells with these receptors are suspended in serum and then washed there is little IgG bound to their surface. These receptors are inefficient at binding ligand, except in the presence of large immune complexes. In that situation, multiple receptors can be occupied by the antigen–antibody lattice leading to cellular activation. In the mouse, FcγRIV is of somewhat higher affinity than the lowaffinity receptors and, presumably, is a typical activating receptor. As mentioned, FcγRII in humans includes both the activating receptor, FcγRIIA, and inhibiting receptor, FcγRIIB. These receptors are almost identical in their extracellular region and, therefore, bind ligand similarly but, as mentioned, the crosslinking of these receptors has exactly the opposite

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Table 19.1 In vivo phenotypes. Deleted gene: FcgRIIB Enhanced skin and lung Arthus reaction Enhanced IgG- and IgE-induced systemic anaphylaxis Enhanced anti-GBM (glomerular basement membrane) antibodyinduced glomerulonephritis Enhanced bleeding in anti-aIIBb3 antibody-induced ITP Enhanced collagen-induced arthritis Development of Goodpasture syndrome Spontaneous development of glomerulonephritis in B6 background Deleted gene: FcgRIII Attenuated skin and lung Arthus reaction Attenuated anti-GBM antibody-induced glomerulonephritis Attenuated cryoglobulin-induced vasculitis Reduced sensitivity to autoimmune hemolytic anemia Reduced sensitivity to hypothermia and bleeding in ITP Reduced sensitivity to anti-GPI antibody-induced arthritis

effect on cells. These, like FcγRIII, are low-affinity receptors and require crossligation to have their effect. Mice have been generated by targeted gene deletion that are missing each of the receptors or the gamma chain common to the activating receptors. In general, mice missing the activating receptors have a decreased immunopathologic response in all of the models of immunologic disease in mice (Table 19.1) (Schmidt & Gessner 2005). Thus, immune complex deposition in the skin, with resulting vasculitis, a phenomenon called the “Arthus reaction,” is decreased in these animals. Similarly, they have attenuated immune complex-induced glomerulonephritis. They have attenuated cryoglobulin-induced vasculitis, reduced sensitivity to antibody-induced autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura (ITP), and reduced development of antibody-induced arthritis. As might be expected, mice deficient in the inhibitory receptor, FcγRIIB, on the other hand have an enhanced immunologic reaction in each of these areas, including Arthus reactions in the skin and lung, glomerulonephritis, ITP, collagen-induced arthritis, Goodpasture-like syndrome, and indeed they even have enhanced IgG- and IgE-induced systemic anaphylaxis. This large group of studies with similar findings suggests that Fc receptors are of critical importance in the development of autoimmunity, not only in animals, but also likely in humans. In the past several decades, there have been additional studies in animals and humans of the efficiency of the mononuclear phagocyte system in the removal of immune complexes from the circulation. In these studies, the definition of immune complexes has varied. In initial studies that we performed years ago, we radiolabeled animal or patient red cells with chromium-51, sensitized them with antibody, and followed their rate of clearance from the circulation after sensitization of the cells with a known amount of antibody (Frank et al. 1977, 1983). In animals and humans we studied

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the effect of IgG and IgM antibody. We had available guinea pigs deficient in the classical pathway protein C4. As the antibodies studied did not appear to activate the alternative pathway, we could study the effect of IgG and IgM Fc fragment-mediated clearance and complement-related clearance independently. It was found that IgM coating of red cells did not cause clearance of the red cells in C4-deficient animals; complement was responsible for the clearance of cells coated with IgM. In this case, the IgM-coated cells were cleared by the liver. IgM plus complement on a target cell is a weak stimulus for phagocytosis and these cleared cells were never phagocytosed efficiently, unless the phagocytes were activated by various inflammatory cytokines. In the normal resting state, the cleared cells remained stuck to the surface of Kupffer cells for a period of time, but then were released back into the circulation as the C3 on their surface became degraded to a nonopsonic form by mechanisms discussed later, and they circulated as Coombs-positive erythrocytes with normal survival. In striking contrast, IgG is a strong stimulus for phagocytosis, and in the presence of IgG antibody the cells were efficiently removed from the circulation and phagocytosed. Interestingly, about 100 or 200 complementactivating IgM antibody sites per red cell were required for removal of the red cells from the circulation, while only one or two complement-fixing sites were required for removal of the IgG-coated cells from the circulation. Perhaps the fact that they had less C3 on their surface is the reason the IgGcoated cells tended not to be cleared by the liver, but by the spleen. As 50% of the cardiac output goes to the liver, one must assume that the IgG- and complement-coated cells pass through the liver to ultimately be cleared by the spleen. Presumably, this occurs because there is insufficient complement on their surface to be recognized and bound by Kupffer cells, and the Fcγ receptors, partially inhibited in function by plasma concentrations of IgG, require an extensive array of IgG bound to antigen for effective binding and clearance. In humans, it was also possible to show that IgM-coated erythrocytes required complement for clearance, and the clearance mechanisms and survival of the cells seemed very similar to that in guinea pigs. For IgG-coated erythrocyte clearance, our studies used anti-Rh antibody. Rh antibody has the major advantage that it does not activate complement efficiently and, therefore, any removal of Rh IgG-coated cells from the circulation requires IgG Fc receptor activity. We showed that these Fc receptors cause progressive removal by the spleen of the red cells from the circulation. Moreover, we found a defect in such clearance in patients with lupus erythematosus and various other autoimmune diseases associated with the deposition of immune complexes in tissues. We hypothesized that the Fc receptor defect might delay the removal of the complexes from the circulation and thereby facilitate the ability of the complexes to escape from the circulation and be deposited in tissues, where they could cause inflammation and tissue damage.

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Later experiments in both animals and humans by others, studied the removal of antigen–antibody complexes formed with tetanus toxin antitoxin or hepatitis B surface antigen– antihepatitis B antibody (Davies et al. 2002). Unlike the antibody-sensitized red cells, these are true soluble complexes. In this case again, radiolabeled complexes were introduced into the circulation and their clearance was studied. It was found in these studies that patients with lupus erythematosus have more than the defect in splenic clearance that we observed in our studies of Rh-coated red cells. These additional studies showed that in humans the clearance from the circulation of these immune complexes by the liver was hyperfast. Thus, patients with lupus, as well as animals in lupus models, had an increased clearance of the complexes by the liver and a decreased clearance by the spleen. The reasons for this have never been fully explored, but we hypothesize that the difference has to do with the fact that the soluble immune complexes injected into patients or animals activate complement and can interact with both IgG Fc receptors and complement receptors simultaneously, providing much more efficient hepatic sequestration. In patients and animals with autoimmune diseases, this combination of antibody and complement combined in an immune lattice, allows for efficient hepatic sequestration by activated Kupffer cells. In summary, immune complexes are known to form regularly in healthy individuals and to be removed from the circulation without causing any toxicity. In patients with a number of autoimmune diseases, unlike in healthy individuals, immune complexes deposit in tissues to mediate various types of pathology for as yet unclear reasons. It is clear that receptors for antibody and complement are important in immune complex clearance, and it is believed that the receptors are not functioning normally in these patient populations. These various receptors are responsible for cellular activation and inhibition signals and it is thought that ultimately an abnormal signaling process contributes importantly to disease manifestations.

Introduction to complement Complement is a term originally introduced 100 years ago to designate factors in fresh serum that in the presence of specific

Lectin pathway 1990

Classical pathway 1890

antibody are able to kill microorganisms (Frank 1998). Later work showed the killing principle was heat-labile. Over time, it was found that this term is a collective one for a group of about 30 known proteins and protein regulators, some of which circulate in the blood and some of which are cell membrane-bound, which play a role in host defense and innate immunity. In recent years, it has become clear that complement is also important in the afferent limb as well as the efferent limb of the immune response. Phylogenetically, the complement proteins are ancient, being present in primitive animals where they serve a host-defense function, even in the absence of any immune system. The adaptive immune system appears in evolution at the level of the fish and, by this point in evolution, all the various complement proteins are arrayed to produce their regulatory and host-defense functions. Over the years, three pathways of complement activation have been identified (Fig. 19.1) (Walport 2001a,b). The first pathway was defined almost a century ago and for this reason is termed the “classical pathway.” It is this pathway that is usually activated by antibody and that is usually responsible for the killing of microorganisms and cells in the presence of specific antibody. A second pathway, first noted in the 1950s but studied in more detail in the 1970s and 1980s is termed the “alternative pathway” (Thurman & Holers 2006). The alternative pathway is thought to be phylogenetically older than the classical pathway; it does not require antibody to function and predates antibody in phylogenetic development. Although antibody is not required for function, the presence of antibody usually allows this pathway to function more efficiently. A third pathway described in the past two decades is still being defined in detail. This pathway, termed the “lectin pathway,” is also phylogenetically ancient and does not require antibody to function (Jensenius 2005). All pathways proceed through a series of proteins that will be discussed below, to the activation and binding of a plasma protein, C3, which is central to all three pathways. The pathways then proceed together through the binding of an additional series of proteins to the lytic steps in complement action. Complement is generally believed to have three major functions in the effector steps in host defense. The most striking and the first defined is its lytic function. On binding to an antibody-sensitized microorganism or cell, complement

MBL MASP 1,2,3

C1q, C1r, C1s

C4

C2

C4b,2a C3

C5 to C9 lysis

C3(H2O)Bb, P Fig. 19.1 The complement pathways. MBL, mannose-binding lectin; MASP, MBL-associated serine proteases.

Alternative pathway 1970

C3(H2O)

Factors B, D, Properdin (P)

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may cause direct lysis. Lysis is due to the formation by the late-acting complement proteins (C5, C6, C7, C8, and C9) of a doughnut- or cylinder-like structure with a hydrophobic exterior and a hydrophilic center, through which water and electrolytes can pass freely. The hydrophobic exterior allows the doughnut-like structure to insert into the lipid membrane of cells and microbes. It floats in the membrane and is oriented such that the hydrophilic center allows free flow between the interior of the cell or microbe and the exterior environment surrounding the cell. The cell cannot maintain its osmotic equilibrium; it swells and lyses. The second and biologically more important function of complement is the ability to opsonize particles, that is coat the particles with complement-derived protein fragments that allow them to be phagocytosed easily. Phagocytic cells have on their surface specific receptors for complement-derived peptides, often cleavage fragments of C3. When these fragments are deposited on microbes, they can link the microbe to the phagocyte receptors; the adherence facilitates the phagocytic process. The third important effector function of complement is generation of inflammatory peptides. Many of the plasma complement proteins circulate in an inactive form and on activation of the complement system are cleaved, with the formation of a large fragment and a small fragment. In most cases, the large fragment continues the lytic sequence and the small fragment has inflammatory activity. For example, the small fragment of C5, C5a, can cause mast cells to degranulate and release histamine, as if they were coated with IgE and antigen. It can cause migration of phagocytic cells toward the place where the peptide is generated, that is induce chemotaxis. These peptides have a variety of other inflammatory properties including the contraction of smooth muscle, and even may have direct bactericidal activity against a variety of bacteria and other microbes (Hugli 1986; Nordahl et al. 2004). Complement proteins are rarely completely absent because of genetic abnormalities and usually the system is acting appropriately, that is damaging cells and tissues that it recognizes as foreign (Frank 2000; Sjoholm et al. 2006). However, although it is extremely rare to have someone missing a complement protein, it is quite common to have complement causing immunologic damage under inappropriate circumstances. Thus, for example, if one produces an abnormal antibody to the basement membrane of the glomerulus, and the antibody binds to the glomerulus, activates complement, and causes inflammatory damage, the complement is acting normally. It is the antibody that is inappropriate. Complement is found in the kidneys, deposited in glomeruli in glomerulonephritis, and along the glomerular basement membrane in Goodpasture disease. It is found in vasculitic lesions, etc. In all these locations, it is presumed that complement is contributing to local tissue damage because of inappropriate activation. Because of these untoward effects of inappropriate activation, there are control proteins that downregulate

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activated complement proteins at each step in the various cascades of reaction. The importance of these proteins is that they prevent unwanted damage of one’s own tissues and cells. Although the absence of complement proteins is unusual, the absence of control proteins is more common, and patients who have absent control proteins may have ongoing “autoimmune” immunologic disease. Moreover, as mentioned in our discussion of the lectin pathway below, there are a sizable group of patients with allotypic variations of MBL that lead to very low levels of this circulating protein. To this point we have discussed effector functions of complement in host defense and it is this aspect of complement function that has received most attention over the years. It is clear that complement also functions in the afferent phase of the immune response, but here there has been far less study and less information is available (Carroll 2004). One reason that information on this important function of complement is coming to light slowly, is there are so few complementdeficient individuals to study. With the advent of knockout gene technology it has been possible to develop mouse strains missing one or more complement proteins or complement receptors, and it has been found that these animals have defects in development of many aspects of the normal murine immune response (Holers 2000). This aspect of complement physiology is discussed toward the end of this chapter.

The classical pathway The classical pathway is usually activated by antibody. IgM, and IgG subclasses IgG1, IgG2, and IgG3, bind the first component of complement to activate the classical pathway (Walport 2001a,b). C1, the first acting component, exists in serum as a three-part molecule (C1q, C1r, C1s) held together in the presence of ionic calcium. C1q has a central protein core and six radiating arms each ending in a pod-like protein domain that recognizes the Fc fragment of IgG or IgM. Each of the six arms is made up of three intertwined chains C1q A, B, and C, and has a triple helix structure like collagen, providing great flexibility. In the case of IgG, the binding of multiple IgG molecules to an antigenic surface allows binding of multiple arms of C1q, each to an Fc fragment with sufficient affinity of the C1q to allow C1 activation. In the case of IgM, a single molecule bound to an antigenic surface by multiple binding sites, with the availability of five Fc fragments within one molecule, is sufficient to bind C1q and activate the classical pathway. On binding of C1q to antibody, a distortion of the C1q molecule takes place, which in turn causes autoactivation of C1r, which then activates C1s. There are two single-chain molecules each of C1r and C1s associated with each C1q, and activation is associated with cleavage of each of the two molecules of C1r and C1s into a short fragment and a long fragment. In each case the short fragment formed has an active enzymatic site that continues the complement cascade. C1

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requires calcium for selfassociation and, therefore, the classical pathway requires calcium for initiation. One function of active C1 is to bind and cleave C4, the next protein in the activation sequence. As with cleavage of the C1 chains, C4 is cleaved into a large fragment, C4b, and a small fragment, C4a. The large fragment continues the complement cascade and the small fragment, like small fragments of C3 (C3a) and C5 (C5a) later acting proteins in the sequence, has anaphylatoxic activity. All of these fragments are able to cause mast cell degranulation with resulting histamine release. Upon activation of C4, a thioester-containing binding site is exposed on the C4b generated, which allows covalent attachment of the C4b to the target of covalent attack. Binding can be via an ester or an amide linkage. The nature of the binding site on C4 and C3 is similar and is discussed in further detail in the section on the alternative pathway. The C14 site, now bound to a target, allows C2 binding to the C4b portion of the site. On C2 binding to the C14b site, C1s cleaves the C2 also into a large and small fragment. Again the large fragment, C2a, remains bound to the assembling protein complex and the small fragment, C2b, is released. C2 binding to C4 requires the presence of ionic magnesium. The new site, consisting of C4b and C2a, no longer requires C1 for activity. Enzymatic activity resides in C2a. This site, termed the “C3 convertase” of the classical pathway, can bind the next complement protein in the sequence, C3, to continue the complement cascade. C3 is cleaved into a large fragment, C3b, and a small fragment, C3a, which again has inflammatory activity. Just as in the case of C4b, C3b can bind covalently to the target of attack. In many cases it binds directly to the C4b on the target. As mentioned earlier, C3 is the central component of all three complement pathways and is present at high concentration in serum, about 1.2 mg/mL. On the addition of C3b to the C4b, C2a site, a new binding site is created that can bind C5, the next protein in the sequence. Again, C5 is cleaved into a large fragment and a small fragment. The large fragment, C5b, continues the complement cascade, although it does not form a covalent bond with the target; it remains associated with the C3b. The small fragment released, C5a, is one of the most potent inflammatory peptides released by complement activation and has strong neutrophil aggregating activity, strong neutrophil chemotactic activity, and is an excellent anaphylatoxin. Injection of a large amount of C5a into an animal will cause anaphylaxis and death due to neutrophil aggregation in the circulation and massive histamine release (Hugli 1986; Shushakova et al. 2002). The complement cascade continues following C5b binding with the binding of C6, C7, C8, and C9. One molecule of C6 and C7 each bind to C5b on the target surface. If this binding takes place at the surface of a cell or microbe, the introduction of C7 to the binding site leads to an increase in hydrophobicity of the C5–C7 complex and insertion of the complex into the lipid cytoplasmic membrane of the cell. Under these circumstances, the cell is targeted for lysis. With the binding of one

molecule of C8 to the C5–C7 complex, a slow leak in cells such as erythrocytes appears, and with the binding of up to 16 molecules of C9, a cylinder or doughnut-like structure is formed, containing all the proteins C5b–C9, that penetrates the cell membrane leading to rapid lysis. Cells protect themselves from lysis in a variety of ways. The many complement-control proteins are discussed in greater detail below, but also the lytic C5b–C9 complex can be shed from the surface of some cells or internalized and destroyed, as the cell attempts to protect itself from damage. Cells such as erythrocytes with little intracellular protein synthetic machinery to help repair their membranes, rely on the control proteins for protection. Cells such as macrophages and endothelial cells have these extra mechanisms for clearing their membranes of deposited complement proteins.

The lectin pathway The lectin pathway unlike the classical pathway does not require antibody to function and appears to be developmentally more primitive than the classical pathway. Nevertheless, it is quite similar in function (Jensenius 2005). In the classical pathway, the recognition molecule that sees the foreign antigen and induces complement activation is antibody. The lectin pathway does not use antibody but has its own recognition molecule. The pathway is initiated by a plasma protein, MBL, or by related proteins, the ficolins. MBL has a structure rather similar to C1q, with a central core and a series of radiating arms composed of a flexible triple helix, each ending in a binding structure. Unlike C1q, in MBL the helix contains three copies of a single chain. In the case of C1q, the binding structure at the end of the arms recognizes immunoglobulin and it is the binding of the antibody that is the critically important feature of the activation sequence. In the case of MBL, there are three lectin-binding sites at the termination of each of the arms of the MBL. Each lectinbinding site has low affinity for sugars like mannose but with the binding of multiple arms of the MBL, each with three binding sites to, for example, the repeating polysaccharides on the surface of a bacterium, the association is stabilized and the complement pathway is activated. Therefore, the protein that recognizes the foreign structure is not specific antibody, but one of the complement proteins, MBL. MBL circulates as a series of multimers and may have two arms, four arms, or six arms. In general, it is thought that the four-arm structure predominates. Associated with MBL is another group of proteins called MASPs (MBL-associated serine proteases). The functional structure again resembles C1, as C1q, the subunit with collagen-like arms that binds to antibody, also has associated with it serine proteases, C1r, and C1s. In the case of MBL, the associated serine proteases are MASP1, MASP2, and MASP3, as well as some other related molecules. It is believed that

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MASP2 is the principal serine protease involved in continuation of the complement cascade, with MASP1 also active. The function of MASP3 is not known at this time and it is not clear why two MASPs, MASP1 and MASP2, exist with much the same function. Currently, it is thought that the main path of activation following MBL binding is via activation of C4 by MASP2. Thus, lectin pathway activation is very much like classical pathway activation. In the classical pathway, antibody is the recognition molecule followed by C1 activation. In the lectin pathway, the recognition molecule is MBL and MASP2 is the C1-like molecule that cleaves C4 into C4a and C4b. The C4b then binds C2, the C2 is cleaved by MASP2, and the pathway continues to C9, just as in the classical pathway.

The alternative pathway The alternative pathway, probably the oldest of the complement pathways in phylogenetic terms, is more difficult to understand in that it operates by a mechanism which is fundamentally different and more primitive than that of the classical and lectin pathways. In these latter two cases, the pathway is specifically activated by a recognition molecule that binds to the target of attack and activates a serine protease; the protease then continues the complement sequence. In the alternative pathway, C3 is itself the recognition molecule and activation of the pathway is inefficient. C3 is a two-chain molecule, α and β, with an internal thioester joining a cystine at position 988 and a glutamine at position 991 in the α-chain backbone. The tertiary configuration of the molecule protects the internal thioester from cleavage through nucleophilic attack by water; however, even so, it undergoes slow hydrolysis in the circulation. When water penetrates to the thioester bond, the bond undergoes hydrolysis with cleavage of the thioester, leaving a free sulfhydryl at position 988 and a hydrated carboxyl ion at position 991. This is associated with a marked change in tertiary structure and the molecule comes to resemble C3b. Hydrolyzed C3, like C3b itself, is capable of binding factor B, a protein of the alternative pathway very much like C2 of the classical pathway. On binding to hydrated C3 or C3b, factor B can be cleaved by a serine protease, very much like C1s of the classical pathway, termed factor D. Thus, a protein complex is formed consisting of hydrated C3 or C3b and the large fragment of cleaved factor B, termed Bb, with the release of the small fragment Ba. This complex is the C3 convertase of the alternative pathway. It may bind a new molecule of C3 and cleave it into C3a and C3b. The major difference between the C3 convertase (C3 cleaving enzyme) of the alternative pathway and the C3 convertase of the classical and lectin pathways, is that there is no C4b in the convertase of the alternative pathway. C3b itself takes the place of the C4b with factor B acting like C2 and factor D acting like C1. A sec-

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ond difference is that factor D, the molecule that resembles C1s of the classical pathway and MASP2 of the lectin pathway, is not physically bound to the active site, but acts as a fluid phase enzyme. The initial alternative pathway C3 convertase (C3(H2O)Bb) binds and cleaves another molecule of C3. When C3a is cleaved from C3b, the thioester becomes immediately available. If this cleavage occurs close to the surface of a cell or microbe, the carboxyl on the C3b generated can form an ester or amide bond with the surface of the cell or microbe. This target-bound C3b can accept another factor B molecule and in the presence of factor D can cleave more C3 into C3a and C3b, with more C3b becoming target bound. In the case of the classical and lectin pathways, the C42 complex is unstable and slowly decays. In the case of the alternative pathway, the C3bBb complex is also unstable. It rapidly decays and is stabilized in the circulation by yet another protein termed “properdin.” Like the classical pathway convertase, the alternative pathway convertase requires magnesium ions to function. Presumably, the first pathway to develop in the complement system, in terms of phylogenetic development, was the alternative pathway. Because initiation is not directed and requires, by chance, hydrolysis of C3 close to the target of destruction with binding of additional C3 to the target, it is inefficient. It is believed that the lectin pathway evolved to recognize the target more directly, and with the appearance of antibody the target could be even more specifically identified. C3b deposited on a target by the lectin or classical pathway can also engage proteins of the alternative pathway to further amplify C3 deposition. C3b undergoes a complex sequence of degradation steps with each degradation product having different biological activity. Because these steps are regulated by control molecules they are considered in the sections below.

Complement receptors and complement control molecules By definition, complement receptors recognize and bind various complement proteins and fragments. As with other receptors, this may cause cellular activation. However, unlike most cellular receptors, some of the complement receptors also act as control molecules. They may interact with the molecules they bind to allow for further degradation of the bound fragment. In performing this function, the receptors act like the many complement control molecules that also regulate the activity of complement proteins to control their biological function. As has been mentioned, C3 has many functions in the complement system and many of the control molecules regulate C3. Four of the major complement receptors (CR1, CR2, CR3, and CR4 in one terminology) bind fragments of C3. Nevertheless, for clarity we will take each step in the complement cascade of reactions in turn. At virtually each step of the complement cascade, control points are established to downregulate the possibility of untoward complement activation.

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Control of activity of C1 and MASPs

Control of the activity of C4 and C2

In the classical pathway, the activation of C1 with cleavage of C4 is downregulated by C1 inhibitor (Han Lee et al. 2003). This single chain molecule is a serpin, (serine protease inhibitor). Inhibitors of this class present a bait sequence to the enzyme to be inhibited. When an enzyme cleaves the inhibitor at the site of the bait sequence (amino acid 444 of the C1 inhibitor), the inhibitor springs apart uncovering a highly reactive site that forms a covalent bond with the active site on the enzyme. The C1 inhibitor has been termed a “suicide” inhibitor, as it is used up during the inhibition process. It functions to inhibit both activated C1r and C1s. Two molecules of inhibitor interact with each of the two C1r active sites and two molecules of interact with each of the two C1s active sites in C1 as part of the inhibition process. Therefore, four molecules of C1 inhibitor are used up, with each molecule of active C1 inhibited. During the process of C1 inhibition, the C1 molecule is taken apart, the Clr and Cls dissociated, and the C1q left bound to the antibody site. As discussed elsewhere in this volume, C1 inhibitor inhibits enzymes in a number of other mediator pathways in plasma, including the kinin-generating pathway, and patients with abnormalities in even one of the genes for normal Cl inhibitor have hereditary angioedema, a swelling disorder. C1 inhibitor is capable of inhibiting MASPs in exactly the same way as it inhibits C1. Receptors also exist for the C1q fragment of C1. Here there has been considerably less research and there is less understanding of how the several C1q receptors that have been described function in normal physiology and in disease.

The next steps in the classical complement pathway, the interaction of C4 and C2, are also under the control of a circulating protein, C4 binding protein. This protein binds to C4b, preventing its interaction with C2 and accelerating the decay of the C4b,C2a site once formed. It also is capable of binding to C3b when these reactants are present at high concentration. As discussed, the C4b,C2a site is further controlled because it is subject to spontaneous degradation over time, losing its activity. Loss of activity is accompanied by the release of C2a from the C4b site. The C4b site can accept another C2 and, in the presence of the C1, can regenerate the C4b/2a site.

Control molecules and cellular receptors that interact with C3 As an essential component in the lytic pathway, C3 functions in the classical, lectin, and alternative pathways. C3b on the cell surface is also a potent opsonin, aiding the phagocytic process. As C3b, if deposited on tissue cells, can become a focus of tissue damage, its formation and degradation are under tight regulation. It is simplest to describe the steps in degradation and then the receptors (Fig. 19.2). There are a great many circulating proteins and cellsurface receptors that can interact with this protein and the results of the interaction may differ depending on the set of control proteins with which it interacts (Barilla-LaBarca et al. 2002; Kim 2006). Virtually all normal cells have these control molecules. Some of the plasma and cellular membrane-bound =

O

S-C

22.5 kDa Fig. 19.2 The two-chain molecule C3 is shown first. There are no receptors that recognize this molecule. The C3 convertase of the classical or alternative pathway cleaves off C3a, an anaphylatoxin. The remainder of the molecule C3b undergoes a marked molecular rearrangement and now is recognized by CD35 (CR1), as well as by the recently recognized receptor on Kupffer cells, C3Ig. C3b binds factor H and now can be cleaved by factor I to iC3b. iC3b is recognized by CD11b/CD18 and CD11c/CD18. These two-chain receptors are on all phagocytes and on dendritic cells. They aid the processing of antigen. In serum, the cleavage of C3 stops at this point, but when an immune complex is bound to cellular CD35 or when C3 is deposited on a cell with CD35, it is cleaved further by factor I to C3c and C3d. The latter fragment is recognized by CD21 found on B cells and dendritic cells. Antigen with multiple bound C3d molecules can interact with both CD21 and the B-cell receptor, which can augment the immune response. (See CD-ROM for color version.)

C3

39.5 kDa

Receptors

35 kDa

S S

S

S 75 kDa

None

R H O S C C3a+C3b

CD35 (C3bR) C3aR

S S

S

S

H S S S

iC3b

R O C

S

Factors H & I CD11b,c; CD18 (iC3bR) S

H R S O C

CD35 Factor I C3c+C3dg

CD21 (C3dR) S S

S

S

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proteins also act as complement receptors, aiding the phagocytic process or providing signals for cellular activation. Two plasma proteins are critical regulators of C3b, factors H and I. C3b when generated will bind factor H, and the complex of C3b and H can be attacked by the circulating complement enzyme, factor I, which can then cleave the C3b α chain leading to the formation of iC3b or inactivated C3b. Cleaved C3b will no longer function as a C3 or C5 convertase, but it remains cell bound and remains a potent opsonin. The rare patients missing factor I have low C3 in the circulation, because the alternative pathway stays active and cleaves C3, and have urticaria, presumably because they continuously generate the anaphylatoxin C3a. It is interesting that, at this step, there is the possibility of separating self from nonself, one of the cardinal features of the adaptive immune response. During an immune response it is essential to separate self from nonself, to prevent the development of autoimmune disease and damage to one’s own tissues. In the case of the complement system, the system does this, albeit in a fairly primitive way. C3b deposited on one’s own tissues or cells is often close to a sialic acid, present in relatively large amounts on normal tissue and cellular membrane carbohydrates. Factor H activity is facilitated by sialic acid. Any C3b deposited on one’s own cells, therefore, tends to be cleaved by factor I, preventing further complement activation. The microorganism surface usually does not have sialic acid. Factor H function is not facilitated. C3b remains on the organism surface and the C3 convertase of the alternative pathway continues to deposit additional C3b on the microbe. Many pathogens have evolved mechanisms to incorporate sialic acid into surface structures, to protect themselves in part from complement attack. For example, Esherichia coli K1 has developed sialic acid-containing capsules to mimic the surface of the normal cell and thus protect the bacterium from destruction. Four different cellular receptors appear to be important in the phagocytosis of C3-coated particles. CD35 (also termed CR1) recognizes C3b, as does a recently described receptor, present on Kupffer cells, termed CRIg. The β2 integrins (CD11b/CD18 also termed CR3, and CD11c/CD18 also termed CR4) principally recognize iC3b, the product formed by the action of factors H and I, and mediate phagocytosis. Receptors for iC3b are present on all phagocytes and dendritic cells, although they are not present on lymphocytes. The β2 integrins are two-chain molecules (α and β chains) (Staunton et al. 2006). The α chain (CD11b or CD11c) provides the ligand recognition and the β chain (CD18) is required for transport of the two-chain complex to the cell surface. Patients with leukocyte adhesion deficiency have a defect leading to their inability to express these molecules on the cell surface and are highly susceptible to infection. Phagocytes bind iC3b-coated particles by virtue of their β2 integrin receptors, which markedly facilitates the phagocytic process. CD11c/CD18 is the signature receptor used in identification of monocytic

446

dendritic cells. Presumably this receptor, acting through complement bound to antigen, is of critical importance in processing of antigen for presentation to the immune system. CD35 (CR1) is present on erythrocytes, phagocytes, dendritic cells, and all B cells. As mentioned, binding of a particle to a phagocyte surface by CD35 aids in the phagocytic process. However, if an immune complex forms in the circulation and binds C3b, most often it will bind not to the surface of a phagocyte, but to the surface of an erythrocyte by erythrocyte CD35, because of the high density of erythrocytes in the circulation. The immune complex, bound to the surface of the red cell is effectively out of the circulation and cannot easily leave the intravascular space to be deposited in tissues like the kidneys. As the erythrocyte circulates through the liver and spleen, the immune complex comes in contact with the fixed phagocytes in the sinusoids of these organs and is removed from the red cell surface and phagocytosed. The red cell exits the liver or spleen free of the complex and continues to have normal survival. During this process, some of the CD35 is removed from the red cell as the immune complex is removed. The infusion of normal erythrocytes into patients with active systemic lupus erythematosus is followed by those erythrocytes gradually losing their CD35, as the CD35 on the infused erythrocytes binds circulating immune complexes and transports them to the liver and spleen. CD35 itself may act as a cofactor protein for degradation of C3, but its function is different from the proteins listed above. Like C3b that has bound factor H, C3b bound to CD35 can be cleaved by factor I, but the cleavage leads to a different fragmentation pattern. Cleavage of the α chain leads first to the formation of iC3b, but the process does not stop at this step. Further cleavage of the α chain leads to the release from the target bound C3b of the largest part of the iC3b, C3c, with retention of a 40-kDa fragment of the α chain of iC3b, C3dg, bound to the target. This fragment can be further trimmed by proteases to C3d. C3dg and C3d do not bind to CD35 or to the β2 integrins, but do bind to CD21 (CR2), present on all B cells, a T cell subset, and on follicular dendritic cells. As β2 integrins are not on B cells and CD21 is not on most phagocytes, the fragmentation pattern of C3 mediated by the various cofactor proteins can direct targets of attack or antigens to phagocytes, antigen-presenting cells, or B cells. A group of other complement control molecules on the membrane of normal cells also acts to dampen the activity of C3 when it is deposited. Thus, MCP (CD46, membrane cofactor protein) acts as a cofactor for the cleavage of C3b by factor I, just as factor H does. Another molecule present on most cells, bound to the cells by a phosphatidylinositol linkage, DAF (decay accelerating factor, CD55), interacts with both the classical and alternative pathway C3 convertase to increase the rate of degradation of the convertase, destroying its activity. It is interesting that these two molecules, widely distributed on cells of the body, together have much of the activity of CD35 on immune cells and phagocytes. CD35 has

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both decay-accelerating and cofactor activity in the same receptor molecule, and these activities are separated and slightly changed in CD46 and CD55. As discussed, the complement system has been present over much of mammalian evolution and microorganisms have evolved mechanisms for using these proteins as docking sites for entry into cells. Thus, MCP has been shown to be a docking site for measles virus, for certain adenoviruses, and for some Neisseria; CD21 is a docking site for Epstein–Barr virus. Each year the list of control molecules that have been found to be docking sites for various viruses or bacteria grows. Several of the complement receptors are thought to aid directly in cellular activation or inhibition. CD35 has been discussed above as a facilitator of phagocytosis. Particles with bound CD35 will attach to the surface of phagocytic cells. This provides the first signal for phagocytosis to the cell. A second signal must be provided to start the phagocytic process. This can be accomplished by IgG interacting with an IgG Fc-activating receptor, like FcγRIII, or by any of a large number of cellactivating cytokines. The β2 integrins CD11b,c/CD18 are the principal iC3b receptors and like CD35 provide a first signal for the phagocytic process. These receptors are present on all phagocytes and natural killer cells. As mentioned, CD11c/ CD18 is used as an identifying marker of dendritic cells. Follicular dendritic cells, and some T cells have CD21 (CR2) on their surface. This receptor recognizes C3d, C3dg, and polymerized iC3b. It is believed that antigens with C3d on their surface can cross-link CD21 with the B-cell receptor. Cross-linking these two receptors can augment the ability of antigen to activate B cells by as much as a 1000-fold. For the purposes of this chapter, it is important to note that factor H abnormalities have been reported in two important medical situations. Lack of factor H activity has been shown to be important in patients with familial atypical hemolytic– uremic syndrome, namely hemolytic–uremic syndrome usually not associated with bacterial infection and diarrhea (Zipfel et al. 2006). Three molecules, each of which play a role in C3 degradation, have been reported to be abnormal in various subgroups of these patients. The three proteins are factor H, factor I, and MCP. The defects in the proteins can be present in either the heterozygous or homozygous state, probably reflecting the fact that half the normal number of C3 control molecules is not sufficient to protect from untoward immunologic activation. One way of thinking about the pathogenesis of this syndrome is that Shiga toxin from bacteria or other toxins enter the circulation and are deposited on endothelial cells, particularly in the kidney, and on erythrocytes. As the individual makes an immune response to the toxin, in the absence of sufficient control molecules, antibody binds to the toxin and cells with toxin and antibody are destroyed by complement activation. It has also recently been reported that the largest risk factor in the development of macular degeneration in the elderly is an alteration of the amino acid at position 402 in factor

H (Tyr→His) (Haines et al. 2005). It is believed from statistical studies of DNA sequences from pedigrees of families with inherited macular degeneration that approximately 50% of cases are due to this alteration in this one amino acid, although the factor H allele with histidine in position 402 is fairly common in the population and other factors must be involved.

Receptors for the anaphylatoxins C3a, C4a and C5a Of the anaphylatoxins, C5a has been studied in greatest detail (Hugli 1986). It is a potent chemotactic factor, causing the directed migration of phagocytes. It contracts smooth muscle cells and causes mast cells to degranulate in the absence of IgE antibody. It caused neutrophils to adhere to one another and to endothelium in vessels. It clearly plays a part in the damage observed during the course of immunologic lung disease. Mice with a defect in the C5a receptor do not develop all of the manifestations of immunologic or allergic lung disease. It is likely that far more information will become available about this important receptor in the development of asthma. There is less information available on C4a and C3a binding. The membrane receptor for C3a is clearly different from that of C5a and can be triggered to cause mucus secretion in the airways, but its role in immunologic airways disease is still speculative.

Control of the late steps in the complement cascade The later steps in the complement cascade are also under tight control. The site composed of the C3 convertase with bound C5 will decay if it does not bind C6 rapidly, and there are a series of molecules that downregulate the late-acting proteins both in serum and on cells. S protein, a plasma protein, interacts with C7 as the C5–C7 complex forms and becomes hydrophobic (Liszewski & Atkinson 1998). On binding S protein, this complex is neutralized and can no longer bind to cell surfaces. Similarly, clusterin, another plasma protein, binds to the forming C5–C9 complex and prevents its activation and completion. Most cells in the body have membrane-bound CD59, which interacts with the C5b–8 site decreasing the binding of C9 and preventing polymerization of C9. It protects the cell by preventing effective pore formation. All of these control molecules are important in maintaining homeostasis and loss of the control molecules often leads to disease. CD59, like CD55, is linked to cell membranes by a phosphatidylinositol linkage. By not having a transmembrane domain, the protein is free to move rapidly in the fatty hydrophobic plane of the cell membrane to intercept forming C5b–9 and prevent cell lysis. No patient has been described with a genetic defect leading to absence of CD59. However, patients with the disease paroxysmal nocturnal hemoglobinuria (PNH) have an acquired bone marrow defect in which they develop a mutation in bone marrow stem cells of the gene PIG A (phosphatidylinositol glycan enzyme A), the first enzyme in the development of phosphatidylinositol bonds (Luzzatto 2006). This is a gene

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present on the X chromosome and a single gene defect in a bone marrow stem cell leads to an inability to synthesize the first intermediate in the phosphatidylinositol linkage pathway, and therefore the failure to have phosphatidylinositollinked proteins on the cell membrane. A failure to generate hematopoietic cells with CD59 leads to all hematopoietic cells of bone marrow origin derived from the abnormal clone being easily lysed by complement. As mentioned, alternative pathway proteins in the circulation undergo slow activation; CD59 is critical for neutralizing complement proteins when they bind to the body’s own cells. In patients with PNH, this mechanism is defective and patients have a hemolytic anemia, often thrombocytopenia, and often low neutrophil count. As this chapter is written, papers have appeared promoting the use of monoclonal anti-C5 to turn off the late components of complement, thereby preventing lysis while allowing opsonization to proceed. This is the first medication that improves cell survival in this patient group with a disease that has a generally grim prognosis. The role of complement in the generation of immunologic lung disease is of particular interest. For many years, it was taken as gospel that complement plays no role in IgE-mediated lung disease or in asthma. Recent work has suggested that this may not be the case. Complement may play a number of interesting functions in the generation of lung pathology. First, it has been suggested that complement functions importantly in directing immune responses toward Th1 or Th2 type immunity. Th1 immunity is generally considered most important in prevention of infection and Th2 immunity is associated with asthma and other allergic diseases. It is believed that the activation of C5 and the generation of C5a are important in directing the immune response toward a Th1 phenotype and lack of C5 therefore skews the system toward the generation of Th2 immunity (Peng et al. 2005; Kohl et al. 2006). On the other hand, once immunity or allergy is established, it is believed that C5a may be generated during immunologic responses in the lung and, acting as an anaphylatoxin, may cause mast cell degranulation, smooth muscle contraction, etc., contributing to the asthmatic response.

Complement in the afferent limb of the immune response In recent years, there has been considerable interest in the role of complement in the development of immunity (Carroll 1998; Carroll & Holers 2005). The discussion up to this point has focused on the efferent limb of the response, how tissue damage is caused or controlled by complement. As mentioned early in the chapter, the complement system is phylogenetically older than the adaptive immune system, and many of the complement proteins existed as the afferent immune system evolved (Nonaka 1998). It is not surprising, therefore,

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that elements of the complement system were incorporated into the afferent immune system and these elements are only now being slowly identified. As mentioned in an earlier section, the binding of complement to an antigen allowing cross-linking of CD21 and the B cell receptor increases antigenicity by up to 1000-fold. In this case, complement augments the immune response. It is also known that patients deficient in complement, although rare, often have major defects in adaptive immunity. Patients deficient in C1, C4 and, to a lesser extent, C2, have a high propensity to develop SLE (Walport et al. 1998; Manderson et al. 2004). In fact, of the relatively few C1q-deficient patients who have been described, 96% have had systemic lupus. Of the relatively few C4-deficient individuals who have been described, 75% have had lupus. Even heterozygosity of the genes for C4 predisposes individuals to the development of lupus. This propensity to cause SLE seems to be independent of the genetic localization of C4, C3 and factor B in the major histocompatibility locus as class III genes, and therefore their linkage to the MHC. It is known that animals deficient in C1q, C4, C3, and CR1/2 make a poor immune response, particularly to T-dependent antigens, have poor germinal center formation, and have poor immunologic memory. It is known that complement aids in the localization and retention of antigens within the germinal center and it is believed that this localization of antigen to the germinal center facilitates an ongoing immune response. In addition to the above, animals, particularly those deficient in C1q and C4, fail to develop normal tolerance as well, although animals deficient in C3 and CR1/2 do not appear to have this defect. Although these are intriguing findings and have been repeated in many laboratories, it is still not completely clear how complement functions in the afferent limb of the immune response. It is quite likely that this question will be clarified over the next few years.

Complement deficiencies and clinical illness Patients and animals with defects in specific complement proteins have similar phenotypes. Both patients and animals deficient in the classical pathway factors and C3 have an increased propensity to infection, particularly with high-grade pathogenic bacteria like pneumococci, as opposed to viruses (Figueroa & Densen 1991; Sjoholm et al. 2006). Patients with late component defects, that is of C5–9, have a propensity to develop systemic Neisseria infections with Neisseria gonorrhoeae or Neisseria meningitidis. Why opsonization, which only requires complement through C3, is not sufficient to protect against these two organisms is not clear, but repeated infection with either of these two organisms is often an excellent tip to the clinician that a late complement protein deficiency is present. Alternative pathway defects are rarer and, in fact, no factor B deficiency has ever been described (Thurman

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Table 19.2 Consequences of complement deficiency. C3 and factors that control C3 levels

Late-acting proteins: C5–9

Decreased C′ activation in the absence of antibody

Decreased opsonization: if control factors are abnormal, increased C′ effect

Inability to form lytic lesions C5 important in PMN chemotaxis

Increase in infection with high grade pathogens: some increase in Neisseria infections

Marked increase in infection with highgrade pathogens: failure to downregulate C3 associated with hemolytic–uremic syndrome and adultonset macular degeneration

Marked increase in Neisseria infection

Defect

Classical pathway

Lectin pathway

Alternative pathway

Functional consequences

Delayed C′ activation Decreased immune response Poor antibody activation of C′

Decreased activation in the absence of antibody

Clinical consequences

Increased incidence of autoimmune disease: infection with highgrade pathogens (Pneumococcus, etc.)

Infection in the newborn: question of increased rheumatic disease

& Holers 2006). The few patients with factor D deficiency also have a propensity to develop infection, but autoimmunity has not been seen in either animals or patients with defects in this pathway. Defects in the lectin pathway are being defined currently (Jensenius 2005). As discussed earlier in the chapter, MBL has a central core and a series of radiating arms ending in the lectin-binding sites. The radiating arms have the structure of collagen and, like collagen, are composed of three intertwined chains; however, unlike collagen, the chains are identical. It has been noted that single gene defects affecting these chains can lead to improper winding of the chains about one another during the formation of the protein, leading to low levels of MBL. This protein is present at very low levels to begin with (2 μg/mL) and patients, commonly with one of three genetic defects in the MBL gene, even when present in the heterozygous state, have inefficient chain matching, and as little as one-tenth of the normal level of MBL. It is reported from Europe that children with these defects have a high frequency of infection, although few studies have been done to confirm this finding. It is also reported that individuals with abnormalities of MBL often may die early during the course of cystic fibrosis. As patients with cystic fibrosis typically have high titer antibody to their organisms, it is not known why the MBL deficiency should lead to early death. It is also suggested that MBL deficiencies facilitate the pathogenesis of rheumatic disease. All these observations are intriguing and all require considerably more study before we understand both the observations and their meaning (Table 19.2). It should be clear from this brief review that antigen– antibody complexes and complement are capable of having important biological effects and can influence the expression of a wide variety of autoimmune and allergic diseases.

We believe that as we develop a clearer understanding of the complex interactions involved in pathogenesis, we will develop a far more insightful approach to the treatment of these important illnesses.

References Aschoff, L. (1924) Lectures on Pathology. Paul B. Hoeber, New York. Barilla-LaBarca, M.L., Liszewski, M.K. et al. (2002) Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells. J Immunol 168, 6298–304. Berzofsky, J.A. & Berkower, I.J. (1993) Immunogenicity and Antigen Structure. Raven Press, New York. Carroll, M. (1998) The role of complement and complement receptors in induction and regulation of immunity. Ann Rev Immunol 16, 545– 68. Carroll, M. (2004) The complement system in regulation of adaptive immunity. Nat Immunol 5, 981–6. Carroll, M.C. & Holers, V.M. (2005) Innate autoimmunity. Adv Immunol 86, 137–57. Cochrane, C.G. & Koffler, D. (1973) Immune complex disease in experimental animals and man. Adv Immunol 16, 185–264. Coppo, R., Andrulli, S., Amore, A. et al. (2006) Predictors of outcome in Henoch–Schonlein nephritis in children and adults. Am J Kidney Dis 47, 993–1003. Davies, K.A., Robson, M.G., Peters, A.M. et al. (2002) Defective Fc-dependent processing of immune complexes in patients with systemic lupus erythematosus. Arthritis Rheum 46, 1028–38. Dixon, F. (1963) The role of antigen–antibody complexes in disease. Harvey Lectures 58, 21. Fauci, A.S., Frank, M.M. & Johnson, J.S. (1970) The relationship between antibody affinity and the efficiency of complement fixation J Immunol 105, 215–20. Figueroa, J. & Densen, P. (1991) Infectious diseases associated with complement deficiencies. Clin Microbiol Rev 4, 369–95.

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Bradykinin Pathways and Allergic Disease Allen P. Kaplan

Summary Bradykinin is generated by cleavage of low-molecular-weight kininogen (LK) by tissue kallikrein to release kallidin (lysylbradykinin) followed by removal of N-terminal lysine by an aminopeptidase and by activation of a plasma cascade consisting of coagulation factor XII, prekallikrein (PK), and high-molecular-weight kininogen (HK). PK and HK circulate in plasma as a complex. The plasma cascade is initiated by contact with negatively charged macromolecules, including the surface of endothelial cells, leading to factor XII activation, conversion of PK to kallikrein, and digestion of HK to liberate bradykinin. The HK–PK complex binds to endothelial cells by HK interaction with free gC1qR, as well as gC1qR complexed to cytokeratin 1, while factor XII binds to a complex of the urokinase plasminogen activator receptor (u-PAR) and cytokeratin 1. Bypass of factor XII to initiate the cascade can occur along the surface of endothelial cells by direct activation of the PK–HK complex by heat-shock protein 90 and a prolylcarboxypeptidase. Bradykinin is degraded by angiotensin-converting enzyme (ACE) and plasma carboxypeptidase N. Therapy with ACE inhibitors can lead to bradykinin accumulation due to a decreased rate of degradation, resulting in complications including cough and angioedema; the latter can be very severe, including airway obstruction reminiscent of C1 inhibitor deficiency. Hereditary angioedema or acquired C1 inhibitor deficiency can lead to peripheral angioedema, swelling of the face, lips, tongue, pharynx and larynx, and bowel wall edema causing abdominal attacks that can be particularly painful.

and mobilization of arachidonic acid. Kinins also stimulate sensory nerve endings to cause a burning dysesthesia. Thus the classical parameters of inflammation (i.e., redness, heat, swelling, and pain) can all result from kinin formation. Bradykinin is the best characterized of this group of vasoactive substances. There are two general pathways by which bradykinin is generated (Margolius 1998) (Fig. 20.1). The simpler of the two has only two components: an enzyme, tissue kallikrein, and a plasma substrate, low-molecular-weight kininogen (LK) (Jacobson & Kritz 1967; Muller-Esterl et al. 1985a). Tissue kallikrein is secreted by many cells throughout the body; however, certain tissues produce particularly large quantities. These include glandular tissues (salivary and sweat glands and pancreatic exocrine gland) and the lung, kidney, intestine, and brain. The enzyme is processed intracellularly from a precursor, prokallikrein, to produce tissue kallikrein; however, the enzyme responsible for this conversion has not been identified. Tissue kallikrein is secreted and digests LK to yield a 10-amino-acid peptide, lysyl-bradykinin (kallidin), with the sequence Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-PheArg. A plasma aminopeptidase cleaves the N-terminal Lys leaving the 9-amino-acid peptide bradykinin.

Surface factor XII, prekallikrein, HK

Tissue kallikrein LK

Lys-bradykinin

Plasma kallikrein

Aminopeptidase

Introduction Bradykinin

Kinins are low-molecular-weight peptides that participate in inflammatory processes by virtue of their ability to activate endothelial cells and, as a consequence, lead to vasodilatation, increased vascular permeability, production of nitric oxide,

Kininase I Des-arg9-bradykinin

HK Kininase II (ACE) Arg-Pro-Pro-Gly-Phe-Ser-Pro + phe arg

Kininase II (ACE) Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

Arg-Pro-Pro-Gly-Phe + ser pro phe

ACE Arg-Pro-Pro-Gly-Phe + ser pro

Fig. 20.1 Pathways for bradykinin formation and degradation.

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Trace factor XIIa Factor XII

Surface

Prekallikrein Factor XIIa

HK Surface

HK

HK Kallikrein Bradykinin

Factor XI

Factor XIa

Factor XII Intrinsic coagulation

HK

Factor XIIa

Factor XIIf

Autodigestion kallikrein C1

CI

C4 and C2 digestion

The second pathway for bradykinin formation is far more complex and is part of the initiating mechanism by which the “intrinsic” coagulation pathway is activated (Kaplan et al. 1998) (Fig. 20.2). Factor XII is the initiating protein that binds to certain negatively charged macromolecular surfaces and autoactivates (autodigests) to form factor XIIa (Silverberg et al. 1980a; Tans & Griffin 1982). There are two plasma substrates of factor XIIa, namely prekallikrein (PK) (Mandle & Kaplan 1977) and factor XI (Bouma & Griffin 1977; Kurachi & Davie 1977), and each of these circulates as a complex with high-molecular-weight kininogen (HK) (Mandle et al. 1976; Thompson et al. 1977). These complexes also attach to initiating surfaces, and the major attachment sites are on two of the domains of HK, thereby placing both PK and factor XI in optimal conformation for cleavage to kallikrein (plasma kallikrein) and factor XIa, respectively. It is important to note that plasma kallikrein and tissue kallikrein are separate gene products and have little amino acid sequence homology, although they have related functions (i.e., cleavage of kininogens). The main substrate of tissue kallikrein is LK, but it is also capable of cleaving HK. Plasma kallikrein cleaves HK exclusively. The two kininogens have an identical amino acid sequence starting at the N-terminus and continuing to 12 amino acids beyond the bradykinin moiety but differ in C-terminal domains because of alternative exon splicing (Kitamura et al. 1985; Takagaki et al. 1985). This scheme for production and degradation of kinins is shown in Fig. 20.1, and further details of the plasma cascade are given in Fig. 20.2. The enzymes that destroy bradykinin consist of kininases I and II. Kininase I is also known as plasma carboxypeptidase N (Erdos & Sloane 1962), which removes the C-terminal Arg from bradykinin or kallidin to yield des-Arg9-bradykinin or des-Arg10-kallidin, respectively (Sheikh & Kaplan 1986a). It is the same enzyme that cleaves

452

Fig. 20.2 Pathway for bradykinin formation indicating the autoactivation of factor XII, the positive feedback by which kallikrein activates factor XII, cleavage of high-molecular-weight kininogen (HK) to release bradykinin, formation of factor XII fragment, and enzymatic activation of C1. The steps inhibitable by C1 inhibitor are indicated by the black rectangles.

the C-terminal Arg from the complement anaphylatoxins C3a and C5a. Kininase II is identical to angiotensin-converting enzyme (ACE) (Yang & Erdos 1967). Bradykinin and kallidin stimulate constitutively produced B2 receptors (Vavrek & Stewart 1985), whereas des-Arg9-bradykinin or des-Arg10Lys-bradykinin both stimulate B1 receptors (Regoli & Barabe 1980), which are induced as a result of inflammation. Stimuli for B1 receptor transcription include interleukin (IL)-1 and tumor necrosis factor (TNF)-α (Davis & Perkins 1994; Marceau 1995). Kininase II is a dipeptidase that cleaves the C-terminal Phe-Arg from bradykinin to yield a heptopeptide, which is cleaved once again to remove Ser-Pro to leave the pentapeptide Arg-Pro-Pro-Gly-Phe (Sheikh & Kaplan 1986b, 1989a). If the C-terminal Arg of bradykinin is first removed with kininase I, then ACE functions as a tripeptidase to remove SerPro-Phe to leave the above pentapeptide (Sheikh & Kaplan 1986b).

Contact activation The concept of contact activation was originally developed because it was found that addition of blood to a glass tube leads to coagulation. Thus “contact” with the silicate surface appeared to initiate a proteolytic cascade culminating in the conversion of fibrinogen of fibrin. At the same time, bradykinin is generated. The reactions that occur during activation in this fashion are shown in Fig. 20.2. It has been shown that factor XII (Hageman factor) circulates as a β-globulin (molecular weight 80 000) that autoactivates on binding to surfaces bearing negative charges (Silverberg et al. 1980a). Because the zymogen has no detectable enzymatic activity (Silverberg & Kaplan 1982) it has been proposed that trace quantities of the active enzyme

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3

5 BK HMWK Transcription Poly-A site 5 5

3 3 Unprocessed mRNAs

Fig. 20.3 The gene for high-molecular-weight kininogen (HK). The 9 boxes shown in the mature preHMWK mRNA represent the exons coding for the heavy chain of both HK and lowmolecular-weight kininogen (LK). Exon 10 codes for the bradykinin sequence and the light chain of HK, whereas exon 11 codes for the light chain of LK. The mature mRNAs are assembled by alternative splicing events in which the light chain sequences are attached to the 3′ end of the 13-amino acid common sequence C-terminal to bradykinin.

RNA Splicing 5

3 Mature pre-HMWK mRNA

3

5

Poly A

Mature preLMWK mRNA

Poly A

Translation

Heavy chain

BK

Light chain

Heavy chain

Pre-HMWK

(factor XIIa) actually circulate but that digestion of factor XII by factor XIIa occurs only on binding of factor XII to the surface. Thus the surface renders factor XII a substrate (Griffin 1978) for traces of preformed factor XIIa. PK and coagulation factor XI each circulate as a complex with HK (Mandle et al. 1976; Thompson et al. 1977) with stoichiometry of 1 : 1 and 1 : 2 respectively (factor XI is a dimer). The binding sites for PK and factor XI on HK overlap (Tait & Fujikawa 1986, 1987) to such a degree that HK can bind only one molecule of each, but never both. However, HK is present in considerable molar excess. Thus there are separate complexes of PK–HK and factor XI–HK and the percentage bound to HK in each case is 85% and 95%, based on equilibrium considerations (Scott & Colman 1980). HK is a key factor regulating contact activation. It is also the link protein that allows assembly of the kinin-forming cascade along the surface of cells and we therefore consider its structural features in some detail. Human plasma has two kininogens designated HK and LK. They are assembled by alternative splicing of the terminal exons (Fig. 20.3) such that a large portion of their amino acid sequence is identical (Kitamura et al. 1985). HK consists of six domains (Fig. 20.4). At the N-terminus are three domains (encoded by exons 1–9) that are homologous to cystatins and stefans (Kellermann et al. 1986a), including sulfhydryl proteases such as cathepsins B, H, and L; domains 2 and 3 actually retain cysteine protease inhibitory activity (Gounaris et al. 1984; Muller-Esterl et al. 1985b; Higashiyama et al. 1986; Ishiguro et al. 1987). Domain 4 contains the bradykinin sequence plus the next 12 amino acids. Up to this point LK and HK have identical amino acid sequences. Then exon 10, which includes bradykinin plus domains 5 and 6, is added for HK (Fig. 20.3), while exon 11 is added for LK with the attachment at the C-terminus of domain 4. When HK is cleaved by plasma kallikrein to release

BK Light chain

Pre-LMWK

bradykinin (fast cleavage occurs at a C-terminal Arg–Ser bond, followed by cleavage at an N-terminal Lys–Arg bond) (Mori & Nagasawa 1981; Mori et al. 1981), and the kinin-free HK is reduced and alkylated, one can isolate a heavy chain (domains 1–3) and a light chain (the C-terminal 12 amino acids of domain 4 plus domains 5 and 6) (Thompson et al. 1979). Thus the light chain of HK and LK are quite different (Kellermann et al. 1986b) and this accounts for the difference in molecular weight and many of the functional properties of HK not shared by LK. As noted above, plasma kallikrein

HK Domains NH2

COOH

1 6

2 Cysteine protease inhibitor

3

5

Prekallikrein and factor XI binding

Bradykinin Fig. 20.4 The structure of high-molecular-weight kininogen (HK). The heavy-chain region consists of three homologous domains (1–3) of which the latter two are sulfhydryl protease inhibition sites. Domain 4 contains the bradykinin moiety. The light-chain region contains the surface-binding site (domain 5) and overlapping binding sites for prekallikrein and factor XI (domain 6).

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preferentially cleaves HK (Reddigari & Kaplan 1988, 1989) while tissue kallikrein (encoded by a separate gene from that of plasma kallikrein) cleaves both HK and LK, but with more favorable kinetics if LK is the substrate (Lottspeich et al. 1984; Muller-Esterl et al. 1985a). The functions of HK in contact activation, as depicted in Fig. 20.2, are multiple. First, it accelerates the conversion of PK and factor XI to kallikrein and factor XIa, respectively (Griffin & Cochrane 1976; Meier et al. 1977; Wiggins et al. 1977). This acceleration appears due to the ability of PK and factor XI to bind to HK; as a result, each is in a more favorable conformation for activation than when they are tested unbound. In addition, HK provides the attachment to initiating surfaces and brings both PK and factor XI to the surface as a complex. If PK and factor XI bind to the surface in the absence of HK, activation by factor XIIa is markedly inhibited, even when compared with activation in the fluid phase. Thus the conformational effects of binding of PK and factor XI to HK are even more evident when activation along the surface is compared with fluid-phase activation (since factor XII is activated along surfaces, this comparison is made by adding preformed factor XIIa to PK or PK–HK either in solution or bound to a surface). Figure 20.2 also depicts a positive feedback in which kallikrein activates surface-bound factor XII to form factor XIIa (Cochrane et al. 1973; Meier et al. 1977; Silverberg et al. 1980b; Dunn et al. 1982). In fact the bound factor XII undergoes a conformational change that renders it a substrate for factor XIIa (Dunn et al. 1982). Thus autoactivation of factor XII can initiate the cascade once sufficient factor XIIa forms to overcome plasma inhibitors (Silverberg et al. 1980b; Tankersley & Finlayson 1984) and only a few percent conversion to factor XIIa is required. Then the kallikrein formed activates the remaining surface-bound factor XII at a much more rapid rate. This positive feedback is also accelerated by the presence of HK (Griffin & Cochrane 1976; Meier et al. 1977; Silverberg et al. 1980b). The factor XIIa formed remains attached to the initiating surface; further digestion of factor XIIa by kallikrein (Fig. 20.2) yields a 32.5-kDa factor XII fragment (factor XIIf) (Kaplan & Austen 1970, 1971; Dunn & Kaplan 1982), which retains the active site of factor XIIa but lacks the binding site to the surface. It is a doublet on SDS gel, with bands at 30 and 28.5 kDa. Factor XIIf consists of the light chain of factor XIIa, containing the active site, disulfide linked to small C-terminal fragments of the heavy chain of 2000 or 500, corresponding to the two SDS bands. Factor XIIf lacks the binding site for the surface and is released into the fluid phase where it can continue to activate PK until it is ultimately inactivated by plasma protease inhibitors. Factor XI is activated by factor XIIa in the presence of HK, but factor XIIf possesses only 2– 4% of the coagulant activity of factor XIIa and HK does not augment its reaction rate. Thus factor XIIf can be important for bradykinin formation, but not for intrinsic coagulation.

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The mechanism by which HK catalyzes factor XII activation is multifactorial and indirect since HK does not increase the enzymatic activity of kallikrein, nor does it interact with factor XII to render it a better substrate (Silverberg et al. 1980b). Its main effect is to allow dissociation of kallikrein from its complex with HK so that it can enzymatically activate factor XII along the cell surface (Cochrane & Revak 1980). Kallikrein bound directly to the surface is much less effective and cannot disseminate the reaction (Silverberg et al. 1980b). Since HK is required for the formation of kallikrein, i.e., activation of PK, the amount of kallikrein is increased when HK is present. Thus the effective ratio of kallikrein/factor XII in this enzymatic reaction is significantly augmented when HK is present. The percentage augmentation of contact activation in the presence of a surface plus HK is estimated to be 3000–12 000 fold (Tankersley & Finlayson 1984; Rosing et al. 1985). If one considers the rate of factor XI activation, HKdeficient plasma is almost as abnormal as factor XII-deficient plasma in a coagulation assay. HK increases the rate of formation of kallikrein, facilitates factor XII conversion to factor XIIa by kallikrein, and facilitates factor XI activation by factor XIIa. For comparison, it is of interest to consider the rate of factor XI activation in PK-deficient plasma where the kallikrein feedback activation of factor XII is not possible, and the only role of HK is in conversion of factor XI to factor XIa. In this case contact activation of coagulation is very slow, but if the time of incubation of citrated plasma with the surface is increased prior to recalcification, the clotting time approaches normal (Wuepper 1973; Saito et al. 1974; Weiss et al. 1974). This is due to gradual conversion of factor XI to factor XIa as a result of factor XII autoactivation on the surface. The next step, conversion of factor IX to factor IXa by factor XIa, is dependent on calcium, and thus prolonged incubation allows the amount of factor XIa to increase toward normal. It should be evident from Fig. 20.2 that plasma deficient in factor XII, PK, or HK cannot generate bradykinin via contact activation. Any bradykinin formed is dependent on tissue kallikrein activation of LK, or for factor XII-deficient plasma a bypass that requires endothelial cells, PK, and HK (see below). A detailed discussion of the structure of each protein, transcriptional and translational events involved in the synthesis of each protein, and mechanistic details regarding activation of factor XII, PK, and factor XI has been presented in Kaplan et al. (1997) and the reader is referred to this publication for further details.

Assembly on cell surfaces Binding of HK to human umbilical vein endothelial cells The first studies of the interaction of proteins of the kininforming cascade with cells were performed initially with platelets (Greengard & Griffin 1984; Gustafson et al. 1986)

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and then human umbilical vein endothelial cells (HUVECs) (Schmaier et al. 1988; van Iwaarden et al. 1988). In each instance, HK was shown to bind to each cell type in a zincdependent fashion. The binding was saturable and reversible, although binding was found to be dependent on domains 3 and 5 (Reddigari et al. 1993a; Hasan et al. 1995; Herwald et al. 1995) so that both heavy chain and light chain were capable of similar ion-dependent interactions (Reddigari et al. 1993a). Factor XII interacts with HUVECs in a similar fashion to HK; the interaction requires zinc ion and factor XII and HK can compete for binding to the cell surface (Reddigari et al. 1993b). The latter observation suggests that they bind to very similar cell-surface proteins with comparable affinity (Fig. 20.5). This binding protein was purified and further characterized by Joseph et al. (1996). These results are summarized below,

120

% 125I-FXII bound

100 80 60 40 Factor XII HK Human lgG

20 0 0

10

(a)

100

1000

10000

1000

104

[Competitor], nM 120

% 125I-HK bound

100 80 60 40 20 0 1 (b)

10

100 Factor XII, nM

Fig. 20.5 High-molecular-weight kininogen (HK) competes with factor XII for the same binding sites on human umbilical vein endothelial cells (HUVECs). (a) HUVECs were incubated with 1 mg/mL [125I]-FXII in triplicate in the presence of incremental concentrations of unlabeled factor XII, HK, or normal human IgG for 120 min and bound ligand was determined. The percentage bound in the presence of a competitor is plotted against the concentration of the competitor. (b) HUVECs were incubated with 1 mg/mL [125I]-HK in triplicate in the presence of increasing concentration of unlabeled factor XII and bound ligand was determined.


and correspond to a p33 endothelial cell protein isolated by Herwald et al. (1996) and identified to be gC1qR, the receptor for the globular heads of C1q discovered by Ghebrehiwet et al. (1994). A solubilized endothelial cell membrane preparation was passed over an HK affinity column in the presence or absence of zinc ion, eluted, fractions neutralized, and an immunoblot performed with biotinylated HK. A prominent increase in HK binding was observed after elution in the presence of zinc. These fractions were then pooled, concentrated, and analyzed by SDS-PAGE. The main feature was the appearance of a new prominent band at 33 kDa that was visible with a Coomassie Blue stain, and ligand blot with biotinylated HK demonstrated binding only to the 33-kDa band. Based on this information, the 33-kDa protein was subjected to N-terminal amino acid sequence analysis and the first 13 amino acids were found to be identical to the known NH2 terminus of gC1qR (Ghebrehiwet et al. 1994). A Western blot using anti-gC1qR monoclonal antibody 60.11 was employed to further assess the identity of these two proteins. Monoclonal antibody 60.11, which interacts with an epitope at the N-terminus of gC1qR, identified the 33-kDa HUVEC-derived membrane-binding protein. The possible binding of factor XII to gC1qR was studied next. HUVEC membrane-purified gC1qR or recombinant gC1qR at 1.0–2.0 μg were applied to nitrocellulose membranes and blotted with biotinylated HK or factor XII in the presence or absence of 50 μmol/L zinc. We found that both HK and factor XII bind to either purified or recombinant gC1qR in the presence of zinc. Addition of excess unlabeled HK reversed the ability of factor XII to bind to gC1qR by over 90% as quantitated by scanning, which suggests interaction with a common domain within the protein. Factor XII only partially (46%) reverses HK binding. This difference may be due to relative affinity of the two ligands for the gC1qR molecule. Nevertheless, these data completely paralleled those observed on binding of factor XII and HK to endothelial cells. Herwald et al. (1996) also demonstrated that gC1qR is a major endothelial cell binding protein for HK used a domain 5-derived peptide from the light chain rather than whole HK as the ligand. In aggregate these data indicate that gC1qR serves as a zinc-dependent binding protein for factor XII as well as for HK, and that binding to HK occurs via the light chain moiety. The specific location within HK for binding to endothelial cells is within domain 5 (Hasan et al. 1995; Schmaier 2003) and this also appears to be the site of interaction with gC1qR. It is also clear that the HK heavy chain also binds to endothelial cells. This interaction has been shown to require domain 3. The methods employed to identify the cell-surface protein that interacts with heavy chain are analogous to those described above for isolation of gC1qR. Later, a second HKbinding protein was identified in HUVECs by affinity chromatography employing HK as ligand (Hasan et al. 1998; Shariat-Madar et al. 1999) and was identified as cytokeratin 1.

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It seemed likely that this protein would contribute to heavy chain interaction with cells and an affinity column was prepared by covalently coupling peptide LDC27 sequence to the matrix; this is a 27-amino acid peptide derived from domain 3 that has been identified as an HK-binding site (Herwald et al. 1995). When cell membranes derived from HUVECs were applied to the column in the presence and absence of 50 μmol/L zinc and each eluted with 0.1 mol/L glycine-HCl, pH 2.5, a band was noted at 68 kDa in the zinc-containing eluate. A ligand blot with HK confirmed binding to this band. Attempts to sequence the protein failed because the Nterminus was blocked. It was therefore digested with cyanogen bromide and the mixture subjected to mass spectrometry. A major peptide at molecular weight 2721.7 was identified, its sequence determined, and shown to correspond to an internal peptide derived from cytokeratin 1 (see Fig. 20.4) (Joseph et al. 1999a,b). Thus HK binding to HUVECs appears to depend on interaction with two proteins, cytokeratin 1 and gC1qR, with binding to each by domains 3 and 5 of HK respectively (i.e., binding of heavy chain to cytokeratin 1 and light chain to gC1qR). We demonstrated that gC1qR cannot bind heavy chain at all, whereas cytokeratin 1, when tested as a purified protein, can bind both heavy and light chains, although binding to heavy chain clearly predominates. Factor XII is capable of binding to both proteins. To confirm that these proteins are important for binding to endothelial cells, we performed an inhibition experiment in which antibody to gC1qR and antibody to cytokeratin 1 were employed. Antibody to gC1qR inhibited zinc-dependent binding by 65%, antibody to cytokeratin 1 inhibited binding by 30%, while a combination of antisera inhibited binding by 85%. Since 15% binding corresponds to zinc-independent binding, our data suggest that we accounted for most if not all HK binding to endothelial cells by these two proteins. A third protein has been reported to be important for HK binding to HUVECs and this has been identified as the urokinase plasminogen activator receptor (u-PAR) by inhibition of HK binding with antisera to domain 2/3 of u-PAR (Colman et al. 1997). However, our laboratory has not been able to isolate u-PAR from cell membranes (confirmed to contain considerable u-PAR) by HK affinity chromatography. One difference in the experiments is that the studies by Colman et al. employed cleaved HK lacking the bradykinin moiety and it is possible that cleaved HK binds more avidly to u-PAR than does native HK, while native HK binds more avidly to gC1qR and cytokeratin 1 than it does to u-PAR.

Binding of factor XII to HUVECs Early studies demonstrated that factor XII binds to both gC1qR and cytokeratin 1 and that it competes for the same binding sites as HK (see Fig. 20.5). The first study to attempt to identify the binding site on HUVECs (rather than testing purified proteins shown to bind HK) was by Mahdi et al. (2002) in which antisera to u-PAR, cytokeratin 1, and gC1qR

456

Endothelial cell

Zn2+

B2 receptor 2+

Zn

HK FXII

BK

2+

Zn

2C-HK

HK PK

K

FXIIa Fig. 20.6 Generation of bradykinin (BK) along the endothelial cell surface via zinc-dependent interaction of high-molecular-weight kininogen (HK) and factor XII with endothelial cell surface receptors (representing gC1qR, cytokeratin 1, and u-PAR). PK, prekallikrein; K, kallikrein. (See CD-ROM for color version.)

were employed to inhibit cell binding of factor XII. A surprising result was that antibody to u-PAR inhibited best, although the other antisera were contributory. We therefore sought to corroborate the observation by isolation of factor XII-binding proteins directly from HUVEC-derived cell membrane preparations by affinity chromatography employing factor XII as ligand. The major zinc-dependent binding protein was clearly u-PAR; gC1qR was also isolated as well as small amounts of cytokeratin 1 (Joseph, K. and Allen P. Kaplan, unpublished data). A higher avidity of binding of factor XII to u-PAR than to either gC1qR or cytokeratin 1 was demonstrated by competitive displacement of factor XII bound to one protein employing increasing quantities of either of the other two. Thus a model for assembly of the kinin-forming cascade on endothelial cells was developed in which zinc-dependent binding of factor XII is associated predominantly with u-PAR, and HK binds to gC1qR as well as cytokeratin 1 while PK is bound to HK. A diagrammatic representation of such binding is shown in Fig. 20.6, as well as activation of the cascade along the endothelial cell surface to generate bradykinin. Table 20.1 summarizes the interactions of the proteins of the kinin-forming cascade with the endothelial cell-binding proteins for each. How that binding occurs, particularly for HK, depends on the way these proteins are distributed within the cell membrane of HUVECs.

Interaction of gC1qR, cytokeratin 1, and u-PAR within HUVEC cell membranes A dilemma posed by antibody inhibition studies in which all three antisera were employed was that the total inhibition obtained with all three antisera combined exceeded 100%. One possible explanation is that these proteins might interact in some fashion within the cell membrane and a trimolecular complex containing all three was proposed (Colman & Schmaier 1997).

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Table 20.1 Plasma inhibitors of enzymes of contact activation: relative percentage contributions to inhibition in normal human plasma. Enzyme Inhibitor

Factor XIIa

Factor XIIf

Kallikrein

Factor XIa*

C1 inhibitor Antithrombin III† a2-Macroglobulin a1-Protease inhibitor a2-Antiplasmin

91.3 1.5 4.3 – 3.0

93 4 – – 3

52 (84)† ND 35 (16)† ND ND

8 (47) 16 (5) – 68 (23.5) 8‡ (24.5)

* Data given are from kinetic studies and irreversible complexes formed in plasma are given in parentheses. † Data obtained from generation of kallikrein in situ. ‡ Data are for results obtained in the absence of added heparin. ND, not determined separately.

We (and others) have addressed each of these issues. Employing high-titer monospecific antisera to gC1qR, the protein was clearly demonstrated to be at the HUVEC cell surface (Joseph et al. 1999b). Mahdi et al. (2001, 2002) then demonstrated the presence of all these proteins within cell membranes by immunoelectron microscopy. Cytokeratin 1 and u-PAR appeared to be colocalized while gC1qR was present throughout the cell membrane. The interactions of these proteins with each other was then examined. First it was shown that gC1qR binds to cytokeratin 1 but not u-PAR, while u-PAR also binds to cytokeratin 1 but not gC1qR. Thus a trimolecular complex is not possible but two bimolecular complexes seemed feasible. gC1qR and u-PAR were then precipitated from cell membrane preparations and the composition of the precipitated materials was analyzed. Cytokeratin 1 was precipitated with both anti-gC1qR and anti-u-PAR; however, the gC1qR/cytokeratin 1 fraction had no u-PAR, whereas the cytokeratin 1/u-PAR fraction contained no gC1qR. A current view of the assembly of the proteins of the kinin-forming cascade on HUVECs envisions factor XII bound to a complex of u-PAR/cytokeratin 1 while HK binds to a complex of gC1qR/cytokeratin 1. It is not known whether HK binds to the complex by both domain 3 and domain 5 simultaneously or whether binding to one site affects binding to the other. Complicating this assessment is the fact that the number of gC1qR sites within the cell membrane is at least three times that of u-PAR or cytokeratin 1; thus gC1qR unassociated with either cytokeratin 1 or u-PAR is likely present and can bind HK or factor XII. Given the relative affinities of factor XII, HK heavy chain, and HK light chain for gC1qR, we would anticipate preferential binding of light chain (domain 5) of HK to gC1qR. Consistent with this is the very prominent inhibition of HK binding to the cell employing peptide HKH20 derived from domain 5 of the light chain (Nakazawa et al. 2002).

Binding to other cells The interaction of factor XII and HK with other cell types resembles that seen in HUVECs, although there are differences in number of binding sites, affinity of binding, and nature of the binding proteins. We recently reported studies of microvascular endothelial cells derived from skin and lung, and compared binding with that seen in HUVECs since these cells are more likely to be “physiologic” (Fernando et al. 2003). Whereas HUVECs had approximately 710 000 binding sites per cell, dermal microvascular cells had 53 000 per cell and lung microvascular cells 316 000 per cell. The binding affinity was 2.5- and 10-fold higher for dermal and lung endothelial cells, respectively. It should be noted that a very large number of HK-binding sites were demonstrated for HUVECs by two separate methods, including a fluid-phase based assay where 850 000 binding sites per cell were documented. It has been suggested that values in the 107 range (Motta et al. 1998) may have been due to ligand binding to the plates used rather than to the cells coating the plates, and thus the cell surface represented only a fraction of the total binding seen (Baird & Walsh 2002, 2003). However, the inhibition of such binding employing antisera to the cellsurface ligands suggests some other interpretation (Mahdi et al. 2003), and our values in the fluid phase (696 427 binding sites/cell) are in close agreement with binding studies within microtiter plates (771 666 binding sites/cell). Binding of HK and factor XII to neutrophils and platelets has also been studied. HK interacts with neutrophils in a zinc-dependent fashion analogous to that seen with other cell types, although the protein with which it interacts is MAC-1 (C3bi receptor; CD11b/CD18) (Wachtfogel et al. 1994). Zinc-dependent HK binding to platelets is dependent on interaction with glycoprotein 1b (Bradford et al. 1997; Joseph et al. 1999c). Since both PK and factor XI circulate as complexes with HK, HK–factor XI and HK–PK should bind to

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HUVECs in an analagous fashion (Shariat-Madar et al. 2001). However, Baird and Walsh (2003) reported that although preformed HK–PK binds to endothelial cells as a complex, HK–factor XI does not. This is in contrast to studies in which HK is bound and factor XI is added separately, in which case factor XI will bind to HK. In response, Mahdi et al. (2003) agree that HK–PK predominates, but that HK–factor XI can bind with lower affinity. Binding to platelets differs because factor XI can interact with platelets in the absence of HK, and there appear to be separate receptors for factor XI and factor XIa (Sinha et al. 1984). Platelets also possess an intrinsic protein with factor XI activity that cross-reacts with plasma factor XI immunologically but differs in molecular weight and isoelectric point (Hsu et al. 1998). This form of factor XI is present even in patients deficient in plasma factor XI (Tuszynski et al. 1982) and has been shown to be an alternatively spliced form of factor XI in which one exon is missing (Hsu et al. 1998). During blood coagulation by the extrinsic (tissue factor) pathway, these forms of factor XI are more likely activated by thrombin feedback (Gailani & Broze 1991; Naito & Fujikawa 1991; Baglia & Walsh 2000; Baglia et al. 2002) than by factor XIIa to augment clot formation. This has been shown to occur as a late event within a fibrin matrix (Rand et al. 1996; Bouma & Meijers 2000). Thus activation on platelets involves factor XII-dependent (Walsh & Griffin 1981; Brunnee et al. 1993) and factor XII-independent (thrombin) pathways (von dem Borne et al. 1994), and factor XI may be attached to the platelets via HK or by separate receptors. LK, which has a separate light chain from HK, interacts with platelets (as is true of endothelial cells) and, of necessity, does so solely via domain 3 (Jiang et al. 1992a,b; Herwald et al. 1995) since the light chains of the two kininogens are completely different.

Activation of the kinin cascade: the role of endothelial cells Activation by binding to the cell surface Factor XII has been shown to slowly autoactivate when bound to endothelial cells and addition of kallikrein can digest bound HK to liberate bradykinin at a rate proportional to the kallikrein concentration, with a final bradykinin level dependent on the amount of bound HK (Nishikawa et al. 1992). Thus, activation of the cascade along the endothelial cell surface is likely; bradykinin is liberated and then interacts with the B2 receptor to increase vascular permeability. Bradykinin can also stimulate cultured endothelial cells to secrete tissue plasminogen activator (Smith et al. 1985), prostaglandin I2 (prostacyclin), thromboxane A2 (Crutchley et al. 1983), and nitric oxide and can thereby modulate platelet function and stimulate local fibrinolysis. To determine whether factor XII binding to gC1qR is capable of initiating this cascade, purified factor XII was incubated with a wide dose

458

range of gC1qR (0–100/μL) for 30 min and replicate samples were incubated in the absence of zinc ion. We found that the rate of PK conversion to kallikrein increased as the concentration of gC1qR increased (Joseph et al. 2001a,b) and there was no activation if zinc was eliminated from the reaction mixture. Purified cell membrane (native) gC1qR yields a response that is indistinguishable from a recombinant protein, indicating that gC1qR glycosylation does not affect its “surface” properties. If gC1qR is incubated directly with PK or with PK plus HK, there is no conversion of PK to kallikrein , again emphasizing the requirement for factor XII. This appears to be a physiologic phenomenon controlled by C1 inhibitor and α2-macroglobulin. This may be one source of the minute quantities of factor XIIa that escape inhibition that are requisite for contact activation in plasma or during pathologic processes and may be operative in patients with C1 inhibitor deficiency. Other data employing endothelial cells have corroborated the aforementioned effect of gC1qR when endothelial cells are incubated with normal plasma and the rate of kallikrein formation compared with that seen with plasma deficient in factor XII, PK, or HK. There was no detectable activation in any plasma except normal plasma (Fig. 20.7a) and the activation was inhibited by antisera to gC1qR and cytokeratin I (Fig. 20.7b). However, when the reaction proceeds beyond 2 hours, factor XII-deficient plasma activates, but HK-deficient plasma and PK-deficient plasma do not; thus a cell-dependent activation of PK in the presence of HK but absence of factor XII appears possible.

Factor XII-independent activation of the PK–HK complex Studies have demonstrated that binding of the PK–HK complex to endothelial cells leads to activation in the absence of factor XII (Rojkjaer et al. 1998; Rojkjaer & Schmaier 1999a,b) and that the kallikrein which forms can digest HK to liberate bradykinin and also initiate fibrinolysis (Lin et al. 1997). The latter reaction is dependent on kallikrein activating prourokinase (bound to cell membrane u-PAR) to urokinase, which in turn converts plasminogen to the fibrinolytic enzyme plasmin. Once such a reaction is set in motion, the addition of factor XII leads to a marked increase in reaction kinetics as a result of the conversion of factor XII to factor XIIa by kallikrein. These observations raise two important questions. First, what is the nature of the PK activator? Second, when factor XII is present (the normal circumstance), is the cascade initiated by factor XII autoactivation or is PK first activated by some cell-derived factor and kallikrein then activates factor XII? The cell-derived protein(s) responsible for PK activation in the absence of factor XII has been purified and characterized. The activity was present in the cell membrane fraction as well as the cytosol derived from endothelial cells and we chose to isolate it from the cytosol. The PK-activating moiety appeared to be inhibitable by corn trypsin inhibitor (CTI) (as

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Pooled normal plasma

Anti-gC1qR

PK-deficient plasma

Anti-cytokeratin 1

FXII-deficient plasma

0.20

Pooled normal plasma

0.25

HK-deficient plasma

Absorbance (OD 405 nm)


0.25


0.15

0.10

0.05

Anti-gC1qR

0.20

anti-cytokeratin 1

0.15

0.10

0.05 0

20

40

60

80

100

(b)

Incubation time (min)

(a)

0

120

20

40

60

80

100

120

Incubation time (min)

Fig. 20.7 Prekallikrein activation on endothelial cells. (a) Endothelial cells were incubated with normal, prekallikrein-deficient, factor XII-deficient or highmolecular-weight kininogen (HK)-deficient plasmas for 1 hour at 37°C. After incubation, the cells were washed with HEPES-buffered saline containing 50 mmol/L zinc chloride and prekallikrein activation was monitored by the cleavage of a kallikrein-specific substrate, S2302 (0.6 mmol/L) at 405 nm. (b) Endothelial cells were preincubated with antibodies to cytokeratin 1, gC1qR, or a combination of both for 30 min before addition of normal plasma.

is factor XIIa) and a CTI affinity column was shown to bind the activity and it was recoverable by eluting the column. A single-step purification followed by sequence analysis of suspect bands seen on SDS gel electrophoresis ultimately determined that heat-shock protein (HSP)-90 is responsible for the activity seen. Thus when cloned HSP-90 was incubated with PK and HK, the PK was converted to kallikrein and HK was cleaved to liberate bradykinin (Fig. 20.8) (Joseph et al. 2002a,b). This is also demonstrable by binding PK and HK to

0.9

HK+PK+HSP90+Zn HK+PK+HSP90

0.8

PK+HSP90+Zn


0.7

PK+HSP90

0.6 0.5 0.4 0.3 0.2 0.1 0 0

20

40

60 Time (min)

80

100

Fig. 20.8 Prekallikrein (PK) activation on heat-shock protein (HSP)-90. Purified HSP-90 (2 mg) was incubated with PK (20 mmol/L), highmolecular-weight kininogen (HK) (20 mmol/L), zinc (50 mmol/L), and S2302 (0.6 mmol/L) and chromogenic activity was monitored. Controls were performed in the absence of either zinc or HK, or both.

120

endothelial cells and assessing the rate of conversion of PK to kallikrein. Both HK and zinc ion are requisite and the rate is fast. However, this is in contrast to the very slow and factor XII-dependent activation seen when whole plasma is employed (Fig. 20.7). The reaction is readily demonstrable in the fluid phase as well as by assembly of components along the cell surface, although it differs strikingly from that seen with factor XIIa. The most critical difference is that PK is not activated unless HK is present. Factor XIIa readily activates PK, although the presence of HK does augment the reaction rate. Second, the reaction is stoichiometric, i.e., the amount of PK activated has a 1 : 1 molar ratio to the amount of HSP90 present. When the structural features of HK required for binding were determined, the individual heavy and light chains were inactive, and cleaved HK, with bradykinin removed (i.e., two-chain rather than single-chain HK), lost about 70% of the activity (Fig. 20.9). Thus native HK is required. Addition of a peptide that prevents the interaction of PK with HK also completely inhibits the effect of adding HSP-90. HSP-90 is therefore a stoichiometric activator of the PK–HK complex and not a PK activator as is factor XIIa. One of the interesting questions we might consider is whether HSP-90 has enzymatic activity, with the PK–HK complex as substrate. HSP-90 does have ATPase activity but it is not known to be a proteolytic enzyme. The PK-activating activity can be inhibited by diisopropylfluorophosphate (DFP) but it has been difficult to characterize the active site. Although DFP inhibits the reaction, we have been unable to incubate DFP with individual components, dialyze it out, and inhibit the reaction. In fact, if DFP is added to a mixture of HSP-90, PK and HK, it inhibits conversion of PK to kallikrein; however, if DFP is then dialyzed out of the mixture, PK activation

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Activation when factor XII is present

0.6


0.5

0.4

0.3

0.2

0.1

0 SC-HK

2C-HK

LK

HC-HK

LC-HK

Fig. 20.9 Effect of high-molecular-weight kininogen (HK) on prekallikrein activation. Cytosol (20 mg) was incubated with 20 nmol/L of HK, lowmolecular-weight kininogen (LK), cleaved HK (2C-HK), purified heavy chain of HK (HC-HK), or light chain of HK (LC-HK) in the presence of 20 nmol/L prekallikrein, 50 mmol/L zinc and 0.6 mmol/L S2302. After 2 hours the chromogenic activity was measured at 405 nm.

proceeds normally. Thus DFP behaves as a reversible inhibitor instead of as an irreversible inhibitor, and is not phosphorylating an active site serine as is its usual effect on serine proteases. Alternative possibilities were considered, for example autoactivation of PK within the PK–HK complex on addition of HSP-90, or even that HK becomes an enzyme that converts PK to kallikrein when HSP-90 binds, but the mechanism is thus far unknown. Other studies have isolated yet another protein with very similar functional capability. Shariat-Mader and colleagues isolated a membrane protein that converts PK to kallikrein within the PK–HK complex and identified it to be prolylcarboxypeptidase (Motta et al. 2001; Shariat-Madar et al. 2002). This is an exopeptidase which, if its enzymatic capability is relevant, is behaving as an endopeptidase. It is said to be active along the cell surface, but not in the fluid phase, which is different from HSP-90; otherwise, its mechanism of action is strikingly similar. Both require the presence of HK and zinc ion, the reaction in each case is stoichiometric, and each is inhibited by DFP. Although the polylcarboxypeptidase is assumed to be the enzyme that activates PK within the PK– HK complex, we suspect that some other mechanism may be operative, perhaps common to both. The prolylcarboxypeptidase provides an interesting link from the kinin-forming cascade to the biology of angiotensin since its function, when originally isolated, was to convert angiotensin II to angiotensin III, which inactivates it. Thus a molecule that can generate bradykinin, a vasodilator, inhibits another that is a vasoconstrictor. HSP-90 is also of particular interest since this is a protein that is constitutively present yet upregulated with tissue stress such as hypoxia or during an inflammatory response.

460

Since the endothelial cell participates in the activation of the bradykinin-forming cascade, when all three components are present, factor XII might be activated by autoactivation on gC1qR, requiring trace amounts of factor XIIa present in plasma, or factor XII might be activated by kallikrein. In the latter scenario, the source of kallikrein would be the stoichiometric interaction of PK–HK with HSP-90 and/or prolylcarboxypeptidase. Formation of factor XIIa then markedly accelerates activation of PK–HK since this reaction has typical Michaelis–Menten kinetics. Of course all these may be occurring simultaneously, but the evidence thus far suggests that the rate of activation of the PK–HK complex exceeds that of factor XII autoactivation. Thus it is possible that the initiation of the cascade on the surface is actually kallikrein, while factor XII has the role of an accelerator. Some favor this possibility, employing endothelial cells and purified protein constituents (Schmaier et al. 1987; Schmaier 1997, 1998; Rojkjaer et al. 1998). On a quantitative basis, the cascade remains factor XII-dependent. However, the data in Fig. 20.7 suggest that factor XII may truly initiate when whole plasma is studied, i.e., minimal dilution in the presence of all the plasma inhibitors.

Inhibition of contact activation Regulation of factor XII-dependent pathways occurs by both intrinsic and extrinsic controls. Cleavage of factor XIIa to XIIf (see Fig. 20.2) is one example of an intrinsic control. The factor XIIf produced is not surface bound and is a very poor activator of factor XI. At the same time, the heavy chain moiety, which has no enzymatic activity, retains the surfacebinding site and can compete with factor XII and HK for binding to the surface. Thus, the conversion of factor XIIa to factor XIIf will reduce the rate of the surface-dependent reactions of coagulation, whereas bradykinin generation via fluid-phase activation of PK continues. Similarly, digestion of kinin-free HK by factor XIa has been reported to limit its coagulant activity (Scott et al. 1985), although in this case the kinetics appear to be too slow to be of physiologic importance (Reddigari & Kaplan 1988). Extrinsic controls are provided by plasma inhibitors for each enzyme. Table 20.1 indicates the major inhibitors of each active enzyme and, where known, their relative contributions to the total inhibition in plasma. Inhibition of the contact activation proteases is clearly different from that of the rest of the coagulation pathways in that antithrombin III (ATIII) appears to play only a minor role. Instead, contact activation appears to be limited mainly by C1 inhibitor, which is not active against any of the other clotting factors except for inhibition of factor XI. C1 inhibitor is cleaved by the protease it inhibits and attaches to the active site in a covalent complex. It may remain in the stable form of the

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acyl enzyme intermediate that characterizes the normal serine protease mechanism (Travis & Salvesen 1983). Thus, after a protease has reacted with C1 inhibitor, it cannot digest protein substrates or hydrolyze small synthetic substrates, and the reaction of the active site serine with DFP is abolished. C1 inhibitor is the only major plasma inhibitor of factor XII and factor XIIf (de Agostini et al. 1984; Pixley et al. 1985). Although ATIII can inhibit activated factor XII (de Agostini et al. 1984; Cameron et al. 1989), its contribution to factor XIIa inhibition in plasma is apparently only a few percent of that due to C1 inhibitor (de Agostini et al. 1984; Pixley et al. 1985). Disagreement exists over the effect of heparin on the inhibition of activated factor XII by ATIII. Some investigators have observed little enhancement of the rate of factor XIIa inhibition (Pixley et al. 1991), whereas others have observed a significant increase (Cameron et al. 1989). Heparin can act as an activating surface for contact activation, and factor XII and factor XIIa can bind to it (Hojima et al. 1984; Silverberg & Diehl 1987). This binding is a factor in the inhibition by ATIII since inhibition of factor XIIf, which lacks the surfacebinding site, is not augmented in the presence of heparin as much as that of factor XIIa (Cameron et al. 1989). Curiously, α2-macroglobulin, although thought of as a “universal” protease inhibitor, does not significantly inhibit either form of activated factor XII. The two major inhibitors of plasma kallikrein are C1 inhibitor and α2-macroglobulin (Gigli et al. 1970; Harpel 1973; Harpel et al. 1985). Together they account for over 90% of the kallikrein inhibitory activity of plasma, with the remainder contributed by ATIII (Schapira et al. 1982a; van der Graaf et al. 1983a). When kallikrein is added to plasma, approximately half is bound to C1 inhibitor and half to α2macroglobulin (Harpel et al. 1985). α2-Macroglobulin does not bind to the active site of kallikrein but appears to trap the protease within its structure so as to sterically interfere with its ability to cleave large protein substrates. The degree of inhibition is greater than 95%, but the residual activity is detectable when assayed for lengthy incubation periods. In contrast, digestion of small synthetic substrates is much less affected, and approximately one-third of the starting activity is retained. When a surface such as kaolin is added to plasma so that kallikrein is generated in situ, close to 70 or 80% of it is bound to C1 inhibitor (Harpel et al. 1985). The reason for the difference between the patterns of inhibition of added kallikrein and of endogenously produced kallikrein is unknown. Interestingly, at low temperatures, most of the inhibition of added kallikrein is accounted for by α2-macroglobulin (Harpel et al. 1985); C1 inhibitor appears to be ineffective in the cold (Cameron et al. 1989) and this may underlie the phenomenon of “cold activation” of plasma. The inhibition of kallikrein by ATIII is also enhanced by heparin and may therefore become significant in heparinized plasma. The inhibition profile of factor XI is complicated by the


involvement of several factors. In kinetic studies of purified components, α1-antiproteinase inhibitor (α1-antitrypsin) appears to be the most significant inhibitor of factor XIa (Heck & Kaplan 1974; Scott et al. 1982a), whereas α1-antitrypsin is not a major inhibitor of other coagulation factors. However, when the generation of irreversible enzyme inhibitor complexes was assessed in plasma, C1 inhibitor was found to be the key inhibitor (Wuillemin et al. 1995), with approximately equal contributions by α2-antiplasmin and α1-antiproteinase inhibitor. ATIII is also an inhibitor of factor XI with potential for augmentation by heparin, the magnitude of which is unclear (Scott et al. 1982b; Beeler et al. 1986). The predominant role of C1 inhibitor in the regulation of contact activation in human plasma is underscored by the fact that it alone is an efficient inhibitor of activated factor XII, kallikrein, and factor XIa. In plasma from patients with hereditary angioedema, in which C1 inhibitor is absent, the amount of dextran sulfate required to produce activation is reduced tenfold compared with normal plasma (Cameron et al. 1989); similar results are obtained in cold plasma. Because some surface was still required for activation under these conditions, we may surmise that the other inhibitors which are active against the contact factors do serve to limit their reactions, but that in normal plasma it is inhibition by C1 inhibitor that forms the barrier to the initiation of contact activation. The plasma concentration of C1 inhibitor is approximately 2 μmol/L, and it is remarkable that its inhibition is ever overcome. That surfaces are able to induce activation must reflect the protection of the proteases at the surface from inhibition. It has also been proposed that kallikrein bound to HK is protected from inactivation by C1 inhibitor (Schapira et al. 1981, 1982b) and α2-macroglobulin (Schapira et al. 1982b; van der Graaf et al. 1983a,b) and that factor XIa is similarly protected from α1-antiproteinase inhibitor (Scott et al. 1982a); however, this mechanism has been ruled out in the case of kallikrein and C1 inhibitor (van der Graaf et al. 1983a; Silverberg et al. 1986).

Inactivation of bradykinin Bradykinin is an exceedingly potent vasoactive peptide that can cause venular dilatation, activation of arterial endothelial cells, increased vascular permeability, hypotension, constriction of uterine and gastrointestinal smooth muscle, constriction of the coronary and pulmonary vasculature, bronchoconstriction, and activation of phospholipase A2 to augment arachidonic acid metabolism. Its regulation is of prime importance, and a variety of enzymes in plasma contribute to kinin degradation. Carboxypeptidase N (Erdos & Sloane 1962) removes the C-terminal Arg from bradykinin to leave an octapeptide, des-Arg9-bradykinin (Sheikh & Kaplan 1986a), which is then digested by ACE, acting as a tripeptidase, to separate the tripeptide Ser-Pro-Phe from the

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pentapeptide Arg-Pro-Pro-Gly-Phe (Sheikh & Kaplan 1986b). Enzymes that have not been characterized rapidly digest Ser-Pro-Phe to individual amino acids and more slowly convert the pentapeptide to Arg-Pro-Pro plus Gly and Phe. The final products of bradykinin degradation are the peptide Arg-Pro-Pro, plus 1 mol each of Gly, Ser, Pro, and Arg, and 2 mol of Phe (Sheikh & Kaplan 1989b,c). The change from bradykinin to des-Arg9-bradykinin formed by this initial cleavage retains some but not all the various activities of bradykinin (Marceau & Bachvarov 1998). It can, for example, interact with B1 receptors (Regoli & Barabe 1980) induced by inflammation (e.g., IL-1, TNF-α) in the vasculature and cause hypotension, but bradykinin’s activities on the skin and the contraction of other smooth muscles are abolished. Bradykinin interacts with constitutively expressed B2 receptors to mediate all its functions. Selective B2 and B1 receptor antagonists have been synthesized (Vavrek & Stewart 1985; Beierwaltes et al. 1987; Stewart et al. 1999). When blood is clotted and serum is studied, all the reactions for bradykinin degradation occur as described, but the rate of the initial Arg removal is accelerated fivefold compared with plasma (Sheikh & Kaplan 1989b). This is probably due to the action of a plasma carboxypeptidase that is distinct from carboxypeptidase N and is expressed (activated) as a result of blood coagulation. It has been designated “thrombin activatable fibrinolysis inhibitor” (TAFI) (Bajzar et al. 1995, 1996). It should also be noted that bradykinin degradation in vivo occurs largely along the pulmonary vasculature and that endothelial cells there have carboxypeptidase as well as ACE activities. In the pulmonary circulation, the initial cleavage may occur by ACE acting as a dipeptidase to first remove Phe-Arg and then Ser-Pro (each of which is next cleaved to free amino acids), leaving the pentapeptide Arg-Pro-Pro-GlyPhe. This is then metabolized further. The cough, wheeze, and angioedema sometimes associated with use of ACE inhibitors for treatment of hypertension or heart failure is likely due to inhibition of kinin metabolism leading to increased levels of bradykinin (Nussberger et al. 1998). Because bradykinin is a peripheral vasodilator, it has been considered to be a counterbalance to the vasopressor effects of angiotensin II. It is clear that the two peptides are also related in terms of metabolism, because ACE cleaves His-Leu from the C-terminal of angiotensin I, a decapeptide, to leave the octapeptide angiotensin II. Thus, ACE creates a vasoconstrictor and inactivates a vasodilator. The prolylcarboxypeptidase discussed above does the reverse: it can produce bradykinin, a vasodilator, and inactivate angiotensin II, a vasoconstrictor.

Considerations in human diseases Bradykinin mediates its effects by interaction with receptors termed B1 and B2. B2 receptors were actually discovered first

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and exhibit high affinity for bradykinin and Lys-bradykinins (kallidin) and mediate effects such as vasodilatation, increased vascular permeability, and contraction of smooth muscle. A B2 receptor antagonist (Icatibond or HOE 140) is being evaluated for possible clinical utility, but has been used in animal models for many years. Once a ligand interacts with B2 receptors, there is rapid internalization and desensitization (Marceau et al. 2002). The B1 receptor, in contrast, is not present constitutively but is induced during an ongoing inflammatory process by cytokines such as IL-1 and TNF-α. This receptor is selectively activated by des-Arg9-bradykinin and des-Arg10-Lys-bradykinin, the products of carboxypeptidase removal of C-terminal Arg from bradykinin and kallidin, respectively. At least one additional cleavage, typically by ACE, is required to inactivate the system. Both B1 and B2 receptors signal via G proteins compled to them. However, the B1 receptor remains at the cell surface, once activated, and continues to function as long as ligands are available.

C1 inhibitor deficiency Although C1 inhibitor was defined as an inhibitor of the activated first component of complement, it is clearly a key control protein of the plasma kinin-forming cascade. The pathogenesis of the swelling in C1 inhibitor deficiency is dependent on the plasma kinin-forming pathway rather than complement; however, it is germane to review the history of the complement data and to point out how it was first thought to be the key, and then to present the more recent data that suggest otherwise. Intracutaneous injection of C1 into normal individuals was reported to cause the formation of a small wheal reaction whereas injection into patients with hereditary angioedema yielded localized angioedema, i.e., an augmented response because of low C1 inhibitor (Klemperer et al. 1968). A kinin-like peptide was isolated from such patients and its formation appeared to be inhibited in C2deficient plasma. Thus C2 was considered to be the source of the pathogenic peptide (Donaldson 1968). However, direct demonstration of such a kinin-like peptide on interaction of activated C1 and C4 and C2 or with C2 alone is lacking. Although it was originally reported that cleavage of C2b by plasmin generates a kinin (Donaldson et al. 1977), attempts to confirm this experiment have all failed (Fields et al. 1983). The only identifiable kinin seen in subsequent studies was bradykinin (Fields et al. 1983). On the other hand, the amino acid sequence of C2b is known, and Strang et al. (1988) synthesized peptides of various lengths and tested each for kinin-like activity. One such peptide was shown to cause edema when injected intracutaneously, reminiscent of the C2 kinin originally described. However, this peptide has not been shown to be a cleavage product of C2b, nor has it been shown to be present during attacks of swelling in patients with hereditary angioedema. Thus, at this point it seems unlikely that a kinin-like molecule is derived from C2b as a result

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of enzymatic cleavage. On the other hand, the presence of bradykinin has been documented, as described below, and it is the likely cause of the swelling. In fact when one of the proponents of the C2 kinin reexamined kinin formation in the plasma of patients with hereditary angioedema, only bradykinin was found (Shoemaker et al. 1994). It should be noted that 24-hour urine histamine excretion may also be increased during attacks of angioedema, suggesting that C3a, C4a, or C5a is being generated. Although the plasma levels of C3 and C5 are normal in this disorder, C3 turnover is clearly enhanced. The lesions, however, are not pruritic, and antihistaminics have no effect on the clinical course of the disease. Thus, complement activation is undoubtedly occurring, perhaps even during quiescent periods, leading to a low level of C4, but the vasoactive consequences of the augmented complement activation that occurs during attacks of hereditary angioedema do not appear to be the cause of the swelling. C1 inhibitor inhibits all functions of factor XIIa and is one of the two major plasma kallikrein inhibitors (Gigli et al. 1970), the other being α2-macroblobulin (Harpel et al. 1985), and all functions of kallikrein are thereby inhibited including the feedback activation of factor XII, the cleavage of HK, and the activation of plasma pro-urokinase (Ichinose et al. 1986) leading to plasmin formation. C1 inhibitor also inhibits the fibrinolytic enzyme plasmin, although it is a relatively minor inhibitor compared with α2-antiplasmin or α2-macroglobulin. Patients with hereditary angioedema appear to be hyperresponsive to cutaneous injections of kallikrein, as they are to C1, and elevated levels of bradykinin and cleaved kininogen have been observed during attacks of swelling (Nussberger et al. 1998). There is also evidence that C1 activation observed in hereditary angioedema may also be factor XII dependent (Donaldson 1968). Thus, a factor XII-dependent enzyme may be initiating the classic complement cascade. Plasmin is capable of activating C1s and may represent one such enzyme (Ratnoff & Naff 1967). Ghebrehiwet et al. (1981, 1983) demonstrated that Hageman factor fragment (factor XIIf) can directly activate the classical complement cascade by activating C1r and to a lesser degree C1s. This may represent a critical link between the intrinsic coagulation–kinin cascade and complement activation (see Fig. 20.2). The presence of kallikrein-like activity in induced blisters of patients with hereditary angioedema supports this notion, as does the progressive generation of bradykinin on incubation of hereditary angioedema plasma in plastic noncontact-activated test tubes (Fields et al. 1983) and the low PK and HK levels seen during attacks (Schapira et al. 1983). More recent data support these indirect observations, favoring bradykinin as the critical pathogenic peptide for hereditary angioedema and, probably, acquired C1 inhibitor deficiency as well. One unique family has been described in which there is a point mutation in C1 inhibitor (Ala443→Val), leading to inability to inhibit the complement cascade but normal inhibition


of factor XIIa and kallikrein (Zahedi et al. 1995, 1997). No family member of this type II mutation has had angioedema. In recent studies, plasma bradykinin levels have been shown to be elevated during attacks of swelling in patients with hereditary and acquired forms of C1 inhibitor deficiency (Cugno et al. 1996; Nussberger et al. 1998) and local bradykinin generation has been documented at the site of the swelling (Nussberger et al. 1999). The role of fibrinolysis also needs to be considered in the pathogenesis of the disease since antifibrinolytic agents such as ε-aminocaproic acid and tranexamic acid appear to be efficacious (Lundh et al. 1968; Frank et al. 1972; Sheffer et al. 1972) and plasmin is generated during active disease (Cugno et al. 1993). Although kallikrein, factor XIa, and even factor XIIa have some ability to activate plasminogen directly, the plasma pathway via the pro-urokinase intermediate appears to be the major factor XII-dependent fibrinolytic mechanism (Fig. 20.10). Among the functions of plasmin are activation of C1s, the ability to cleave and activate factor XII as kallikrein does (Kaplan & Austen 1971), and digestion of C1 inhibitor (Wallace et al. 1997). Each of these would serve to augment bradykinin formation and further deplete the levels of C1 inhibitor. Thus, the formation of plasmin may in this fashion contribute to the pathogenesis of the disease.

Regulation of blood pressure The possible relationship of the contact system to blood pressure regulation is an intriguing question. As already seen, ACE creates the hypertensive peptide angiotensin II, and plays a major role in inactivating the hypotensive product of contact activation, bradykinin. An unfortunate circumstance has dramatized the kinin-forming capacity of factor XIIf: trauma patients given plasma protein fractions as plasma expanders that were contaminated with factor XIIf showed profound hypotension (Alving et al. 1978, 1980). The mechanism by which infused factor XIIf causes hypotension has been demonstrated to be due to bradykinin formation. A more tenuous connection exists between blood pressure regulation and the contact system in the factor XII-dependent activation of plasma prorenin. Prorenin is activated to renin by cold treatment of plasma or acidification to pH 3.3. With acid treatment, most of the renin activity is produced after reneutralization; this alkaline-phase activation is mediated by kallikrein (Derkx et al. 1979; Sealey et al. 1979), as is the cold-induced activation (Brown & Osmond 1984). Kallikrein is able to activate purified prorenin (Yokosawa et al. 1979), but when added to plasma it does not cause prorenin activation in the absence of an acidification step. Although it has been supposed that acid treatment serves to destroy kallikrein inhibitors, prorenin is not activated in plasma deficient in C1 inhibitor or α2-macroglobulin (Purdon et al. 1985). Thus, some other unknown event must occur on acidification. The physiologic significance of this reaction is uncertain.

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Factor XII Prekallikrein HK Contact activation Activation along cell surface

Plasmin kallikrein Prorenin

Angiotensinogen

HK

Renin pH

HK Angiotensin I

ACE

Prekallikrein

Bradykinin

Prolylcarboxypeptidase

Angiotensin II

Angiotensin III ACE

Glucose

mRNA plasma prekallikrein

Plasma kallikrein

AT1 receptor B2 receptor upregulation

Kinins and ACE inhibition Severe angioedema often involving the face, tongue, or both is seen as a complication of the use of ACE inhibitors. It appears that this swelling is also a consequence of elevated levels of bradykinin (Nussberger et al. 1998); however, the accumulation of bradykinin is due to a defect in degradation rather than excessive production. ACE, being identical to kininase II, is the major enzyme responsible for bradykinin degradation (see Fig. 20.1) and although it is present in plasma, the vascular endothelium of the lung appears to be its major site of action (Alabaster & Bakhle 1992). The action of ACE always leads to the formation of degradation products with no activity, whereas kininase I alone yields the des-Arg products, which are capable of stimulating B1 receptors. The excessive accumulation of bradykinin implies that production is ongoing or that some event leads to activation of the plasma cascade or release of tissue kallikrein, and then faulty inactivation leads to swelling. Continuous turnover of the cascade is implied by data demonstrating activation along the surface of cells and cellular expression or secretion of a PK activator other than factor XII.

Kinins and allergic rhinitis Early studies suggested activation of the plasma bradykininforming cascade in allergic rhinitis on the basis of finding tosylarginine methyl ester esterase activity in the secretions, indicative of production of plasma kallikrein (Naclerio et al. 1983). Assessment of kinins by means of high-performance liquid chromatography demonstrated the presence of both lysyl-bradykinin and bradykinin during the immediate phase and the late phase of allergen-induced rhinitis (Proud et al.

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Arg-Pro-Pro-Phe + Ser-Pro + Phe-Arg

Fig. 20.10 Diagrammatic representation of the many interconnections between the renin–angiotensin pathway and the bradykinin-forming pathway. HK, highmolecular-weight kininogen.

1983; Naclerio et al. 1985). The presence of lysyl-bradykinin indicated release of tissue kallikrein, whereas bradykinin can be derived from lysyl-bradykinin by the action of plasma aminopeptidase or it can be produced directly by plasma kallikrein. Both HK and LK were present, and therefore the preferred substrate for each type of kallikrein was present (Baumgarten et al. 1985). Chromatographic assessment of the secretions demonstrated that both tissue kallikrein and plasma kallikrein are produced (Baumgarten et al. 1986a,b). Bradykinin can produce hyperemia, rhinorrhea, and nasal congestion; however, it has not been possible to assess its contribution to symptoms because a potent and selective bradykinin receptor antagonist that can be administered in vivo has not been available.

Bradykinin and asthma Bradykinin has many effects on the airway including both bronchoconstriction and bronchdilatation, stimulation of cholinergic and sensory nerves, mucus production, and edema (Barnes et al. 1998). Inhalation of bradykinin provokes prominent bronchoconstriction in asthmatics (Fuller et al. 1987; Polosa & Holgate 1990). Nevertheless, bradykinin causes bronchial relaxation in rodent models of asthma via prostaglandin E release if there is pretreatment of the muscle preparation by cholinergic agonists such as carbachol (Li et al. 1998). Studies of asthmatics have revealed the presence of kinins in bronchoalveolar lavage fluid. Early studies demonstrated tissue kallikrein in bronchoalveolar lavage fluid (Christiansen et al. 1992), but there has been no definitive assessment of the plasma bradykinin-forming cascade in bronchoalveolar

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lavage fluids or within lung parenchyma. Bradykinin challenge of asthmatic subjects leads to symptomatic and physiologic changes similar to those seen in the natural disease (Fuller et al. 1987), and allergen challenge of asthmatic subjects leads to an increase in kinin levels in conjunction with histamine release during both the early and late-phase time intervals (Liu et al. 1991; Christiansen et al. 1992). Of particular interest is that Icatibant (HOE 140), a B2 receptor antagonist, inhibited histamine-induced bronchial hyperresponsiveness induced by allergen in allergic rhinitis, and more prolonged treatment improved pulmonary function in asthmatic subjects followed for 1 month (Akbary et al. 1996; Turner et al. 2001). In rats a B1 receptor antagonist diminished airway hyperresponsiveness, suggesting induction of B1 receptors as a result of the inflammatory process (Huang et al. 1999). A role for kinins in general is suspected as mediators of bronchial hyperreactivity, but this need not be restricted to bradykinin or lysyl-bradykinin and may involve neurokinins secreted from type C sensory nerve fibers, such as substance P, neurokinin A, or vasoactive intestinal polypeptide. Bradykinin many be a stimulus for secretion of these neuropeptides (Geppetti 1993).

Bradykinin and anaphylaxis The hypotension of anaphylaxis is clearly multifactorial, including the effects of multiple vasoactive mediators leading to hypovolemia and edematous tissues. Bradykinin is certainly one of those capable of causing hypotension, and one study of insect-sting anaphylaxis demonstrated cleavage of HK concomitant with changes in blood coagulation and fibrinolytic parameters indicative of disseminated intravascular coagulation (Smith et al. 1980). Thus massive release of bradykinin seems likely, although kinin levels were not measured directly. It is of interest that in a rhesus monkey model of endotoxic shock, infusion of a monoclonal antibody to factor XII leads to a 50% improvement in blood pressure, although disseminated intravascular coagulation was unaffected.

Bradykinin formation The mechanism of bradykinin formation in any IgEmediated reaction is multifactorial, including not only tissue kallikrein secretion (see Fig. 20.1) but also direct activation of the plasma cascade by secreted heparin (Hojima et al. 1984; Brunnee et al. 1997), activation along the surface of endothelial cells, or exposure to connective tissue proteoglycans. Any local dilution of plasma constituents decreases the effect of protease inhibitors and leads to an increased rate of the enzymatic reactions to augment kinin formation. Plasma leakage can initially be the result of other permeability factors (e.g., histamine or leukotriene C4/D4), which is then augmented as bradykinin is produced. Bradykinin may be formed (and rapidly degraded) in virtually every type of inflammation. However, its contribution to disease manifestations can only


be assessed when we have high-potency oral agents or parenteral monoclonal antibodies that inhibit bradykinin formation or block its effects at the receptor level.

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Chemokines James E. Pease and Timothy J. Williams

Summary

Introduction

A basic cell function is the ability to detect a chemotactic gradient and move along it. This function is especially important in leukocytes. Leukocyte chemoattractants, such as C5a and formyl-methionyl-leucyl-phenylalanine (fMLP), have been known for a long time, but their lack of specificity did not explain how specific leukocyte types are able to localize to distinct sites under basal conditions or how these cells are recruited to sites of inflammation. This was a particular issue in allergic inflammation, which is characterized by Th2 lymphocyte recruitment and activation, and the accumulation of large numbers of eosinophils. The discovery of a large family of small proteins, the chemokines, in the 1980s began to throw light on the mechanisms involved in leukocyte recruitment. In humans, around 40 chemokines have been identified, small proteins that signal through 18 different G protein-coupled receptors. There has been extensive research into the differential expression of these receptors on different leukocyte types, and on what chemokines are generated in the context of different types of inflammatory reaction. The mechanisms involved in this cell–cell signaling are complex, as cell types tend to express more than one chemokine receptor and a particular chemokine often can act on more than one receptor. Moreover, chemokine receptor expression is a dynamic process, different receptors being upregulated or downregulated during the life history of the cell. Despite these difficulties, we now have a comprehensive knowledge of chemokine biology. Complementing this, smallmolecule antagonists have been produced that can render a particular cell type insensitive to the attraction of a defined chemokine. All this information can be utilized for the design of new drugs aimed at blocking leukocyte recruitment and, thus, the pathology associated with the activation of this cell once in the inflamed tissue. Choice of the chemokine receptor target has proved to be a major challenge for those trying to produce new therapeutic agents.

The ability to sense a chemical gradient and respond accordingly by migration is crucial to the functioning of many cells, whether they be cells from higher order organisms such as humans, or unicellular organisms such as the slime-mould Dictyostelium discoideum. This property, chemotaxis, is critical for reproduction, the organization of cells during development and growth, and repair. In higher order organisms, it is chiefly recognized because of its importance in localization of immune cells under basal conditions, and in their recruitment at sites of inflammation. The study of this process in Dictyostelium has shed much light on the basic mechanisms involved (Van Haastert & Devreotes 2004). Dictyostelium detects cAMP in its immediate environment using a G protein-coupled receptor (GPCR). The cAMP is released by the organisms themselves during times of nutrient deprivation, and coordinates the directed migration of cells toward one another. There, they cluster to form an aggregate known as a “fruiting body,” with the sole aim of maintaining spores until conditions are appropriate for their germination. The sensing of the cAMP gradient requires that the organism is able to detect tiny differences in concentration between the front of the cell and its rear, which direct its subsequent migration, a process known as chemotaxis. The GPCR for cAMP sits in the cell membrane and acts as a transducer, with the external cAMP gradient reflected as the degree of G-protein activation inside the cell. Subsequent activation of the enzyme phosphatidylinositol 3-kinase (PI3K) results in the generation of an intracellular gradient of secondary messengers such as phosphatidylinositol 3,4,5-trisphosphate (PIP3), with its highest concentration at the region of the cell membrane associated with the greatest amount of G-protein activation. This is further polarized by the activity of the enzyme PTEN, which is displaced from this region to the rear of the cell membrane where it inhibits the activity of PI3K by breaking down PIP3 into phosphatidylinositol 4,5bisphosphate (PIP2). This results in the amplification of the external polarizing signal and is followed by locomotion of the organism along the cAMP concentration gradient, which requires both activation of adhesion molecules at the front of the cell and their inactivation at the rear.


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Inflammatory Cells and Mediators receptors themselves are members of the GPCR superfamily there is cause for optimism, as over the last few decades successful generation of small molecule agonists and antagonists of these proteins has been productive and GPCR-selective drugs account for around half of the drugs currently prescribed (Fredriksson et al. 2003).

Chemokines In mammals, the family of chemokines (chemotactic cytokines) has been recognized as being particularly important in guiding cellular migration. Their discovery was a result of investigations by several laboratories of the molecules responsible for mediating inflammation. These proteins have an essential role in host defense, organizing the localization of leukocytes to specialized tissues, such as the thymus, under basal conditions, and also coordinating their recruitment and activation in response to inflammatory stimuli. Until the discovery of chemokines almost 30 years ago, the mechanisms by which specific subpopulations of leukocytes were selectively recruited to distinct sites in the body was poorly understood. The so-called classical chemoattractants such as C5a and fMLP that were known at the time were able to recruit several different leukocyte subtypes and did not explain the selective recruitment observed in vivo, for example the recruitment of eosinophils to the allergic lung. The identification of chemokines and their specific leukocyte cell-surface receptors has greatly enriched our understanding of the process of leukocyte recruitment. Since the excessive production of chemokines has been linked with the pathogenesis of several clinically important diseases, including asthma, considerable efforts have been undertaken to understand how chemokines and their receptors function at the molecular level, with a view to selective blockade as a means of modifying undesirable immune responses such as those seen in allergy. As the chemokine

Relationship between chemokines, cytokines, and adhesion molecules Before we look in detail at the molecules themselves, it is worth taking an overall view of the links between the cytokines, chemokines (a subdivision of cytokines), and cellular adhesion molecules to gauge the context in which chemokines exert their effects. Figure 21.1 depicts the recruitment of effector cells (in this case eosinophils) to sites of allergic inflammation. Th2 lymphocytes typical of allergic inflammation are activated by antigen-presenting cells (APCs) and release the cytokines interleukin (IL)-4, IL-13, and IL-5, able to induce synthesis of the chemokine CCL11/eotaxin-1 in airway epithelial cells. Eosinophils, like other leukocytes, form loose interactions with the luminal surface of the venular endothelium, via low-affinity binding of selectin adhesion molecules so that the cells tend to roll along the endothelial surface. CCL11 acts on CCR3 receptors on eosinophils within venules, and induces the upregulation of integrins on the eosinophil cell surface such as integrin α4β1. The integrins bind to complementary receptors on the venule wall resulting initially in the arrest of eosinophils, subsequently followed

Allergen

Epithelium APC (Mo/DC) IL-5

IL-4 IL-13 Th2 cell

Priming, survival

CCL11/eotaxin-1

Adhesion

Rolling Selectins

a4b1

Emigration

VCAM Venule

Eosinophil

Endothelium

472

Fig. 21.1 An overview of chemokines, cytokines, and cells in allergic inflammation. This diagram illustrates the many links between the cytokine, chemokine, and cellular adhesion systems, which mediate the recruitment of effector cells at sites of allergic inflammation typified by eosinophils. The role of the chemokine CCL11/eotaxin-1 is highlighted as inducing firm adhesion to the endothelium via the activation of integrin a4b1, and also the subsequent emigration of the eosinophils to the tissues. DC, dendritic cell; Mo, monocyte; VCAM, vascular cell adhesion molecule. (See CD-ROM for color version.)

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by their chemotactic migration through the vessel wall and to the source of CCL11 production, in this case the airway epithelia. The cytokine IL-5 increases the survival of accumulated eosinophils and also primes them for enhanced responses to diverse chemoattractants such as leukotriene (LT)B4 (Sehmi et al. 1992), fMLP (Sehmi et al. 1992) and CCL11 (Shahabuddin et al. 2000). Similarly, chemokine-mediated signaling can influence the downstream signaling pathways of other cell surface receptors. For example, although the chemokine CCL11 alone is unable to induce IL-4 production by basophils, it can readily potentiate IL-4 production following their stimulation with an allergen such as cat dander (Devouassoux et al. 1999).

Classification of chemokines In the human, around 40 chemokines have been identified through a combination of proteomic and genomic efforts. Chemokines are typically small proteins of around 8–10 kDa and, despite often low sequence homology, share the same common protein fold as deduced by NMR and crystallographic studies. The tertiary structure has both common elements of secondary structure and contains a “Greek key” motif, in which three antiparallel β-pleated strands overlie a C-terminal α helix (Fig. 21.2). With a few rare exceptions, four conserved cysteine residues within chemokines form intermolecular disulfide bonds, which serve to stabilize the overall conformation, typically in the order Cys1–Cys3 and Cys2–Cys4. The cysteine residues also allow the convenient subdivision of chemokines into four families, based on the arrangement of two cysteine residues within the aminoterminal region of the protein (Fig. 21.3). The majority of chemokines fall into either the CC or CXC classes, where the

Fig. 21.2 Secondary structural elements of chemokines as typified by the CC chemokine CCL5/RANTES. The three antiparallel b-pleated sheets (the so-called “Greek key” motif) overlay a C-terminal, a-helical domain. The basic residues located in the region known as the “40s loop” and implicated in glycosaminoglycan binding are shown as ball-and-stick representations. (See CD-ROM for color version.)

two amino-terminal cysteines are either adjacent or have a single amino acid inserted between them (Zlotnik & Yoshie 2000). Two further classes of chemokine have also been described sharing only three members between them; namely the C class featuring a single amino-terminal cysteine residue and the CX3C class in which the two cysteines are separated by three residues. This latter class is quite intriguing as the sole member, a chemokine called CX3CL1 or fractalkine, is expressed as a membrane-bound form on the end of a

CC chemokines

C chemokines

NH2

NH2

C C C CC

C

C

COOH

Fig. 21.3 Subdivision of the chemokine family. Shown are the four classes of chemokines based upon their arrangement of amino-terminal cysteine residues. Conserved cysteine residues are shown as black spheres with disulfide bonds depicted as dashed lines. The sole CX3C ligand is presented as a bound form attached to a mucin-like stalk decorated with oligosaccharides (diamonds).

CXC chemokines

CX3C chemokines NH2

NH2 C

C

C C

COOH

C C C

C

COOH

COOH

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mucin-like stalk. This allows it to serve as an effective adhesion molecule, allowing leukocytes expressing the respective cognate receptor to bind. CX3CL1 can also be released from the cell surface as a soluble chemokine by the action of metalloproteinases such as ADAM17 (Garton et al. 2001). Likewise, the CXC chemokine CXCL16 is also expressed in both stalk and soluble forms. The majority of CXC and CC chemokines have been shown by NMR and crystallographic studies to form dimers and higher order oligomers, although the biological significance of this dimerization is unclear. The concentrations of chemokines employed for these studies are typically in the millimolar range, several orders of magnitude higher than those observed in vivo. Earlier studies of the chemokine IL-8 suggested that at physiologic concentrations, the dimer readily dissociates to the monomeric form and it is this form which activates its specific receptor (Burrows et al. 1994). In contrast, studies of an amino-terminal deletion variant of monocyte chemoattractant protein (MCP)-1, which is able to form heterodimers with wild-type MCP-1, was found to act as a dominant negative, inhibiting monocyte chemotaxis in response to MCP-1 (Zhang & Rollins 1995).

Nomenclature of chemokines Like many areas of molecular immunology, intensive research in the chemokine field has been undertaken by several different laboratories worldwide, with the result that many chemokines were discovered simultaneously and given quite different colloquial names, usually descriptive of their activity. An extreme example is the chemokine known as PARC (pulmonary and activation-regulated chemokine), MIP-4 (macrophage inflammatory protein-4), AMAC-1 (alternative macrophage activation-associated CC chemokine-1), and DC-CK1 (dendritic cell chemokine-1). This created a sense of confusion even among those in the field, and consequently a systematic nomenclature was devised and introduced (Zlotnik & Yoshie 2000). Chemokines are now given the prefixes CCL (CC ligand), CXCL (CXC ligand), CX3CL (CX3C ligand), and XCL (C ligand), together with an identifying number. Table 21.1 shows the chemokines identified to date in the human, together with their previous colloquial numbers where given. This nomenclature has been embraced by the research community and is used throughout the remainder of this chapter.

Interaction of chemokines with glycosaminoglycans Chemokines bind readily to proteoglycans, molecules expressed on the surface of many cells such as endothelial cells and

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which consist of a core protein and glycosaminoglycan (GAG) side chains. These GAG chains typically contain repeating disaccharide units and can often be up to 200 molecules in length. The presence of carboxyl groups and high levels of sulfation results in a high density of negative charge, which can interact electrostatically with the basic chemokine (Witt & Lander 1994; Hoogewerf et al. 1997; Kuschert et al. 1999). These interactions are typically of micromolar affinity, although some chemokines such as CXCL4/platelet factor 4 can bind to GAGs with nanomolar affinity (Loscalzo et al. 1985), indeed CXCL4 was discovered by virtue of its affinity for the GAG heparin (Deuel et al. 1977). The interaction of chemokines with endothelially expressed proteoglycans has been proposed to immobilize a high concentration of chemokine locally upon the luminal surface of the venular endothelium, maintaining chemokine concentrations under conditions of shear flow (Handel et al. 2005). The GAG binding domains of a few CC chemokines have been determined by a combination of structural studies and site-directed mutagenesis and they are typically constructed of a cluster of basic residues located within a region known as the “40s loop” (Fig. 21.2). A mutant CCL5 molecule in which thee basic residues within this cluster were mutated to alanine was observed to be unable to bind to GAGs yet still maintained its in vitro chemotactic activity (Proudfoot et al. 2001). In contrast, the same mutant chemokine was found to be unable to recruit cells when administered intraperitoneally to mice, suggesting that GAG binding is essential for in vivo activity of certain chemokines (Proudfoot et al. 2003).

Chemokine receptors Like many other cytokines, chemokines mediate their effects upon leukocytes by engagement with cell surface receptors. These belong to the superfamily of GPCRs, which is thought to make up almost 5% of the coding portion of the human genome and number over 1000 members (Fredriksson et al. 2003). Chemokine receptors are typically in the region of 350 amino acids long, and belong to the class A of rhodopsin-like receptors, with seven putative hydrophobic regions thought to take the form of α helices that span the cell membrane in a serpentine fashion, leaving the amino terminus extracellular and the carboxyl terminus intracellular (Fig. 21.4). To date, 19 human chemokine receptors have been formally identified, with 10 binding CC chemokines, seven binding CXC chemokines, and solitary receptors for the CX3C and C chemokines (Murphy et al. 2000; Murphy 2002). Nomenclature of the receptors is similar to that of the chemokines with CCR, CXCR, CX3CR used to depict the class of chemokine receptor and a number used to distinguish it from other members of the family (Table 21.2). XCR is used to describe

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Table 21.1 Human chemokines. The systematic names of chemokines, together with their most common colloquial names and receptor agonist activity are shown, although some human chemokines appear to be missing from the list, e.g., CCL6. In such instances, while a chemokine of that name has been identified in the mouse, no human ortholog has been documented. Systematic name

Colloquial name

Receptor usage

Systematic name

Colloquial name

Receptor usage

CCL1 CCL2 CCL3 CCL3L1 CCL4 CCL4L1 CCL5 CCL7 CCL8 CCL11 CCL13 CCL14 CCL15 CCL16 CCL17 CCL18

I-309 MCP-1/MCAF MIP-1a/LD78a LD78b MIP-1b LAG-1 RANTES MCP-3 MCP-2 Eotaxin MCP-4 HCC-1 HCC-2/Lkn-1/MIP-1d/MIP5 HCC-4/LEC TARC DC-CK-1/AMAC-1/ MIP5/PARC/MIP-4 MIP-3b/ELC/Exodus-3 MIP3a/LARC/Exodus-1 SLC/6Ckine/Exodus-2 MDC/STCP-1 MPIF-1 MPIF-2/eotaxin-2 TECK Eotaxin-3 CTACK/ALP/ILC/ESkine MEC

CCR8 CCR2 CCR1,5 CCR1,5 CCR5 CCR5 CCR1,3,5 CCR1,2,3 CCR3 CCR3 CCR2,3 CCR1 CCR1,3 CCR1 CCR4 Unknown

CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL16

GROa/MGSA-a GROb/MGSA-b GROg/MGSA-g PF-4 ENA-78 GCP-2 NAP-2 IL-8 Mig IP-10 I-TAC SDF-1a /b BLC/BCA-1 BRAK/Bolekine SR-PSOX

CXCR2 CXCR2 CXCR2 CXCR3B CXCR2 CXCR1,2 CXCR2 CXCR1,2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR5 CXCR6

CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CCR10,3

XCL1 XCL2

Lymphotactin/SCM-1a/ATAC SCM-1b

XCR1 XCR1

CX3CL1

Fractalkine, neurotactin

CX3CR1

CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28

the sole receptor for the C class chemokines and is so named to distinguish it from the complement receptors which have the prefix CR. Ligand binding by chemokine receptors is typically of low nanomolar affinity and is class restricted, in that CXC receptors generally bind only chemokines of the CXC family and, likewise, CC receptors bind only chemokines of the CC family. Receptors are generally promiscuous and bind several different ligands, with a distinctive “pecking order” in terms of affinity for receptor, and potency and efficacy in in vitro assays such as chemotaxis and intracellular calcium flux.

Structural determinants of chemokine binding and receptor activation The amino termini of chemokine receptors are typically negatively charged, containing numerous acidic side chains. This is thought to play a part in tethering the basic chemokine to

the receptor with high affinity. This interaction may be facilitated further by glycosylation or sulfation of additional sidechain residues within the receptor amino terminus (Farzan et al. 1999; Bannert et al. 2001). Experiments in which the amino termini of chemokine receptors were exchanged to generate chimeric constructs suggest that activation occurs in two stages. Following initial binding of the chemokine to the amino terminus of the receptor, the chemokine is subsequently “delivered” to the remaining extracellular regions of the receptor (Monteclaro & Charo 1996; Pease et al. 1998). This second interaction most likely induces a conformational change within the intracellular regions of the receptor, increasing the affinity of the receptor for heterotrimeric G proteins and initiating intracellular signaling. Structural information regarding the activation of chemokine receptors is scant, as only one GPCR has had its conformation solved by

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N

N

C

b g PI3K

PLC

PIP2

a

DAG

MAPK

+ 2+ i[Ca ]

IP3

Akt

CHEMOTAXIS Fig. 21.4 Chemokine receptors and downstream signaling. A diagram depicting the binding of chemokines by their receptors and the subsequent activation of downstream signaling cascades. Chemokines (black) are thought to be tethered to the amino-terminus of the receptor, allowing the chemokine N-terminus to activate the receptor by disruption of interhelical interactions. This facilitates the recruitment of heterotrimeric G proteins to the receptor, which induce a series of intracellular signals resulting typically in chemotaxis. DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

crystallographic techniques, namely rhodopsin (Palczewski et al. 2000). As chemokine receptors belong to the same class A group of GPCRs as rhodopsin, homology modeling approaches using rhodopsin as a template allow structures to be generated that can be examined by site-directed mutagenesis. Conserved between the majority of class A GPCRs is an aspartate–arginine–tyrosine motif (Vincent et al. 1991) at the cytoplasmic end of the third transmembrane α helix, which is thought to act as an ionic lock, holding the receptor in an inactive state prior to its activation by ligand (Ballesteros et al. 2001). On ligand binding, a conformation change in the GPCR is believed to result in rearrangement of the intracellular loops, facilitating the recruitment of G protein. The importance of the DRY motif in chemokine receptor structure and function was shown in a collaborative study from

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our own group, where nonconservative mutagenesis of this domain in the receptor CCR3 led to a dramatic loss of function and greatly reduced cell-surface expression (Auger et al. 2002). Homology modeling and mutagenesis studies of the receptor CCR5 have given insights into chemokine receptor activation, highlighting an interaction between the N-terminus of the chemokine and transmembrane helices of the receptor (Blanpain et al. 2003; Govaerts et al. 2003). In the inactive receptor, the side chains of helix II and helix III are believed to make contact via hydrophobic interactions, the perturbation of which by the chemokine N-terminus, is thought to induce the conformational changes needed for receptor activation (Fig. 21.4). This fits nicely with experimental data describing the truncation of the chemokine N-terminus. Truncation of the chemokine CCL5/RANTES by eight residues to generate CCL5 (9–68) produces a receptor antagonist as deduced by several assays, blocking responses to ligands that use the same receptor (Gong et al. 1996). Likewise, extension of the CCL5 N-terminus with a single methionine residue (Met-CCL5/RANTES generates a potent receptor antagonist (Proudfoot et al. 1996).

G protein coupling of chemokine receptors and downstream signaling Like other members of the GPCR super family, chemokine receptors relay signals from the exterior to the interior of the cell, by interacting with heterotrimeric G proteins which comprise three subunits named α, β, and γ. While around 20 α subunits have been identified in mammals, experiments employing pertussis toxin suggested that Gαi proteins were primarily responsible for signaling downstream from chemokines, as physiologic responses such as chemotaxis are readily inhibited by preincubation of cells with the toxin (Thelen et al. 1988). The toxin ADP-ribosylates a cystine residue within the carboxyl-terminus of the Gαi subunit, rendering it nonfunctional. The crystal structure of a Gαi βγ heterotrimer has been solved and suggests that the carboxyl-terminal helix of the α subunit binds to the third transmembrane domain of the GPCR, and perhaps the receptor carboxy terminus (Wall et al. 1995). Once the heterotrimer is recruited to the activated GPCR, a guanosine disphosphate (GDP) bound within the α subunit is replaced by a molecule of GTP, resulting in the displacement of the βγ from the Gα subunit and the release of both subunits. These can then go on to activate various effectors and multiple downstream signaling events. Activation of the Gα subunit is terminated by an intrinsic GTPase activity and results in the reassociation of the α subunit with the βγ dimer. Activation of G proteins is thought to occur catalytically, with a single activated GPCR able to activate several G proteins (Janetopoulos et al. 2001). A rapid occurrence following binding of chemokine to its receptor is a rise in the intracellular calcium concentration

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Table 21.2 Chemokine receptors and their principal ligands. Listed are the principal ligands of chemokine receptors and their distribution over a range of cell types. Also listed are the chromosomal locations of each receptor. Although there are reports in the literature of chemokine receptors on nearly every type of cell, those shown above represent the commonly agreed attributions.

Receptor

Principal ligands

Chromosomal location

Receptor principally expressed by

CCR1

CCL3/MIP-1a CCL5/RANTES CCL7/MCP-3

3p21

Mo, DC, Eo, Bs, T, PMN, NK

CCR2

CCL2/MCP-1 CCL7/MCP-3 CCL8/MCP-2 CCL13/MCP-4

3p21

Mo, DC, T, Bs

CCR3

CCL11/eotaxin-1 CCL13/MCP-4 CCL24/eotaxin-2 CCL26/eotaxin-3

3p21

Eo, T, Bs, Mc

CCR4

CCL17/TARC CCL22/MDC

3p24

DC, T, Bs, NK

CCR5

CCL3/MIP-1a CCL4/MIP-1b CCL5/RANTES

3p21

Mo, DC, T

CCR6

CCL20/MIP-3a

6q27

DC, T

CCR7

CCL21/MIP-3b

17q12

DC, T, B, NK

CCR8

CCL1/I-309

3p22

Mo, T, NK

CCR9

CCL25/TECK

3p21.3

T

CCR10

CCL27/CTACK

17q21

T

CXCR1

CXCL1/Groa CXCL6/GCP-2 CXCL8/IL-8

2q35

N, Mo

CXCR2

CXCL1/Groa CXCL2/Grob CXCL3/Grog CXCL5/ENA-78 CXCL6/GCP-2 CXCL8/IL-8

2q35

N, Mo

CXCR3

CXCL9/Mig CXCL10/IP-10 CXCL11/I-TAC CXCL4/PF-4

Xq13

T, B

CXCR4

CXCL12/SDF-1a /b

2q21

T, B, DC, Mo

CXCR5

CXCL13/BCA-1

11q23

B, T

CXCR6

CXCL16

3p21

T, NK

XCR1

XCL1/lymphotactin XCL2

3p21

T, NK

CX3CR1

CX3CL1

3p21

T, NK, DC, Mo

B, B lymphocyte; Bs, basophil; DC, dendritic cell; Eo, eosinophil; Mc, mast cell; Mo, monocyte; N, neutrophil; NK, natural killer cell; PMN, polymorphonuclear leukocyte; T, T lymphocyte.

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within the cell ([Ca2+]i) and this has commonly been used in the laboratory as a measurement of receptor activation. This is mediated by the βγ subunits, which activate phospholipase Cβ leading to hydrolysis of phosphatidylinositol to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is able to induce the release of Ca2+ from intracellular stores and the elevated calcium levels subsequently induce protein kinase (PK)C activation. This activity is necessary for leukocyte responses such as the neutrophil respiratory burst (Li et al. 2000). The βγ subunit also activates PI3K. The use of PI3K inhibitors in vitro has demonstrated a significant role for PI3K in the process of leukocyte chemotaxis (Turner et al. 1995) and has been further corroborated by studies using mice deficient in various PI3K isoforms. Mice deficient in PI3K-γ show impaired neutrophil recruitment in a model of peritonitis and defective leukocyte chemotaxis in response to CCL3 (Li et al. 2000), while mice deficient in PI3K-δ exhibit attenuated allergic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge (Lee et al. 2006). In T lymphocytes at least, a more complicated signaling mechanism may be apparent as PI3K has been reported to be dispensable for the chemotaxis of Th2 cells in response to the CCR4 ligands CCL22 and CCL17 (Cronshaw et al. 2004). These studies identified a pathway involving small GTPases and, more specifically, Rho-associated coiled-coil forming protein kinase (ROCK), as being the critical PI3K independent pathway for T lymphocyte migration. ROCK is an effector of Rho, a small GTPase that is also activated by the βγ G protein subunit via the other GTPase Ras/Rac that has been previously shown to be associated with cytoskeletal rearrangements, effecting cellular responses such as shape change, adhesion, and chemotaxis (Bokoch 1995).

Regulation of chemokine receptor expression In order to respond to a particular chemokine, leukocytes must express the cognate receptor at the cell surface. To compound the issue of promiscuity, whereby receptors typically bind several chemokines, leukocytes also generally express several different chemokine receptors. These receptors have been broadly divided by others into two groups, those constitutively expressed by leukocytes and those whose expression is dynamic and induced by external stimuli, such as observed during inflammation (Proudfoot 2002). The former are especially involved in homeostasis for example the receptor CCR7 and its ligands CCL19 and CCL21 are vital for directing B and T lymphocytes to the secondary lymphoid organs, while the latter are important for the recruitment of leukocytes to inflamed tissues. A good example of the dynamic manner in which chemokine receptors can be expressed is the differential expression pattern observed at both the mRNA and protein level on Th1 and Th2 subsets of T lymphocytes following their polarization (Bonecchi et al. 1998; Sallusto et al. 1998). Th1 lymphocytes selectively express

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CCR1, CCR5, and CXCR3, whereas CCR3, CCR4, and CCR8 are found on Th2 lymphocytes. This fine tuning of receptor expression enables the cells to respond to a variety of different chemokines in vivo, for example, enabling them to leave one tissue compartment and migrate to another. It is likely that such flexibility is vital for a focused adaptive immune response. It should be noted that the division of chemokine receptors into either inducible and inflammatory subsets of receptors is not perfect, as some receptors can be involved in both the basal homing of leukocyte to tissues and also their recruitment in inflammation. For example, the eosinophil receptor CCR3 mediates both the recruitment of eosinophils to the gut under noninflammatory conditions and also their recruitment to the lung following allergen challenge (Gurish et al. 2002; Ma et al. 2002). The number of chemokine receptors on the leukocyte surface results from a balance between the rate of internalization and the rate of replacement (recycling and synthesis of nascent receptor). Following ligand binding, the carboxyltermini of chemokine receptors undergo phosphorylation by protein kinases at a series of serine and threonine residues, a process known as “desensitization.” This facilitates the recruitment of β arrestins to the carboxyl-terminus, rendering the receptor unable to interact with G proteins and thereby switching the receptor off. Desensitization can be described as homologous or heterologous, dependent on the ligand observed to induce the process. Homologous desensitization occurs following repeated exposure of leukocytes to the same ligand and is mediated by specific G protein-coupled receptor kinases (GRKs) following occupation of the receptor by ligand. In contrast, heterologous desensitization does not require direct activation of the chemokine receptor itself and can be mediated by protein kinases, such as PKA and PKC, following the activation of downstream signaling pathways by other receptors (Ali et al. 1999; Bohm et al. 1997). These can be either distinct chemokine receptors expressed on the same leukocyte, or other GPCRs such as those for fMLP and C5a which cross-desensitize signals via the chemokine receptor CXCR2 on human neutrophils (Sabroe et al. 1997). The recruitment of arrestin also initiates the endocytosis or internalization of receptors by binding to clathrin with the receptor–arrestin complex subsequently sequestered in clathrin-coated pits. This pathway is often considered a default system for the degradation and recycling of GPCRs (Pelchen-Matthews et al. 1999; Shenoy & Lefkowitz 2003). Although the rate of internalization of a receptor is an important factor in determining its level at the cell surface, the rate of recycling and the rate of synthesis of new receptors are also important. The concept of two different classes of receptor (as distinguished by their recycling) has been introduced recently, in which class A receptors traffic to recycling endosomes and are rapidly returned to the cell surface (Shenoy & Lefkowitz 2003). In contrast, class B receptors are dephosphorylated in endosomes, followed by slow recycling back to

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the plasma membrane. Sequentially, the receptors pass through late endosomes and the Golgi and, finally, are transported back to the cell surface. Quite unexpected was the finding from Fong and colleagues that T and B lymphocytes from mice deficient in β arrestin-2 had impaired chemotactic responses to the CXCR4 ligand CXCL12 (Fong et al. 2002). Until this publication, as their name implies, arrestins were thought to provide solely a stop signal to impede G protein activation and facilitate endocytosis. Following allergen-challenge, the same β arrestin-2-deficient mice also exhibit reduced T lymphocyte accumulation in the airways (Walker et al. 2003). In light of these findings, it is now believed that arrestins additionally function as an adapter scaffold, allowing the docking of kinases such as JNK-3 (McDonald et al. 2000), Hck, or c-Fgr (Barlic et al. 2000). These latter two molecules have been shown to associate with β arrestin to induce neutrophil degranulation following treatment with the chemokine CXCL8. The additional finding that arrestins can modulate histone acetylation suggests that these molecules may also play a role in the transcriptional profiles of leukocytes following their recruitment to inflamed tissues (Kang et al. 2005).

Modulation of chemokine function Proteolytic processing of chemokines As mentioned earlier in this chapter, the N-terminus of the chemokine is instrumental in inducing activation of the receptor and subsequent signaling. This permits a means of regulating signaling by means of proteolysis (reviewed in Struyf et al. 2003). The type II membrane protein CD26/ DPP IV has an aminopeptidase activity, which is known to cleave dipeptides selectively from the N-terminus of proteins with a proline or alanine residue at the second position of their sequence, an attribute common to chemokines. Indeed, several CC chemokines have greatly impaired responses at their receptors following CD26 processing, including CCL5 (Oravecz et al. 1997), CCL22 (Proost et al. 1999), and CCL11 (Struyf et al. 1999). This latter chemokine, a potent eosinophil attractant, has its chemotactic activity for eosinophils reduced 30-fold on truncation by CD26/DPP IV (Struyf et al. 1999). Several matrix metalloproteinases (MMPs) also have been implicated in the modulation of chemokine activity. For example, CCL7 is processed by MMP2/gelatinase A resulting in the removal of four N-terminal residues and the generation of a receptor antagonist with activity both in vitro and in vivo (McQuibban et al. 2000). In addition, MMPs are also able to process the chemokines CCL2, CCL8, and CCL13 (McQuibban et al. 2002; Overall et al. 2002). Such a strategy has not been lost on the hookworm Necator americanus, which secretes metalloproteases to degrade CCL11 and thereby inhibits eosinophil recruitment (Culley et al. 2000).

Chemokines

Chemokine scavenging by the silent receptors D6 and DARC The chemokine receptor D6 was originally identified by Nibbs and colleagues following RT-PCR analysis of murine spleen with the human ortholog being subsequently amplified from human genomic DNA (Nibbs et al. 1997a,b). D6 is an extremely promiscuous receptor, binding with high affinity a large number of inflammatory CC chemokines, including CCL2, CCL3L1, CCL4, CCL5, CCL7, CCL11, and CCL16. However, despite considerable efforts, no cell signals have been documented to be transduced following chemokine binding, leading the receptor to be described as “silent.” D6 is expressed at high levels on the surface of lymphatic endothelial cells and on syncytial trophoblasts of the placenta (Nibbs et al. 2001), and undergoes rapid constitutive internalization enabling it to rapidly remove chemokines from the endothelial cell surface (Fra et al. 2003; Bonecchi et al. 2004; Galliera et al. 2004) which suffer a proteolytic fate while the receptor is recycled back to the cell surface (Weber et al. 2004). This has led to the suggestion that D6 acts as a “gatekeeper” preserving the integrity of lymphoid tissue by the scavenging of inflammatory chemokines (Young et al. 1955; Fra et al. 2003; Nibbs et al. 2003). This has been supported by recent studies using mice deficient in D6. Exacerbated inflammation was observed following application of phorbol ester to the skin, with the histology of lesions reported to resemble those of human psoriasis (Jamieson et al. 2005; Martinez de la Torre et al. 2005). These results support the notion that D6 is involved in the resolution of the inflammatory response. Similarly, the Duffy antigen receptor complex (DARC) is thought to act as a scavenger of both CC and CXC chemokines and is expressed primarily on erythrocytes. Although DARC was originally defined serologically in the 1950s as a minor red blood cell antigen, it was not until 1990s that it was identified at the cDNA level. It was quickly noted that the molecule had considerable homology to chemokine receptors and it was subsequently shown that DARC could bind the chemokine CXCL8 with high affinity (Chaudhuri et al. 1993; Horuk et al. 1994; Neote et al. 1994; Peiper et al. 1995). In addition to its expression on erythrocytes, DARC has also been identified on the postcapillary venule endothelial cells of several organs (Horuk et al. 1997) and on subsets of neurons in the central nervous system (Dawson et al. 2000). Studies of mice in which the DARC gene has been deleted suggest that it functions as a biological “sink” for chemokines, with both an antiinflammatory role and antiangiogenic role (Neote et al. 1994). As is the case with D6, chemokine binding by DARC does not appear to result in signal transduction, either in erythrocytes or transfectant systems. This is thought to be primarily due to the lack of a DRY motif in the putative third transmembrane helix, present in the majority of signaling chemokine receptors, and thought to play a critical role in maintaining GPCR conformation (Ballesteros et al. 2001).

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Chemokines and their receptors in allergic inflammation Having described in some detail the biology of chemokines and their receptors, in the following sections we attempt to address which chemokines and which chemokine receptors are implicated in the pathogenesis of allergic disease. We will do this on a cell-by-cell basis, looking at the individual subsets of leukocytes implicated in the allergic response and in doing so, we will dissect relevant in vitro and in vivo data and, where applicable, the translation of this basic research into the clinic setting. It should be stated at this juncture that it would be erroneous to view chemokines in isolation of other in vivo chemoattractant systems, as leukocytes are also equipped with receptors for chemoattractants other than chemokines. These molecules also have a potentially important role in the pathogenesis of allergic reactions. Although non-chemokine chemoattractants typically exhibit low specificity for leukocytes, they may be particularly important in the early stages of an allergic response, to be replaced by the more specific chemokines as the response develops. For example, C5a, a fragment of the complement component C5, generated during complement activation, is a potent chemoattractant for neutrophils and other subsets of leukocytes. C5a has been shown to appear before CXCL8 in certain inflammatory models (Ivey et al. 1995). Likewise, the lipid mediators LTB4 and prostaglandin (PG)D2 are potent leukocyte chemoattractants and have been postulated to “grease the way” of T lymphocytes to allergic airways, cooperating with chemokines in a sequential manner (reviewed by Luster & Tager 2004). LTB4 is produced rapidly by activated mast cells and recent research by ourselves and others has highlighted its potential to recruit mast cell progenitors (Weller et al. 2005) and lymphocytes (Goodarzi et al. 2003).

DCs downregulate CCR6 expression and consequently lose responsiveness to CCL20. Instead, they upregulate expression of the receptor CCR7 (Yoshida et al. 1997), which makes them responsive to the chemokines CCL19/MIP-3β (Dieu et al. 1998; Sozzani et al. 1998) and CCL21/SLC (Saeki et al. 2001). This maturation is accompanied by the production of chemokines such as CCL22/MDC which will recruit activated T cells expressing the receptor CCR4 (Tang & Cyster 1999). Initial studies employing mice deficient in either CCL19 and CCL21 (Gunn et al. 1999) or CCR7 (Forster et al. 1999; Ohl et al. 2004) revealed an impaired capacity for the recruitment of DCs to draining lymph nodes. This was subsequently supported by studies in which a mouse with severe combined immunodeficiency (SCID) was reconstituted with peripheral blood mononuclear cells from allergic humans following the neutralization of CCL21 by antibodies (Hammad et al. 2002). In this model, impaired DC homing to the draining mediastinal lymph nodes was observed following antigen challenge, with a resultant decrease in both Th2 cytokine production and T cell recruitment. At the genomic level, regulation of this key chemokine receptor appears to be mediated by the transcription factor Runx3 (Fainaru et al. 2005). Mice deficient in this transcription factor exhibit enhanced expression of CCR7 on alveolar DCs, and subsequently display increased migration to the lymph nodes draining the lung. Interestingly, the resultant accumulation of activated DC within the lymph nodes is associated with features typical of asthma, including increased serum IgE levels and AHR to the nonselective muscarinic receptor agonist methacholine. Of note is a point made by the authors of that study, namely that the gene encoding Runx3 lies within a region of chromosome 1p36 previously linked by another group to asthma and atopy in a scan of the human genome for susceptibility genes (Haagerup et al. 2002).

Chemokines as recruiters of T lymphocytes Chemokines as recruiters of dendritic cells Dendritic cells (DCs) play a crucial part in the allergic response by presenting antigen to other leukocytes. In their role as a sentinel cell, they need to be able to traffic to tissues where they can survey the environment and following encounter with antigen, return to the lymph nodes as an activated APC. Chemokines carefully regulate these migratory steps via a selection of different CC chemokines and receptors. The recruitment of immature DCs to inflamed tissues is facilitated by the receptors CCR2, CCR5, and CXCR4, which recognize a variety of different ligands, although use of these receptors appears to be selective depending on whether the cells have myeloid or plasmacytoid origins (Penna et al. 2001). The chemokine receptor CCR6 was originally identified as being expressed by lung-derived DCs (Power et al. 1997), and mediates chemotaxis in response to CCL20/MIP-3α which is expressed by epithelial cells from a variety of tissues (Greaves et al. 1997). On maturation,

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T lymphocytes Naive T lymphocytes As is the case for DCs, T lymphocytes also have a critical requirement for directed migration to and from secondary lymphoid organs. Migration to the secondary lymphoid organs is also controlled by the CCR7 ligands CCL19 and CCL21, and plt mice deficient in both chemokines exhibit a failure in naïve T cells homing to lymph nodes (Gunn et al. 1999). The receptor for both ligands, CCR7, also appears to determine, at least in part, T cell exit from peripheral tissues, as T cells from CCR7-deficient mice are unable to leave the lung or skin following allergen challenge and enter the draining lymph nodes (Bromley et al. 2005; Debes et al. 2005). In addition to CCR7, naive T lymphocytes also express the receptor CXCR4 (Bleul et al. 1997). In a well-characterized murine model of allergic airway disease, neutralizing antibodies to both CXCR4 and its ligand CXCL12 were observed to reduce lung eosinophilia and AHR (Gonzalo et al. 2000),

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and treatment of mice with the small-molecule antagonist of CXCR4 resulted in a significant reduction in AHR, eosinophilia, and the production of Th2-associated cytokines following allergen challenge (Lukacs et al. 2002). Collectively these data suggest that the CXCR4–CXCL12 axis may play a role in the pathology of allergic disease.

Activated T lymphocytes As we have already mentioned, T cells polarized in vitro to either the Th1 or Th2 subset can dynamically regulate their cell surface levels of several different chemokine receptors, allowing them to respond to a variety of signals (Sallusto et al. 2000). Th2 cells are distinguished from Th1 cells by their expression of the chemokine receptors CCR3, CCR4, and CCR8. Such polarization has also been observed in vivo, with IL-4 producing cells recovered by bronchoalveolar lavage (BAL) shown to preferentially express CCR3 and CCR4 (Morgan et al. 2005). We and others have shown in vitro that CXCR3 ligands are natural antagonists of CCR3-mediated responses (Loetscher et al. 2001; Xanthou et al. 2003) and we postulated that this counterplay between Th1- and Th2associated chemokines and their receptors might result in the fine tuning of leukocyte recruitment in vivo. Subsequent studies have shown that CXCR3 ligands are upregulated following allergen challenge in both the human (Bochner et al. 2003) and murine lung (Fulkerson et al. 2004) and, in support of our postulate, intravenous administration of low doses of CXCL9 was demonstrated to inhibit allergen-induced eosinophil recruitment (Fulkerson et al. 2004). Consistent with a role for the CCR4 and CCR8 axes in T-cell recruitment to the allergic lung, CCL22 and CCL17 have also been observed to be upregulated in the human lung following allergen challenge (Bochner et al. 2003; Pilette et al. 2004). Neutralization of both CCL11 and CCL22 by specific monoclonal antibodies has been reported to block early stage recruitment of Th2 cells to the murine lung following allergen challenge with the long-term blockade of Th2 cell recruitment, following repeated antigen stimulation only inhibited by CCL22 blockade, suggesting roles for either chemokine in the initial and late stages of T cell recruitment, respectively (Lloyd et al. 2000). Coexpression of CCR4 and CCR8 on a significant percentage of T cells in the allergic lung as revealed by immunohistochemical study of bronchial biopsies was reported in an early study (Panina-Bordignon et al. 2001). A more recent study using flow cytometry found an increased percentage of CCR4+ CD4+ T cells recovered in the BAL following allergen challenge, although the percentage of CD4+ or CD8+ T cells expressing CCR4 was unchanged (Thomas et al. 2007). As the authors of this later study suggest, this may be due to differences in either the methodologies employed for staining or in cell isolation from different tissue compartments (BAL compared with bronchial biopsy). The lack of commercially available CCR8-specific antibodies that work efficiently in

Chemokines

immunostaining (Fox et al. 2006) has not assisted the elucidation of the role of CCR8 in T cell trafficking to the allergic lung, although Thomas et al. (2007) recently observed that CCR8 mRNA was found at similar levels in both CD4+ peripheral blood and BAL T cells following allergen challenge. This is not supportive of an active role for this receptor in cell recruitment, although elevated levels of the ligand have been recently reported to be significantly elevated in the BAL fluid of asthmatics compared with normal controls (MontesVizuet et al. 2006). Attempts to demonstrate an absolute requirement for CCR4 or CCR8 in rodent models of allergic inflammation have met with mixed results. Following challenge with ovalbumin, CCR4-deficient mice revealed little difference from their wild-type counterparts allergic airways inflammation (Chvatchko et al. 2000), although attenuation of chronic AHR has been reported in CCR4-deficient mice challenged with Aspergillus fumigatus spores (Schuh et al. 2002). Likewise, our own groups reported that monoclonal antibody neutralization of CCR4 in guinea pigs was ineffective at modulating the allergic response in the lung following challenge with ovalbumin (Conroy et al. 2003). Mice deficient in CCR8 exhibit similar airway inflammation to wild-type mice following ovalbumin challenge (Chung et al. 2003; Goya et al. 2003), and neutralization of the CCR8 ligand CCL1 reduced eosinophil migration to the murine lung, but had no effect on Th2 cell recruitment following allergen challenge (Bishop & Lloyd 2003). In contrast to both these reports, defects in Th2 responses have been reported in CCR8-deficient mice following both ovalbumin and cockroach antigen-induced airway inflammation (Chensue et al. 2001). Thus, like CCR4, the role of CCR8 in allergic inflammation remains less than clear-cut. The roles of the chemokine CCL2 and its receptor CCR2 in allergic disease appear to be quite complicated. Studies using CCL2-deficient mice suggest that the chemokine is critical for the in vitro polarization of T cells to the Th2 subclass as ovalbumin challenge of deficient mice led to reduced IL-4 and IL-5 production and an inability to undergo immunoglobulin class switching (Gu et al. 2000). Supportive of this, depletion of CCL2 in murine models of allergic airways disease has been demonstrated to reduce AHR (Gonzalo et al. 1998; Campbell et al. 1999). Likewise, mice deficient in CCR2 exhibit reduced pulmonary granuloma formation following the injection of Schistosoma egg antigen (Warmington et al. 1999). However, in contrast, studies by other groups have shown enhanced Th2 responses to ovalbumin (Kim et al. 2001) and Aspergillus (Blease et al. 2000a) in CCR2-deficient mice. A more recent study comparing both CCR2- and CCL2deficient mice has reported intact Th2-mediated responses and lung fibrosis in both animals following challenge with Aspergillus (Koth et al. 2004). In the context of the other studies, it thus appears that there is considerable variation in the importance of either chemokine or ligand in allergic

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pathology depending on the experimental model employed, making the relative importance of either CCL2 or CCR2 in human allergic disease difficult to gauge.

Regulatory T cells IL-10 is a potent antiinflammatory cytokine that works in part by the inhibition of APC function and cytokine production by DCs and macrophages. In the asthmatic setting, IL-10 can inhibit the production of cytokine by both Th2 cells and mast cells and also modulate eosinophil function (reviewed in Hawrylowicz & O’Garra 2005). CD4+CD25+ regulatory T cells (Tregs) are a major source of IL-10 and recent studies have focused on these cells as a means of suppressing allergic inflammation. CD4+CD25+ Tregs purified from peripheral blood make up around 10% of the CD4+ population and have been reported to express CCR4 and CCR8, which may facilitate their migration toward APCs and activated T cells allowing them to inhibit APC function and suppress responding T cells (Iellem et al. 2001). In vitro, CD25 + Tregs have been reported to suppress the differentiation of murine CD4+ T cells towards Th2 cells via a contact-dependent mechanism (Stassen et al. 2004) and defects in the ability of human Tregs to suppress allergen-driven T cell activation have been proposed to result in asthma (Bellinghausen et al. 2003; Ling et al. 2004). CD4+ Tregs differentiated from cell lines derived from the blood and skin of patients presenting with allergic contact dermatitis to nickel, have been shown to express CCR3, CCR4, CCR5, CXCR3, and CCR8 (Sebastiani et al. 2001). Curiously, functional responses to most ligands were lost on activation, with the exception of responses to the chemokines CCL17, CCL2, and CCL1. Similarly, in mice immunized with Schistosoma mansoni egg antigen-coated beads in order to elicit a Th2 response, CD4+CD25+ IL-10-producing cells were shown to selectively express CCR8, and their ability to infiltrate the resulting granulomas coincided with the production of CCL1, the main CCR8 ligand (Freeman et al. 2005). Natural killer T cells In contrast to Tregs, which regulate the adaptive arm of the immune system, natural killer T cells (NKT cells) regulate the innate immune system and are a subpopulation of αβ T cells, defined by the expression of a conserved T-cell receptor (V Vα24Jα 18-Vβ11 in humans) which recognizes the glycolipid α-galactosylceramide (α-GalCer) (Dellabona et al. 1994). Flow cytometric analysis suggests that the majority of NKT cells express the chemokine receptors CCR1, CCR2, CCR5, CXCR3, CXCR4, and CXCR6 (Kim et al. 2002; Motsinger et al. 2002). Unlike other T cells, NKT cells have low levels of CCR7, which is required for effective migration to secondary lymphoid organs. As a consequence, they are preferentially recruited to extra lymphoid tissues by the production of chemokines such as CCL2, CCL3, and CXCL10. As for CD4+ T cells, NKT subsets can be further divided based on their cytokine expression. A CD4+ IL-4/IL-2-producing subset, has

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been reported to express CCR4, while CD8– and CD4–CD8– low cytokine-producing subsets expressed the receptors CCR1, CCR6, and CXCR6 (Kim et al. 2002). As is the case for the differential expression of chemokine receptors on Th1 and Th2 lymphocytes, it is envisaged that such flexible programs of expression allow the fine tuning of NKT cell recruitment in vivo. Mice deficient in CD1 also lack NKT cells and in a ragweedinduced model of allergic airways disease, in vivo stimulation with α-GalCer has been reported to result in reduced CCL11, IL-4 production and subsequent eosinophilia, when compared with their wild-type counterparts. This suggests that NKT cells may play a proinflammatory role in allergic airways inflammation. This finding is in contrast with a reported suppressive role in bleomycin-induced models of pulmonary fibrosis, where administration of α-GalCer resulted in increased survival of mice (Kimura et al. 2004). Patients presenting to clinic with allergic asthma have been documented as having elevated levels of peripheral blood NKT cells compared with healthy volunteers (Sen et al. 2005). These NKT cells express significant levels of CCR9 and are subsequently responsive to CCL25 in vitro. Histochemical analysis identified NKT cells as having infiltrated the bronchi mucosa of asthmatics and, following purification, these cells were identified as having the potential to drive in vitro cocultures of CD3+ T cells into the expression of IL-4 and IL-13, unlike those isolated from healthy volunteers which drove interferon (IFN)-γ production. This ability to induce a Th2 bias was dependent on cross-talk between activated CCR9 and CD226, as shown by independent blockade of CD226 expression by shRNA and transfection of NKT cells from nonasthmatics with CCR9 cDNA, suggesting that blockade of CCR9 may prove to be fruitful in the treatment of asthma (Sen et al. 2005). Despite an initial study suggesting that approximately 60% of the pulmonary CD4+CD3+ cells in patients with moderate-to-severe persistent asthma were NKT cells (Akbari et al. 2006), two subsequent studies have shown comparatively few NKT cells in allergen-challenged lung compared with control (Thomas et al. 2006; Vijayanand et al. 2007), with the stringency of the flow cytometry parameters employed in the original study a source of much debate (Ho 2007). Thus, at the time of writing, despite strong evidence that NKT cells play a proinflammatory role in murine models of allergic airways inflammation, such findings have not been translated to humans.

Chemokines as recruiters of mast cells Mast cells are principally recognized for their effector functions in allergic reactions and express FcεR1 receptors that are capable of binding IgE with high affinity. Recognition of a polyvalent antigen triggers receptor cross-linking, resulting in the release of degranulation products such as histamine and the de novo synthesis of mediators with potent inflammatory activity such as prostaglandins and cytokines (Marshall

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2004). Mast cells characteristically express the receptor c-kit and, accordingly, its ligand SCF induces their chemotaxis (Boyce et al. 2002), in addition to its other important effects such as proliferation, differentiation, and inhibition of apoptosis. Several independent studies have described a repertoire of chemokine receptors expressed by mast cells or mast cell lines. CCR1 (Juremalm et al. 2002), CCR3 (Ochi et al. 1999; Romagnani et al. 1999), CCR4 (Juremalm et al. 2002), CCR5 (Ochi et al. 1999), CXCR2 (Ochi et al. 1999), CXCR3 (Brightling et al. 2005), and CXCR4 (Ochi et al. 1999; Juremalm et al. 2000) have all been described as being expressed by mast cells. In vivo studies employing blocking antibodies to chemokines or mice deficient in receptors have suggested a role for the CCR1–CCL3 signaling pathways leading to mast cell degranulation (Toda et al. 2004). Following costimulation of both FcεRI and CCR1, reduced chemotaxis was observed, which was postulated to focus the inflammatory response by maintaining cells at the site of allergen accumulation. Subsequent in vivo experimentation using a murine model of allergic conjunctivitis has expanded these findings and shown that costimulation of both receptors optimizes their capacity for degranulation, a phenomenon absent in CCR1deficient mice and which can be impeded in wild-type mice by neutralization of CCL3 (Miyazaki et al. 2005). Antagonism of CCR3 by small molecules in the same model is also reported to be beneficial with an impaired early phase reaction, again thought to be due to the inhibition of mast cell degranulation (Nakamura et al. 2005). CCR3 is expressed on both mature human mast cells and their progenitors (Ochi et al. 1999), although attempts to describe a definitive role for CCR3 in mast cell trafficking have proved difficult, with studies often throwing up apparently conflicting results. CCR3-deficient mice infected with the helminth Trichinella spiralis were reported to exhibit a normal jejunum and cecum mast cell hyperplasia and unaffected worm expulsion (Gurish et al. 2002), while intraperitoneal sensitization of the same mice, followed by aerosol challenge, resulted in increased numbers of tracheal intraepithelial mast cells and increased AHR compared with wild type (Humbles et al. 2002). Work from our own group has demonstrated that immature murine and human mast cells express the receptor BLT1, unlike their mature counterparts, which suggests that circulating mast cell progenitors can migrate along a gradient of LTB4 generated by the activation of mast cells in tissues (Weller et al. 2005). This provides a potential component of mast cell hyperplasia associated with allergy. CXCR2deficient mice have been reported to have reduced numbers of intestinal mast cell progenitors as determined by limiting dilution assays, suggesting that mediators such as CXCL1/KC and CXCL2/MIP-2 may also be involved in trafficking, in contrast to mice deficient in CCR2, CCR3, and CCR5, which show no reduction in the numbers of mast cell progenitors (Abonia et al. 2005).

Chemokines

Chemokines as recruiters of basophils Like mast cells, following their activation, basophils can also release histamine and are a source of leukotrienes and cytokines with the potential to amplify inflammation. Hence, the mechanism by which they are recruited to sites of allergic inflammation is of interest. The question of which chemokine receptors are expressed by basophils was originally addressed at the mRNA level with transcripts for CCR1, CCR2, CCR3, and CCR5 detected, although curiously migration was only observed in vitro to the chemokines CCL2 and CCL11 (Iikura et al. 2001). In a model of cutaneous inflammation in which humanized SCID mice received autologous human skin grafts, CCL11 was reported to be a potent inducer of basophil migration (Fahy et al. 2001). Likewise, CCL5, a ligand for the receptors CCR1, CCR3, and CCR5 has been reported to play a role in recruiting basophils into the nasal mucosa of allergic patients following allergen challenge (Kuna et al. 1998). When injected intradermally into rats, CCL5 has been reported to induce basophilic cell recruitment and induction of histidine decarboxylase mRNA transcripts, with the potential to further exacerbate inflammation by elevating histamine production (Conti et al. 1997). Transcripts for CXCR4 have also been reported in basophils coupled with functional data linking CXCL12-induced [Ca2+]i flux and chemotaxis, although there has been debate as to whether the receptor is present on resting cells (Jinquan et al. 2000; Iikura et al. 2001). Several chemokines have been shown to induce basophil degranulation, including CCL2 (Kuna et al. 1992; Iikura et al. 2001), CCL3 (Alam et al. 1992), CCL7 (Dahinden et al. 1994), and CCL13 (Garcia-Zepeda et al. 1996), although CCL11 is without effect in this respect. CCL11 can, however, potentiate the production of IL-4 production by basophils, mediating this effect via CCR3 (Devouassoux et al. 1999). CCL13 has been reported to induce a biphasic response in assays of basophil shape change, with the receptors CCR2 and CCR3 apparently cooperating to coordinate responses across the concentration range examined (Heinemann et al. 2000), which is reminiscent of the actions of CXCR1 and CXCR2 on neutrophils to coordinate responses to CXCL7 (Ludwig et al. 1997). In humans, intradermal injection of CCL11 has been reported to induce an acute wheal and flare reaction, most likely mediated by the degranulation of mast cells (MenziesGow et al. 2002). Supportive of this, numbers of detectable mast cells were also observed to decrease in the 24 hours following injection of CCL11, and the ability of the basophil to produce CCL3 following IgE cross-linking suggests that it may constitute a positive-feedback mechanism for amplification of leukocyte recruitment in allergy (Li et al. 1996).

Chemokines as recruiters of eosinophils Although eosinophils are characteristically found in large numbers at sites of allergic inflammation, defining a precise role for the cells in the pathogenesis of asthma has been difficult.

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Neutralization of IL-5 in vivo or the use of IL-5-deficient mice has been shown to suppress eosinophil production, suggestive of a link between the activation of lung eosinophils and the development of AHR (Eum et al. 1995; Foster et al. 1996). This postulate was dealt a blow with the finding that neutralization of IL-5 in asthmatic patients did not show any effects on airway function (Leckie et al. 2000), although a considerable number of eosinophils remained in the lung tissue (FloodPage et al. 2003a). However, subsequent studies in both mice (Humbles et al. 2004; Lee et al. 2004) and humans (FloodPage et al. 2003b) have suggested a critical role for eosinophils in airway remodeling with eosinophil-derived TGF-β thought to be a key driver of the remodeling process. Whilst CCL5/RANTES was shown to be chemotactic for eosinophils in an earlier study (Kameyoshi et al. 1994), the chemokine is capable of attracting numerous other leukocyte subtypes and it was not until the discovery of a chemokine named “eotaxin” that a highly potent, highly selective eosinophil chemoattractant was described (Jose et al. 1994). CCL11/ eotaxin-1 was discovered following protein purification of BAL fluid from allergen-sensitized guinea pigs. Following the identification of murine and human orthologs, the chemokine was subsequently found to signal via the receptor CCR3 (Ponath et al. 1996). This receptor is highly expressed on human eosinophils at around 50 000 receptors/cell and is very promiscuous, binding upwards of a dozen different chemokines with varying potency and efficacy. Two other chemokines CCL24/eotaxin-2 and CCL26/eotaxin-3 (Forssmann et al. 1997; Patel et al. 1997; White et al. 1997; Kitaura et al. 1999; Shinkai et al. 1999) were subsequently identified as signaling via CCR3 and are so-named because of their chemotactic activity for eosinophils, having little amino acid sequence similarity with CCL11. CCR3 is also expressed by basophils (Uguccioni et al. 1997), mast cells (Ochi et al. 1999), and a subpopulation of Th2 lymphocytes (Sallusto et al. 1997), making it a likely player in the pathogenesis of allergic disease. Several studies have demonstrated that CCL11, CCL24, and CCL26 are generated in allergic reactions and that their production correlates with eosinophil recruitment in both animals and humans (Rothenberg et al. 1995a,b; Humbles et al. 1997; Ying et al. 1997, 1999a,b; Berkman et al. 2001). There is also evidence to suggest that CCL11 can act in concert with IL-5 to prompt release of eosinophils from the bone marrow, leading to peripheral blood eosinophilia (Collins et al. 1995; Palframan et al. 1998a, 1998b). Use of mice deficient in CCL11 has led to conflicting reports in the literature, most likely due to genetic differences between the mouse strains used. Whilst BALB/c mice deficient in CCL11 were reported to show a reduction in ovalbumin-induced lung eosinophilia (Rothenberg et al. 1997), CCL11-deficient mice of the outbred ICR strain exhibited no difference in the numbers of BAL eosinophils following challenge with the same allergen (Yang et al. 1998). Similarly, the ability of mice deficient in the CCL11 receptor CCR3 to

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develop AHR to methacholine following antigen inhalation appears to be dependent on the route of sensitization, be it intraperitoneal (Humbles et al. 2002) or epicutaneous (Ma et al. 2002). A more recent study using an assortment of CCL11/CCL24 doubly deficient mice, CCR3-deficient mice, and eosinophil-deficient Δdbl-GATA mice, reported that airway eosinophil recruitment was ablated following allergen challenge (Fulkerson et al. 2006a). A subsequent study by the same group employed mice deficient in CCR3, which had been backcrossed onto an IL-13 transgenic background in which expression of the cytokine was targeted to the lung. In this model, a 98% reduction in lung eosinophil numbers was observed compared to littermate controls, which correlated with reduced collagen deposition in the airways (Fulkerson et al. 2006b). Expression of CXCR4 mRNA by freshly isolated eosinophils has previously been reported although detection of cell-surface receptor was only apparent following their culture for 24 hours in a medium containing IL-5 (Nagase et al. 2000). This upregulation of CXCR4 is reminiscent of a previously described mechanism for the clearance of senescent neutrophils, which preferentially home to the bone marrow where CXCL12 is produced (Martin et al. 2003). So, although the lack of CXCR4 on freshly isolated eosinophils suggests that the receptor is unlikely to mediate eosinophil recruitment in the context of allergy, its upregulation as eosinophils age may mark the cells for clearance from the circulation. Another receptor with variable expression on eosinophils is CCR1. While our own laboratory has been able to detect CCR3 on the surface of eosinophils from all the donors that we have examined, we found that in approximately 20% of individuals CCR1 was also expressed at high levels (Sabroe et al. 1999; Phillips et al. 2003). This renders the eosinophils responsive to the chemokine CCL3/MIP-1α and suggests that, in a significant proportion of the population, the CCR1– CCL3 axis has the potential to recruit eosinophils in allergic disorders. Indeed, expression of CCL3 in the human asthmatic lung has previously been reported by several other groups (Alam et al. 1996; Holgate et al. 1997; Tillie-Leblond et al. 2000; Zimmermann et al. 2000) with increased serum CCL3 levels also reported in atopic dermatitis patients (Kaburagi et al. 2001). In murine models of allergic airways disease employing challenge with cockroach allergen, administration of a blocking monoclonal antibody to CCL3 resulted in reduced AHR and reduced eosinophil recruitment to the lungs in the initial stages of disease (Campbell et al. 1998). As with the data on AHR in CCR3-deficient mice, the allergen/sensitization protocol used appears to be important, as similar neutralization of CCL3 in an ovalbumin-induced model resulted in only a partial reduction in AHR and eosinophil recruitment to the lung (Gonzalo et al. 1998). Likewise, a role for the CCR1–CCL3 axis in airways remodeling observed during chronic lung inflammation has been put forward (Blease et al. 2000b).

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Following challenge of CCR1-deficient mice with Aspergillus fumigatus, significantly lower levels of Th2 cytokines and fibrosis were observed in the lungs of the deficient mice compared with their wild-type counterparts.

Blockade of the chemokine system as a therapeutic strategy for the treatment of allergy As we have seen, the leukocytes implicated in allergic inflammation express a variety of chemokine receptors that appear to play distinct roles in the pathology of allergic disease. Consequently, blockade of the chemokine system has great appeal as a possible therapeutic strategy. An initial key question in this respect is how selective an approach to take. A broad approach might try to inhibit the activities of several chemokines simultaneously. Since the finding that chemokines defective in their ability to interact with GAGs are inactive in vivo (Proudfoot et al. 2003), this raises the possibility that antagonism of the chemokine–GAG interaction may be a useful therapeutic angle (Handel et al. 2005). Indeed, soluble GAGs have been used by our own group to inhibit the actions of chemokines on eosinophils in vitro by sequestering the chemokine such that it can no longer bind to the specific receptor (Culley et al. 2003). Heparin, produced in vivo exclusively by mast cells, has been shown to inhibit eosinophil recruitment in a guinea pig model of allergic lung inflammation (Seeds et al. 1995), and to attenuate the late asthmatic response to allergen challenge in patients with atopic asthma suggesting that it may represent a natural antiinflammatory agent able to attenuate allergic inflammation in the lung following mast cell degranulation. If a more selective approach is to be taken, the key question is surely which chemokine receptor should be blocked, and typically results from experimental observations about whether a particular cell type expressing a particular receptor plays a key role in the pathogenesis of disease (Wells et al. 2006). As CCR3 is expressed on the cell types implicated in the initiation and maintenance of the allergic phenotype, e.g. eosinophils (Ponath et al. 1996), basophils (Uguccioni et al. 1997), Th2 lymphocytes (Sallusto et al. 1997), and mast cells (de Paulis et al. 2001), the receptor has become a focus for the pharmaceutical industry, with the development of small molecule antagonists of CCR3 a goal of several companies. Initial in vitro and in vivo proof of principle studies suggested that CCR3 blockade was feasible (Sabroe et al. 2000; White et al. 2000; Dhanak et al. 2001a; Naya et al. 2001a; Varnes et al. 2004), and subsequent high-throughput screens identified several potent small-molecule antagonists of CCR3, typically with activities in the low nanomolar range. High-throughput screening of chemical libraries in biotechnology/pharmaceutical companies led to the discovery of a plethora of smallmolecule CCR3 antagonists with in vitro activities typically in the low nanomolar range (Sabroe et al. 2000; White et al. 2000; Dhanak et al. 2001a,b; Naya et al. 2001a,b, 2003; Saeki et al. 2001; Bryan et al. 2002; De Lucca et al. 2002; Wacker

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et al. 2002; Wan et al. 2002; Warrior et al. 2003; Hodgson et al. 2004; Varnes et al. 2004; Anderskewitz et al. 2005; De Lucca et al. 2005; Ting et al. 2005a,b; Fryer et al. 2006; Morokata et al. 2006; Suzuki et al. 2006). Enthusiasm for CCR3 antagonists appeared to wane with the report that IL-5 blockade had little effect on lung function, as recently reviewed by Wells et al. (2006). However, as mentioned in an earlier section, subsequent studies in both humans and animals have highlighted an additional role for eosinophils in the process of airways remodeling associated with asthma. Deletion of the murine IL-5 gene was observed to suppress both lung eosinophilia and tissue remodeling, with a decrease in the growth factor TGF-β1 (Cho et al. 2004) correlating with clinical data in which anti-IL-5 blockade resulted in a reduction in the numbers of airway eosinophils expressing mRNA for TGF-β1, and in the deposition of extracellular matrix proteins in the reticular basement membrane of bronchial biopsies (Flood-Page et al. 2003b). The recent generation of mice deficient in eosinophils has allowed the role of the cell to be probed further, with data derived from allergen challenge implicating the eosinophil in both airway remodeling (Humbles et al. 2004) and AHR (Lee et al. 2004). Consequently, interest in CCR3 as a therapeutic target has been rekindled, particularly as it is also expressed by basophils, Th2 cells, and mast cells, and blockade on these cells may also be therapeutically beneficial. An often insurmountable obstacle is that finding that limited homology between human and rodent orthologs of chemokine receptors means that potent, efficacious inhibitors of a human receptor often have little if any activity at their rodent ortholog. This makes target validation impossible and reports describing the in vivo efficacy of such compounds generally lag well behind in vitro studies. However, a flurry of recent papers have described activities of CCR3 antagonists in a variety of animal models. The Yamanouchi Pharmaceutical Company have recently described compounds with efficacy in an assay of eosinophil recruitment to the macaque lung following bronchoprovocation with CCL11 (Morokata et al. 2006), and in a murine model of cutaneous inflammation (Suzuki et al. 2006). Likewise, Abbot Laboratories have described the antagonist A-122058, which was effective in reducing the number of eosinophils following intraperitoneal injection of CCL11 into mice (Warrior et al. 2003). To date, the only small-molecule antagonist of CCR3 to have entered phase II trials has been the GSK766994 compound from GlaxoSmithKline and, following a lack of efficacy in a study of allergic rhinitis, there are no plans to assess the efficacy of this molecule in the asthmatic setting (Murdoch 2006). This follows on from the reports of efficacy of their GW701897B compound in a model of vagally mediated bronchoconstriction in antigen-challenged guinea-pigs (Fryer et al. 2006). One point worth making is the fact that chemokine antagonists, like many agonists and antagonists of GPCRs,

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exert their effects by binding to the transmembrane helices (de Mendonca et al. 2005), a region often highly conserved between different receptors. Thus, it is not surprising that some of these compounds have selectivity for more than one receptor, such as the compound UCB 35625 (a trans isomer of BANYU J113863) which has nanomolar activity at both CCR1 and CCR3 (Sabroe et al. 2000). It may prove beneficial to target one or more receptors with such compounds to provide a more broad-spectrum approach to chemokine receptor blockade.

Acknowledgments We are grateful to Asthma UK and the Wellcome Trust for their support of our research in this field.

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Chemokines

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Neurotrophins Wolfgang A. Nockher, Sanchaita Sonar and Harald Renz

Summary Neurotrophins, namely nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), neurotrophin (NT)-3, NT-4, and NT-5, were first discovered as target-derived growth factors responsible for the survival and maintenance of specific subsets of central and peripheral neurons. Additional neurotrophin-responsive cells are now known to include immune cells such as mast cells, lymphocytes, and eosinophils, as well as other ectodermal-derived cells such as keratinocytes and epithelial cells. Moreover, these cells are also shown to synthesize neurotrophins, especially after activation during an inflammatory response. Therefore, neurotrophins not only modulate various cell to cell signaling within an inflamed tissue, such as neuroimmune interactions, but also interactions with structural tissue cells. Initial observations have revealed elevated levels of systemic neurotrophin levels in allergic inflamed tissues. The pathophysiology of allergic diseases is characterized by an initial immune response as well as alterations of the local innervation and the integrity of tissue cells. Neurotrophins have been shown to modulate the sensory innervation of allergic inflamed tissues resulting in an enhanced neuronal activity. In allergic airway diseases such as asthma or rhinitis, neurotrophins account for major clinical symptoms such as bronchial hyperreactivity or rhinorrhea and sneezing reflex. Furthermore, in allergic skin diseases, neurotrophins have been shown to modulate cutaneous neuroimmune interactions resulting in sensory hyperreactivity and enhanced pruritus. More recent data indicate that neurotrophins not only account for changes in neuronal density and activity, but also influence the function of immune and structural cells during allergic inflammation. Neurotrophins promote the activation and survival of infiltrating and resident immune cells, e.g., eosinophils and mast cells, and studies of various allergic diseases reviewed over the last two decades indicate that neurotrophins behave as inflammatory cytokines delivering and activating survival signals to effector cells of the allergic response. In addition, it has also been shown


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that neurotrophins may also modify diverse functional activities of structural tissue cells, e.g., fibroblasts and keratinocytes. While most knowledge comes from studying NGF, the most prominent member of the neurotrophin family, the contribution of other neurotrophins like BDNF, NT-3, and NT-4 remains to be more intensively elucidated. In various animal studies, local inhibition of neurotrophin signaling significantly abrogated development or magnitude of the allergic inflammation and airway hyperreactivity. Therefore, novel strategies for therapy of allergic diseases may result from a precise understanding of the molecular mechanisms of neurotrophin functions as well as unraveling differential activities of single members of the neurotrophin family.

Neurogenic inflammation Allergic diseases are characterized by an activation of the immune system resulting in an induction of cellular and humoral immune response followed by eosinophilia and synthesis of IgE antibodies. Recent studies indicate an extensive communication between cells of the nervous and the immune system during such an inflammatory process. The direct potentiation of local inflammation by directional pathways connecting neurons and immune cells has led to the concept of “neurogenic inflammation.” The classical concept of this hypothesis defined an alteration of the inflammatory response by the local nervous system (Barnes 1992). Stimulation of an afferent nerve results in the propagation of the action potential not only to the spinal cord but also to a retrograde activation of synaptic endings in the tissue resulting in a release of neuropeptides and neurotransmitters (axon reflex). These neurotransmitters then modulate functional activity of immune cells. However, this concept may also work in the opposite direction, namely that inflammatory cells influence the structure and activity of tissue nerves. Thereby, inflammation-induced alterations in the local neuronal function can lead within a vicious cycle to increased release of proinflammatory neuropeptides on stimulation (Fig. 22.1). Neuroimmune interactions have been well described in local allergic inflammation of the skin or the airways, such as atopic

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Neurotrophins

Stimulation of an afferent nerve

Epithelial barrier

Fig. 22.1 Axon reflex and neurogenic inflammation in allergic airway diseases. Neurotrophins potentiate release of neuropeptides resulting in enhanced vessel and immune cell reactivity. Eos, eosinophil. (See CD-ROM for color version.)

Vessel: Vasodilation Extravasation of plasma proteins Immune cells: Chemotaxis Activation

dermatitis or asthma (James & Nijkamp 1999; Darsow & Ring 2001). One important feature of this neuroimmune interaction is the alteration of the local innervation by growth and activation factors like interleukins and cytokines liberated during the allergic inflammation by immune cells. Vice versa, the inflammatory process represented by infiltrated immune cells, is modulated by neurotransmitters and neuropeptides that are released by the local nervous system. Neurogenic inflammation may also be initiated by activation of sensory nerves, inflammatory mediators or irritants invading the skin or the airways. The cross-talk between neurons and immune cells is then mediated via cytokines, neuropeptides and their receptors. Neuropeptides, the main neurotransmitters of sensory nerves, such as tachykinins (substance P and neurokinin A and B) or calcitonin gene-related peptide (CGRP), are transferred via axonal transport not only to presynaptic axons endings in the spinal cord but also to peripheral nerve endings. On stimulation with mechanical, thermal, chemical or inflammatory stimuli, tachykinins are released and act in a dual fashion, as afferent neurotransmitters to spinal cord motor neurons as well as efferent neurosecretory mediators diffusing into the peripheral tissue (Barnes 1996). Many studies indicated that tachykinins as well as other neuromediators released from sensory nerve endings participate in allergic airway and skin inflammation. Substance P, the most prominent member of the tachykinin family, modulates a broad range of functional responses of immune cells including lymphocytes, eosinophils, mast cells, macrophages, and nonimmune cells such as microvascular endothelial cells, fibroblasts, keratinocytes, and epithelial cells. These functional responses include cellular development, growth, differentiation, chemotaxis, vasoregulation, and wound healing. Neurogenic inflammation is mainly involved in allergic airway diseases, such as asthma or rhinitis, contributing to the inflammatory response to allergens, infections, and various irritants. Following irritation, activation of sensory airway nerves leads to an axonal response that acts as an immediate protective mucosal defense mechanism resulting in coughing and sneezing. Coughing, sneezing and other protective mechanisms clear the upper and lower airways from offend-

Neurotrophins

Eos

Tissue Vessel T cell

Retrograde propagation of the action potential

Neuropeptides

Propagation of the action potential to the spinal cord

ing agents. However, dysfunction of sensory nerves due to an enhanced and/or chronic activation results in an overexpression of neuropeptides. Increased concentrations of neuropeptides modify the functional activity of other nerves, structural cells, e.g., smooth muscle cells, as well as resident and invading immune cells. While the immunoregulation of neuropeptides has been intensively studied, an important question is related to the mechanism of neurotransmitter induction during the inflammatory process. It is well known that various interleukins like IL-1 mediate signals from the immune to the nervous system and activate tachykinin synthesis; however, communication between neurons and immune cells is not exclusively restricted to these “classical” cytokines. Recent studies have indicated that neurotrophins, a protein family of growth factors originally found in the nervous system, trigger neuroimmune interactions. Neurotrophins exert their signals through specific cell-surface receptors, which are found on various cell types within and outside the nervous system. Neurotrophins have been reported to influence the intensity and duration of a local immune response through the regulation of neuropeptide synthesis in the local nervous system. As neuropeptides are short-lived signaling molecules that are rapidly degraded at the site of synthesis, their action is therefore mainly restricted to the site of synthesis. Neurotrophins, however, are produced constitutively by various tissue and immune cells during the allergic inflammation. Therefore, acting as long-term modulators, neurotrophins potentiate interactions between the nervous and immune systems (Fig. 22.2). In addition to controlling neuropeptide synthesis, neurotrophins modulate the number and activity of peripheral nerves resulting in neuronal hyperplasia. Recent data suggest that neurotrophins exert direct effects via their receptors on immune cells and also modulate biological activities of structural tissue cells, such as fibroblasts, keratinocytes, epithelial, and smooth muscle cells. During allergic tissue inflammation, structural cells respond morphologically and functionally resulting in increased proliferation, cytokine, and mediator release, and synthesis of extracellular matrix components. Enhanced neurotrophin expression triggered

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Inflammatory Cells and Mediators are well defined: BDNF, NT-3, and NT-4/5. NT-4 was first discovered in Xenopus laevis and it supports the survival of chick neurons, whereas NT-5, thought to be the mammalian equivalent of NT-4, does not. Whether or not NT-4 and NT-5 are different members of the neurotrophin family, or the same protein in different species is presently unclear. Today the affiliation of additional factors (e.g., NT-6 and NT-7) to this protein family is under discussion. Translated neurotrophins contain a signal sequence for secretion and are produced as precursor proteins (proneurotrophins) of 30–35 kDa. Following proteolytic C-terminal cleavage, a biologically active 12–14 kDa protein is released. The mature proteins are very well conserved and approximately 50% of the amino acids are common to all neurotrophins, indicating a functional homology. Within the cells, the neurotrophins are stored in vesicles and secreted as biologically active protein dimers associated by noncovalent bonds. However, the unprocessed 30–35 kDa proforms can also be released and there is growing evidence that the proneurotrophins are not only intracellular precursor molecules, but may also possess special functions in signal transduction outside the cell. There is also some evidence that NGF and pro-NGF differentially activate pro- and antiapoptotic cellular responses through differential activation of neurotrophin receptors, and the physiologic significance of proneurotrophin signaling is still under investigation. All members of the neurotrophin family use a common group of specific high-affinity cell-surface receptors: the tropomyosin-related tyrosine kinase (Trk) receptors (Fig. 22.4). Additionally, neurotrophins also signal through a common low-affinity neurotrophin receptor, the p75NTR (Pattarawarapan & Burges 2003). The p75NTR is a member of the tumor necrosis factor (TNF) receptor/Fas/CD40

Neurotrophins

Neuropeptides

Chronicity of the disease (t) Fig. 22.2 Potentiation of neurogenic inflammation by neurotrophins. (See CD-ROM for color version.)

by inflammatory cytokines integrates structural tissue cells into the neurotrophin signaling network during allergic inflammation (Fig. 22.3).

Biology of neurotrophins and neurotrophin receptors Neurotrophins are a family of homologous proteins that have similarities in structure, and receptor utility as well as physiologic activities. While the first member of the protein family, NGF, was discovered about half a century ago, in the following decades additional neurotrophins have been reported and the discovery of additional members remains open. Today, at least three additional members of this family

NEUROTROPHINS: NGF, BDNF, NT-3 Excitability Structural cells: Epithelia, Fibroblasts NGF induction CYTOKINES: IL-1, TNF, etc.

Synthesis

Biological effects

NEUROPEPTIDES: Substance P, Neurokinin Immune cells: Macrophages Lymphocytes Eosinophils Mast cells

Airway remodeling

Airway inflammation

• Subepithelial thickening • ECM deposition • Proliferation of ASMC

• Activation and survival of eosinophils in the lung • Accumulation and mediator release of lung mast cells

496

Airway nerves

Activity

Airway hyperresponsiveness • Unspecific irritation • Low stimulation threshold • Enhanced contractility

Fig. 22.3 Neurotrophin signaling modulates the activity of neurons, and immune and structural cells during allergic inflammation of the airways. ASMC, airway smooth muscle cells; ECM, extracellular matrix. See text for definition of other abbreviations. (See CD-ROM for color version.)

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NGF BDNF NT-3 NT-4

p75NTR Low-affinity

NGF

trkA

BDNF NT-4

trkB

NT-3

trkC

High-affinity

Fig. 22.4 Signaling of neurotrophins by cell-surface receptors. See text for definition of other abbreviations. (See CD-ROM for color version.)

superfamily and all mature neurotrophins bind with low affinity to this receptor. In contrast, the Trk receptors bind neurotrophins specifically and with a higher affinity; however, there exists some crossreactivity between the Trk receptors and the neurotrophin ligands. NGF interacts preferentially with TrkA receptors and BDNF binds preferentially to TrkB. NT-3 interacts preferably with the TrkC receptor but can also bind to TrkA and TrkB. NT-4/5 overlaps with BDNF for TrkB receptor usage. Following specific binding of neurotrophins to their respective Trk receptors, they induce receptor homodimerization, which initiates kinase activation and subsequent transphosphorylation. The Trks are receptor tyrosine kinases that utilize a complex set of intracellular substrates and adaptor proteins to activate a defined secondary signaling cascade required for neurotrophin signal transduction. Classical signaling molecules such as the phosphatidylinositol 3-kinase pathway and the mitogen-activated protein (MAP) kinase cascade, have been identified as the primary downstream cellular events following Trk activation. However, the specificity of downstream Trk receptor-mediated signaling is controlled by the expression of intermediate molecules in these pathways as well as membrane trafficking that regulates the localization of different signaling components. As a consequence, in various cell types Trk receptor activation may result in different biological effects. While activation of the Trk receptor system generally induces cell differentiation, proliferation, and survival, signaling pathways activated by p75NTR appear to promote survival as well as apoptosis of cells. Thus, the signaling of neurotrophins via their complex receptor systems is not thoroughly and completely understood. In the absence of Trk receptors, p75NTR mostly induces apoptosis in conjunction with non-Trk receptor complexes such as sortilin. However, coexpression of p75NTR and TrkA may form high-affinity NGF binding sites by the interaction of transmembrane and cytoplasmic domains of both receptor molecules, and, thus, the p75NTR enhanced NGF-mediated TrkA receptor activity. In this way, it seems that the traditional view of an independent signaling of p75NTR and the Trk receptors may change to provide a more interactive cooperation during neurotrophin signaling.

Neurotrophins

Cellular sources and effects of neurotrophins The important role of neurotrophins as neuronal growth factors has been well known for about five decades, and the contribution of the peripheral nervous system to the pathogenesis of allergic diseases has also been well recognized. However, it is now well established that the expression as well as the signaling of neurotrophins via their specific receptors is not only restricted to the nervous system but also found in various other cell compartments (Fig. 22.5). These “other cell compartments” comprise also cells of the immune system that are able to express both neurotrophins and their corresponding receptors. These observations provide growing evidence that neurotrophins play a key role in influencing the developing allergic immune response. As the pathophysiology of allergic diseases depends on the progression of allergic inflammation, the biological functions of neurotrophins within the immune system are very important and will be discussed in detail. In addition, recent findings in asthma research lead to the suggestion that neurotrophins may also influence tissue remodeling processes, which are hallmark events that accompany chronic asthma. Moreover as discussed, in addition to cells of the nervous and immune system, neurotrophins are also expressed by structural tissue cells such as epithelia, fibroblast, and smooth muscle cells. Preliminary data indicate that neurotrophins may have autocrine and/or paracrine effects on functional activities of resident tissue cells. Based on all these observations, it is obvious that neurotrophins are not only growth factors modulating the peripheral nervous system, but also could play a more general role as cytokines mediating/delivering signals within and/or between various cell systems (Nockher & Renz 2006b). In this way, the definition of neurotrophins as “neurotrophic factors” seems to reflect a scientific relic rather than the biological tissue specificity of these signaling molecules (Fig. 22.5).

Peripheral nervous system Disturbances of neuronal activities within the allergic inflamed tissues are well known. For example, pruritus in atopic dermatitis and airway hyperresponsiveness in allergic asthma are essential features of these diseases and mainly contribute to the morbidity of these diseases. Therefore, unraveling the pathophysiology of neuronal changes in allergic inflammation is a key to the development of clinical strategies in the treatment of allergic diseases. The neurotrophins were originally identified as essential factors for the development of the vertebrate nervous system (Lewin & Barde 1996). They regulate the survival, death or the differentiation of neurons in the embryonic and postnatal stages, as well as neuronal maintenance later in life. While many sympathetic neurons depend on NGF for survival into adulthood, postnatal sensory neurons cease to rely on this

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Sources

Targets

Effects Neuropeptide secretion Activity

Airway epithelium Neurons Keratinocytes

NGF BDNF NT-3 NT-4

Proliferation

ASM

Eos

Lym

MF MC

Activation Proliferation Mediator release

Inflammatory cells

factor. Somatic sensory neurons have their cell bodies within the dorsal root ganglia and their processes project to the spinal cord and to the periphery via the dorsal nerve root. Sensory ganglion cells are heterogeneous with regard to cell body size, fiber type and sensory receptor subtype. Moreover, they vary in neuropeptide content and signaling properties. In general, large-fiber sensory neurons are attached to muscle spindles and mediate proprioception. Neurons with mediumsized fibers subserve mechanoreceptors, which are located in joints as well as in the deep cutaneous layers of the skin, and mediate touch, pressure and vibratory sensations. In contrast, small-fiber neurons have terminals in the superficial skin, and mediate pain and temperature sensation. In general, larger axonal diameters are associated with larger cell bodies of the neurons and this relationship, although only approximate, has enabled researchers to correlate specific neurotrophin receptor expression with functional aspects of neurons within the dorsal root ganglion. The NGF receptor TrkA is expressed predominantly on small-diameter neurons, some of which are nociceptors. In contrast, TrkB, the high-affinity receptor for BDNF, is mainly expressed on medium-sized sensory neurons, which generally function as mechanoreceptors. The NT-3 receptor TrkC is found on large-diameter sensory neurons, which mediate proprioception. In chicken embryos, about 75% of large-diameter muscle afferents express TrkC, whereas only about 7% of large-diameter cutaneous afferents express this protein. The corresponding neurotrophin ligands NGF, BDNF and NT-3 were expressed in the appropriate target tissues that are innervated by these neurons, e.g., superficial epidermis, deep dermal layers, and muscle fibers. Thus, neurotrophins are target-derived neuronal growth factors that regulate neuronal outgrowth towards their place

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Fig. 22.5 Cellular sources and responders to neurotrophins in allergic inflammation. ASM, airway smooth muscle; Eos, eosinophil; Lym, lymphocyte; MC, mast cell; Mf, macrophage. See text for definition of other abbreviations. (See CD-ROM for color version.)

of synthesis (Ernfors 2001). Overexpression of neurotrophins in peripheral body tissues results in hyperinnervation as demonstrated in genetically modified animals. Transgenic mice with constitutive NGF overexpression in the clara cells of the lung exhibit an increased number of tachykinincontaining sensory as well as sympathetic nerve fibers in the airways. Vice versa, homozygous mice with disruption of the NGF gene showed extensive sensory and sympathetic neuronal cell loss. In addition, mice that lack expression of the p75NTR were shown to have a deficit in sensory innervations. On the other hand, mice lacking BDNF expression develop severe sensory deficits, while survival of sympathetic neurons is not affected. While visceral sensory neurons require neurotrophins for survival during development, these neurons do not further depend on neurotrophins for their survival throughout adulthood. Nevertheless, neurotrophins influence the function of peripheral sensory neurons after development, such as upregulation of neuropeptide synthesis (Lindsay & Harmer 1989), increase in excitability, establishment of functional synapses, or peripheral axonal branching. Therefore, enhanced tissue expression of neurotrophins results in functional changes and hyperactivity of the peripheral nervous system.

Immune system While the allergic inflammation is the initial trigger of the development and magnitude of all allergic diseases, the contribution of neurotrophin signaling to the local and systemic inflammatory response is of outstanding interest. The beststudied neurotrophin within the immune system is NGF, the first discovered and most prominent member of the neurotrophin family. Nevertheless, for many cells of the immune

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Differentiation

Mediators (IL-4, EPX) Cytotoxicity Chemotaxis

NT-3

Survival

Th2 Th1

Eos

NGF BDNF NT-3

? Survival

T-naive NGF

B cell

Survival Phagocytosis NO-synthesis

M NGF NT-3

MC

Priming Maturation Degranulation Survival

Fig. 22.6 Stimulatory effects of neurotrophins on various biological functions in the immune system. While the role of NGF has been most intensively studied, actions of other neurotrophins are less well characterized. Eos, eosinophil; EPX, eosinophil peroxidase; MC, mast cell; Mf, macrophage; NO, nitric oxide. See text for definition of other abbreviations. (See CD-ROM for color version.)

system the production of one or more neurotrophins and/or the expression of various neurotrophin receptors has been described. Consequently, neurotrophins mediate autocrine as well as paracrine signaling, which results in diverse biological effects. These effects besides cell survival, differentiation, and/or proliferation, also include other functions such as activation and cytokine or mediator release (Fig. 22.6).

Monocytes, macrophages, and dendritic cells Dendritic cells and tissue macrophages are important in the pathogenesis of local inflammatory processes. As antigenpresenting cells, they are mainly involved in the initiation and acceleration of the allergic immune response. While the involvement of dendritic cells and tissue macrophages within the cytokine signaling network of an inflammatory process is well established, their contribution to neurotrophin signaling is, so far, less well resolved. Whereas some data about neurotrophin expression are available for the monocytes/ macrophage system, detailed information about neurotrophin signaling in dendritic cells is absent. Within macrophage populations in various body tissues, lung macrophages are the best studied with regard to neurotrophin expression. In a murine model of allergic asthma our group has shown that macrophages found in bronchoalveolar lavage (BAL) fluid produce NGF and BDNF after allergen challenge (Nockher & Renz 2003), and others have shown a constitutive expression of NT-3 and NT-4 in murine alveolar macrophages. While mouse alveolar macrophages express NT-3 and NT-4/5 without their corresponding receptors, interstitial lung macrophages expressed BDNF and NT-4/5 as well as the receptors TrkB and TrkC. This implies that interstitial macrophages show a bidirectional mode of neurotrophin signaling both

Neurotrophins

via autocrine and paracrine mechanisms; however, alveolar macrophages are insensitive for neurotrophins due to receptor deficiency. In humans, macrophage populations are less studied. However, monocytes isolated from human peripheral blood show a constitutive expression of BDNF and NGF, and the release of NGF is significantly enhanced in monocytes from allergic patients compared with those from healthy donors (Rost et al. 2005). Monocyte secretion of BDNF has been shown to be increased by inflammatory cytokines such as IL-6 and TNF-α, which are released by activated cytokines (Schulte-Herbruggen et al. 2005). Neurotrophins function as survival factors for monocytes and macrophages especially after activation. Monocytes express the NGF receptor TrkA and ligand activation protects the cells from apoptosis induced by gliotoxin or UV-radiation. When monocyte-derived macrophages were stimulated with lipopolysaccharide (LPS), their NGF as well as TrkA expression was significantly enhanced while neutralization of endogenous NGF in LPS-activated macrophages induced apoptosis. Therefore, NGF exerts a survival effect on cells of the monocyte/macrophage lineage and activated cells especially required NGF for protection against apoptosis.

Lymphocytes T lymphocytes are important immune cells responsible for directing and maintaining a local allergic inflammatory response by production of Th2-type cytokines. Additionally, the synthesis of neurotrophins has been shown in T cells. Splenic T cells from mice express basal levels of NGF and NT-3, and activation with concanavalin-A upregulates their synthesis. BDNF release occurs after antigen stimulation of cultured splenic T cells from sensitized mice. Interestingly, tissue-specific BDNF production by T cells was observed in this study. T cells isolated from the inflamed lung showed high levels of BDNF synthesis without stimulation, whereas unstimulated T cells from the spleen did not produce detectable levels of BDNF. These differences may result from different T cell populations in spleen and lung, and/or a preactivation state of lung T cells at the site of local inflammation. In humans, CD4+ and CD8+ T cells, as well as CD19+ B cells purified from human peripheral blood lymphocytes, constitutively synthesize increased amounts of BDNF after stimulation. In contrast CD19-sorted B cells obtained from mouse spleen or lungs did not synthesize BDNF even under stimulation. Lymphocytes are not only able to synthesize neurotrophins, but also depend on these factors for maturation and maintenance. The expression of various neurotrophin receptors has been shown in human T cell clones, and in the mouse BDNF/TrkB signaling is necessary for the development of thymocyte precursors. Failure of Trk signaling results in mass apoptosis of lymphocytes, as has been shown in the thymus of TrkB-deficient mice. In the B cell system, NGF acts as an autocrine survival factor for surface γ+/α+ memory B

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lymphocytes but NGF had no effect on IgM→IgG or IgM→ IgA class switching (Torcia et al. 1996). Thus, NGF seems to be essential for the maintenance of specific secondary IgG- and IgA-mediated immune responses. On the other hand, divergent findings were reported with regard to IgE production. In human B lymphocytes, NGF inhibits the production of IgE after induction by IL-4, while in mononuclear cell preparations from spleens of allergic sensitized mice NGF favors IgE synthesis (Nockher & Renz 2003). While allergic inflammation is driven by a Th2-biased immune response, the possible role of neurotrophins in initiating or maintaining this process may be of interest. It has been found that Th1 but not Th2 or Th0 cell clones express the active BDNF receptor gp145trkB, and the Th1 or Th2 cytokine milieu favors expression of different members of the neurotrophin family. Both Th1 and Th2 CD4+ T cell clones produce low amounts of NGF in culture; however, upon mitogen stimulation, only Th2 cells upregulate NGF secretion, indicating a preferential Th2 source for this factor following T-cell activation (Lambiase et al. 1997). Additionally, when naive ovalbumin-specific CD4+CD45RB+ T cells from ovalbumin-specific D011.10 T cell receptor transgenic mice were directed to Th1/Th2 differentiation, the NT-3 receptor TrkC was expressed in Th2 cells but not in Th1 and naive CD4+ T cells (Sekimoto et al. 2003). Consequently, NT-3 synergistically enhanced anti-CD3-induced IL-4 production by Th2 cells but did not affect interferon (IFN)-γ synthesis by Th1 cells. Thus, at least in this mouse model, NT-3 seems to play a critical role in regulating Th1/Th2 balance, favoring a Th2-biased immunity. However, further studies are necessary to clarify if enhanced neurotrophin signaling directly influences the direction of T helper cell maturation during allergic inflammation in humans.

Eosinophils Eosinophils are the most important infiltrating cell population in allergic reactions, especially in allergic airway diseases. Eosinophils produce an array of cytokines and chemokines that contribute to the allergic inflammatory response, and also cell growth factors involved in tissue repair and fibrosis. During the last decade, it has been recognized that although human eosinophils produce NGF and NT-3 both factors are differentially regulated (Kobayashi et al. 2002). Whereas NT3 is constitutively expressed without further regulation, the amount of NGF synthesis depends on cellular activation. Synthesis of NGF is enhanced by Fc-receptor-mediated stimuli and eosinophil stimulation with IL-5 and soluble immune complexes results in an increased immunologically and biologically active NGF within the cells (Kobayashi et al. 2002). Although eosinophils are potentially able to express cellsurface receptors for neurotrophins, receptor expression may depend on the maturation or activation of these cells. In the bone marrow, eosinophils express the TrkB and TrkC receptors for BDNF and NT-3, respectively, but not the NGF

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receptor TrkA as well as the p75NTR. The expression profile of NT receptors in peripheral blood eosinophils is not clear. In one report, expression of all receptors was shown; however, the Trk expression varied both on mRNA as well as on the protein level between single patients. In contrast, in our experiments, circulating blood eosinophils from allergic patients did not show any Trk expression, but, more importantly, eosinophils obtained from BAL fluid after allergen provocation expressed all the neurotrophin receptors (Nassenstein et al. 2003). When BAL fluid eosinophils were cultured in the presence of neurotrophins, an antiapoptotic effect of neurotrophins was observed. Consequently, all members of the neurotrophin family are survival factors for eosinophils obtained from BAL fluid, but not for circulating blood eosinophils of patients with allergic asthma. Thus, the induction of neurotrophin receptors in activated eosinophils makes them susceptible to the antiapoptotic signals delivered by neurotrophins acting in an autocrine or paracrine manner. As cytotoxic activity and production of IL-4 is enhanced by NGF, one may speculate that an increased local neurotrophin synthesis supports maintenance as well as cellular activity of eosinophils within the inflamed tissue.

Mast cells Mast cells are important tissue-resident effector cells of the allergic immune response as they mainly contribute to immediate hypersensitivity reactions when activated through binding of IgE–antigen complexes. Numbers of mast cells are increased in allergic tissues, and recent studies have shown that NGF and NT-3, especially, play a pivotal role in mast cell biology as they mediate survival and differentiation. Reports on neurotrophin receptor expression in mast cells vary in a species- and tissue-dependent way. Mast cells obtained from different sources as well as from different species were all shown to respond to NGF through expression of the specific NGF receptor TrkA (Sawada et al. 2000); however, the expression pattern of other neurotrophin receptors is not unique. In mice, mast cells obtained from neonatal skin express TrkC; mast cells prepared from human lung express TrkB and TrkC but human umbilical cord blood-derived mast cells lack TrkB expression. While NGF alone does not support the survival of cultured mast cells, NGF acts as a cofactor together with stem cell factor (SCF) to prevent apoptosis. This effect is clearly antiapoptotic rather than proliferative as NGF does not stimulate changes in cell cycle progression. A similar finding has been recently observed in NT-3-overexpressing transgenic mice, where increased numbers of mast cells in the skin did not correlate with local mast cell proliferation (Metz et al. 2004). The authors speculate that NT-3 modulates skin mast cell numbers via differentiation of preexisting, immature mast cells and/or by enhancing the migration of circulating mast cells into the skin. At least, NGF has been shown to act as a chemoattractant for mast cells and induces chemotactic

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movement together with morphologic changes within the cytoskeleton (Sawada et al. 2000). Furthermore, NGF is involved in mast cell development such as differentiation from cord blood cell progenitors. NGF, and even more the combination of NGF and SCF, increased or induced expression of typical mast cell markers: IgE-receptor type I (FceRI), chymase, or mast-cell specific tryptase. Additionally, NGF modulates inflammatory cytokine expression such as induction of IL-6 expression and inhibition of TNF-α. NGF also regulates mast cell degranulation and subsequent cytokine and mediator release. In vitro, NGF stimulates the release of serotonin in cultured mast cells. Controversially, NT-3 failed to increase serotonin release in cultured mast cells but induced a significant increase in mast cell degranulation in vivo (Metz et al. 2004). While mast cells respond to NGF and NT-3, expression of NGF, NT-3, as well as NT-4/5, has also been shown. Therefore, neurotrophins are involved in vital functions of tissue mast cells, and mast cells have the capacity to produce these factors in an autocrine manner. Spontaneous NGF release is significantly increased in response to IgE and specific allergen (Xiang & Nilson 2000), and thus NGF could be specifically released by stimuli causing an allergic reaction and mast cells can thereby be a source of NGF in IgE-mediated inflammatory diseases.

Structural tissue cells The neuroimmune cross-talk involved in airway diseases also involves structural cells and their contribution to both neurotrophin production and responsiveness via autocrine and/or paracrine manner. Synthesis of neurotrophins like NGF and BDNF by structural cells such as the airway epithelia has been clearly demonstrated. Various in vitro studies with human lung cells in culture have identified bronchial epithelial cells, primary fibroblasts, and bronchial smooth muscle cells to express NGF (reviewed by Nockher & Renz 2003). Besides bronchial structural cells, human cutaneous fibroblast cell lines, intestinal epithelium, retinal epithelium, human vascular epithelium, and also vesical smooth muscle cells are other potential sources of NGF. Moreover, the synthesis of BDNF and NT-3 has been shown in structural cells like rat thoracic aortic smooth muscle cells, and human keratinocytes were identified to express NT-4/5 (Grewe et al. 2000). In vitro stimulation with IFN-γ, a marker cytokine for atopic eczema, was shown to induce keratinocyte NT-4 production (Grewe et al. 2000). In the same study, IFN-γ-injected skin and prurigo lesions of atopic dermatitis skin were characterized by intense epidermal staining for NT-4. Consistent with the in vitro studies, in vivo studies corroborate very well the expression of NGF by structural cells in the airways. Immunolabeling of bronchial biopsies from healthy patients without asthma show considerable staining of NGF in epithelial cells, bronchial smooth muscle cells, and fibroblasts (OlgartHögland et al. 2002). Immunohistochemistry performed in

Neurotrophins

mouse airways in our laboratory showed epithelial cells lining the airways as potential sources of neurotrophins like NGF, BDNF, and NT-3. The epithelial neurotrophin production was shown to be upregulated by IL-1β, TNF-α and Th2 cytokines. Inflammatory cells like eosinophils express the TrkA and TrkB receptors responsive to NGF and BDNF, respectively, and coculture with airway epithelial cells results in an enhanced epithelial neurotrophin production, which in turn enhances eosinophil survival (Hahn et al. 2006). Although most neurotrophins are clearly produced from structural cells, the relative expressions of each are different. This becomes more relevant in pathologic conditions as differential modulation of neurotrophins could shed more light on their individual relevance and functions, rather than all being referred to equally for the condition. Furthermore, in inflammatory conditions, the infiltrating inflammatory cells further supplement the increases in NT levels. Therefore, besides a physiologic function, their levels could be important in pathologic conditions and the role of each in such a scenario needs further investigation. The increases in neurotrophins could then have an autocrine or a paracrine effect on the structural cells lining the airway. One function for the responsiveness of structural cells to neurotrophins could be proliferation. Studies in human airway cells in culture show that NGF production depends on cell density. This has been shown in human fibroblasts and in bronchial smooth muscle cells (Olgart & Frossard 2001). It has been suggested that NGF is synthesized at higher rates by proliferating cells than by confluent cells. This could be an important factor in disorders like asthma where injury to airway epithelial cells triggers proliferation and therefore the increases in NGF levels. Other factors that lead to an induction in neurotrophin levels by structural cells are cytokines. Proinflammatory cytokines such as IL-1β and TNF-α or transforming growth factor (TGF)-β, increase the synthesis of NGF by airway structural cells. Human pulmonary fibroblasts, airway epithelial cell lines and bronchial smooth muscle cells, have all been shown to respond to proinflammatory cytokines by NGF production (reviewed by Nockher & Renz 2003). The increases in neurotrophin levels by structural cells observed in asthmatic airways and allergies seem to be due to a concerted outcome brought about by varied signals that are also upregulated in inflammatory conditions. Recent studies have also suggested that NGF is capable of directly stimulating structural cells. NGF has been shown to induce contraction and migration of human pulmonary fibroblasts (Micera et al. 2001) and vascular smooth muscle cells. The presence of specific receptors for neurotrophins in structural cells like TrkA and p75NTR further suggests an autocrine effect that could be useful for the cells in pathologic conditions. NGF, however, is also able to induce the proliferation of structural cells (Botchkarev et al. 2004) and can support the regeneration of injured tissue. Clinical studies as well as animal studies support an important role of NGF in wound healing.

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Topical treatment of corneal ulcers with NGF promotes restoring of corneal integrity (Lambiase et al. 1998). In a case report, NGF application reduced the size of pressure ulcers by favoring the increase of epithelial tissue (Bernabei et al. 1999). Moreover, topical administration of NGF into wounds increases the degree of reepithelialization and is able to accelerate the rate of wound healing in the skin (Matsuda et al. 1998).

Pathophysiology of neurotrophins in allergic diseases Initially, elevated neurotrophin synthesis was described in a study of patients with vernal keratoconjunctivitis, a disease with local mast cell activation. Later on, NGF serum levels were found to be increased in patients with various allergic diseases such as allergic asthma, urticaria, and atopic dermatitis (Bonini et al. 1996). Neurotrophin levels were also increased in local body fluids such as BAL fluids or nasal fluids of allergic patients, and local neurotrophin production resulted from enhanced neurotrophin expression of immune as well as structural cells. Consequently, an enhancement of local production of NGF and other neurotrophins has been shown in body fluids and tissue biopsy specimens obtained from allergic patients. However, the exact cellular sources of circulating neurotrophins are still not well defined. While a spill-over from enhanced local production into the bloodsream is noteworthy, direct proof is still lacking. One hint may come from asthmatic patients after immunosuppressive treatment with glucocorticoids, who show a significant decline in circulating neurotrophin levels (Noga et al. 2001). Inhaled glucocorticoid drugs were designed to exert their immunosuppressive effects mostly locally at the site of administration with only negligible systemic effects. Therefore, one may speculate that the reduction of blood neurotrophin levels originates from the suppression of an upregulated neurotrophin synthesis in the airways. Vice versa, increased levels of circulating neurotrophins may originate from enhanced local production within the inflamed tissue. It is well known that human platelets contain large amounts of BDNF, probably accumulated during vascular circulation and which could be released after platelet activation (Lommatzsch et al. 2005a). Recently, it has been shown in asthmatics that plasma and platelet levels of BDNF correlate with parameters of airway hyperresponsiveness (AHR) in steroid-naive but not corticosteroid-treated patients (Lommatzsch et al. 2005b). As corticosteroids were able to suppress BDNF secretion in blood mononuclear cells, this clinical study gives indirect evidence of a role of neurotrophins in the progression of inflammatorymediated airflow limitation in asthma.

Allergic asthma Allergic asthma is characterized by chronic airway inflammation, development of AHR, recurrent reversible airway

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obstruction, and subsequent airway remodeling. It is now clear that neurotrophins mainly contribute to the pathogenesis of inflammation and AHR, as well as tissue remodeling in allergic asthma.

Airway inflammation Allergic inflammation results in elevated serum IgE antibody titers, cellular infiltration of eosinophils and lymphocytes into lung tissue, and high levels of inflammatory cytokines such as IL-4 or IL-5 in BAL fluids. Asthmatic patients display increased levels of neurotrophins in serum and BAL fluid. Increased neurotrophin production has also been reported in response to segmental allergen provocation in the lung. Notably, this upregulation occurred at 18 hours after allergen challenge, when a marked infiltration of immune cells was also evident. This finding implies that inflammatory cells are a major source of neurotrophin synthesis in the allergic lung and eosinophils, lymphocytes, and macrophages have been shown to produce these factors (Nockher & Renz 2003). However, whether neurotrophin expression is upregulated even earlier after allergen challenge has not been investigated in humans. Animal models of allergic airway inflammation have also revealed that T lymphocytes and macrophages represent potential sources of neurotrophin production in the inflamed lung, and in vitro allergen stimulation of mononuclear cells from sensitized animals was shown to result in enhanced NGF synthesis. While immune cells are also able to express neurotrophin receptors, autocrine/paracrine effects of enhanced neurotrophin expression may modulate functional aspects of these cells. On the one hand, infiltrating immune cells such as eosinophils and lymphocytes, a hallmark of acute allergic inflammation, may also be a major source of NGF in the airways. On the other hand, in a murine model of asthma, our group has shown that NGF-overexpressing transgenic mice, which constitutively overexpress NGF in lung epithelial cells, recruited significantly more eosinophils after allergic sensitization compared with wild-type animals (Päth et al. 2002). Likewise, the p75NTR knockout mice exhibit a significant decrease in eosinophilic infiltration and similar effects were found after inactivation of NGF by intranasal application of an anti-NGF antibody during allergic sensitization (Kerzel et al. 2003) (Fig. 22.7). These data clearly show that allergic eosinophilia is associated with enhanced neurotrophin production in the inflamed lung. As has been shown previously, neurotrophins support survival of activated eosinophils and thus enhanced neurotrophin expression by either resident lung tissue cells or invading immune cells supports eosinophil homoeostasis in the airways (Nassenstein et al. 2003). However, whether neurotrophins in the lung besides supporting the survival of invading eosinophils also possesses chemoattractive activities, remains to be determined. Moreover, NGF amplifies the humoral allergic immune response such as stimulation of Th2 cytokine and IgE synthesis.

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Neurotrophins

Effects

Eos

Airway

T cell

Airway inflammation • Cells in the BALF

Fig. 22.7 Effects of NGF overexpression (NGF-tg mice) or depletion of p75NTR expression (p75NTR–/– mice) in a mouse model of allergic airway inflammation. Airway inflammation, characterized by eosinophil infiltration, and activity of sensory nerves, measured by response to capsaicin, are augmented in NGF-transgenic mice and diminished in p75NTR–/– mice. Bronchial smooth muscle activity, characterized by response to methacholine, is not affected by neurotrophin signaling. BALF, bronchoalveolar lavage fluid; Eos, eosinophil; wt, wild type. (See CD-ROM for color version.)

Tissue

Bronchial hyperreactivity • Capsaicin response (sensory nerves) • Methacholine response (smooth muscle)

Mouse:

wt

A Th2-biased immune response is critically important for the development of asthma-associated symptoms, and inhibiting the activity of NGF by anti-NGF treatment inhibits the local production of IL-4, the most important Th2-related cytokine. In addition to infiltrating eosinophils, macrophages, and T cells, tissue-resident mast cells are also involved in neurotrophin signaling. These cells secrete NGF after IgE-mediated stimulation and also depend on NGF as a maturation and survival factor. As mentioned above, NGF and BDNF levels in BALF of asthmatic patients are markedly increased 18 hours after segmental allergen provocation, whereas no change was observed directly (20 min) after the challenge (Virchow et al. 1998). These data may suggest that increased neurotrophin production is only associated with the allergic late phase reaction, which is characterized by a marked infiltration of immune cells. However, abrogation of NGF signaling by intranasal application of neutralizing antibodies inhibits allergen-induced early phase reactions in animal studies (de Vries et al. 2002). Vice versa, in transgenic mice that constitutively overexpress NGF in the lung, the early phase reaction, characterized by allergen-induced bronchoconstriction and serotonin release, was examined compared with wild-type animals. Similarly, other studies suggest a local upregulation of NGF expression in the bronchi as an early event in asthma, as increased transcripts of NGF were found in airway tissue from mild asthmatics following challenges of subclinical doses of allergen without inducing asthma symptoms (Kassel

NGF-tg

p75NTR–/–

et al. 2001). An increase in mast cells in the tissue parallels the upregulation of NGF expression in the airways and these cells were intensively immunostained for NGF. Hence, many immunocompetent cells involved in the allergic reaction, either tissue-resident and acutely reactive or infiltrating during the ongoing inflammation, may contribute to enhanced local neurotrophin expression (Fig. 22.8).

Airway hyperreactivity A key feature of chronic allergic asthma is the development of AHR. Target and effector cells responsible for AHR and airway obstruction include sensory and motor neurons as well as smooth muscle cells. Airway hyperreactivity is characterized by an increased susceptibility to a broad range of physiologic and nonphysiologic stimuli, including methacholine, histamine, hypertonic saline, cold air, and cigarette smoke. One possible mechanism for this is an inflammation-induced hypersensensitivity of sensory neurons that augments neuronal reflex circuits controlling lung function (Braun et al. 2002). During allergic asthma, qualitative and quantitative changes in the functional activity of lung sensory neurons were observed, such as an increase in mechanosensitivity resulting in an exaggerated neuronal excitability. There is growing evidence that changes in sensory airway innervation in the lung are under the control of inflammatory mediators released during allergic inflammation. The detailed mechanisms linking airway inflammation and AHR are not

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Airway Epithelium

Mast cell T cell Macrophage

Neurotrophins Eosinophil

Tissue

Vessel

completely defined, but neurotrophins represent candidate molecules regulating and controlling the cross-talk between the immune and peripheral nervous system (Nockher & Renz 2006a). NGF amplifies inflammatory AHR by upregulation of substance P production in lung sensory neurons and AHR has shown to be blocked by a substance P receptor antagonist (de Vries et al. 1999). In NGF-transgenic mice, airway inflammation is at least partly mediated by neuropeptides, as treatment of allergen-sensitized animals with a neurokinin receptor antagonist prevents eosinophil influx and IL-5 synthesis (Quarcoo et al. 2004). In a human study, the inflammatory cytokine IL-1β induced the release of NGF from isolated bronchi as well as the airway response to a tachykinin NK-1 receptor agonist, demonstrating that NGF mediates AHR induced by inflammation (Frossard et al. 2005). Allergen-challenged animals display an increase in substance P-immunoreactive nodose neurons, and a similar effect was shown following tracheal NGF application. Tracheal injection of NGF also changed substance P expression in mechanically sensitive Aδ-fibers, which do not express this neuropeptide under normal conditions (Hunter et al. 2000). Thus, NGF induced a switch in the phenotype of nodose neurons to become a component of the tachykinergic lung innervation. Recently, it has been shown in allergen-sensitized guinea pigs that inhibition of the NGF-receptor TrkA prevents the development of AHR as well as the increase in lung concentrations of substance P and substance P-positive neurons in nodose ganglia (de Vries et al. 2006). However, NGF also increases the release of stored substance P from sensory nerve endings without the need for neuropeptide synthesis in the neuronal cell body. This is obvious by the observation that NGF can increase AHR in the absence of sensory nerve cell bodies such as in isolated tracheal rings. The contribution of NGF expression on the development of neuronal AHR

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Fig. 22.8 Neurotrophins in the allergic inflamed lung. Immune cells as well as structural lung cells, e.g., epithelia, fibroblasts, and smooth muscle cells, produce neurotrophins constitutively or upregulate neurotrophin synthesis during inflammatory stimulation. Increased production of neurotrophins by inflammatory and structural lung cells results in elevated neurotrophin levels in the blood as well as in the bronchoalveolar fluid. (See CD-ROM for color version.)

is further demonstrated by sensory hyperreactivity in a mouse model of allergic airway inflammation using the NGF overexpressing transgenic mice (Päth et al. 2002). Sensory airway reactivity is measured via reactivity against capsaicin, which acts specifically via vaniloid receptors expressed on sensory neurons. Hyperreactivity caused by capsaicin-induced irritation of lung sensory neurons was markedly increased in allergen-sensitized NGF-transgenic mice compared with allergen-sensitized control animals. Consequently, in transgenic mice depleted for the expression of the pan-neurotrophin receptor p75NTR, the capsaicin-induced sensory hyperreactivity was almost abolished in allergen-challenged animals (Kerzel et al. 2003) (Fig. 22.7). Neurotrophins act via direct mechanisms through neurotrophin receptor signaling such as increasing the capsaicin receptor expression (Zhang et al. 2005) but also via indirect effects such as enhanced neuropeptide synthesis. The latter mechanism is supported by enhanced substance P-positive sensory innervation in the lungs of NGF-transgenic mice and a reduced amount of substance P in the lungs of p75NTR−/− mice (Kerzel et al. 2003). Nonspecific AHR in allergic asthma results in airway narrowing, which depends on an increased reactivity of sensory nerves and on the release of bronchoconstrictor stimuli that modulate airway smooth muscle cells. While reactivity to capsaicin reflects local activity of sensory nerves, methacholine directly induces smooth muscle contraction via muscarinic acetylcholine type 3 (M3) receptor expressed on airway smooth muscle cells. Animal studies have demonstrated that increased NGF expression or altered NGF signaling modulates sensory hyperreactivity; however, no effect of NGF on modulation of methacholine airway reactivity was shown in the same study (Nockher & Renz 2006a). After allergen challenge, no significant differences were found in the induction of an enhanced methacholine response between NGF-transgenic

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and wild-type mice, as well as in p75NTR−/− and wild-type animals (Fig. 22.7). Recently, comparable observations were demonstrated by modulation of BDNF signaling in the allergic lung. While treatment with anti-BDNF antibodies significantly reduced capsaicin-mediated sensory hypersensitivity, the methacholine response was unaltered (Quarcoo et al. 2004). However, using the p75NTR–/– mice, the upregulation of cholinergic AHR by acetylcholine was significantly diminished in these animals compared with wild-type mice (Tokuoka et al. 2001). In contrast to methacholine, which is relatively specific to M3 receptors on smooth muscle cells, the response to acetylcholine also includes reactivity of cholinergic airway neurons via neuronal muscarinic M2 receptors. Thus, the altered acetylcholine response in p75NTR–/– mice indicates that neurotrophins are able to modulate the activity of local cholinergic nerves. This is consistent with the recent observation of an enhanced cholinergic innervation as well as increased cholinergic activity in explanted mouse tracheal segments after culture with NGF (Bachar et al. 2004). Therefore, it seems that neurotrophin synthesis and signaling in the lung modulates local neuronal hyperreactivity of sensory as well as cholinergic neurons, but may not directly affect smooth muscle reactivity (Fig. 22.7).

Airway remodeling Accumulating evidence suggests the potential role of NGF in bronchial remodeling. The final phenotype of AHR and increased subepithelial thickening is a conglomeration of many events and mediators, and NGF seems to be playing a prominent role in mediating these events. Airway remodeling mainly consists of deposition of collagen beneath the epithelial basal lamina, which is associated with an increased number of myofibroblasts, hypertrophy of bronchial wall smooth muscle, and hyperplasia of mucus glands and goblet cells. Edema, vascular dilatation, and increased numbers of blood vessels also contribute to the thickening of the airway wall. The NGF-transgenic mouse (NGF-tg) overexpressing NGF in the clara cells of the lung shows an increased subepithelial thickening in the absence of any ongoing inflammation (Hoyle et al. 1998). NGF stimulates the contraction and migration of pulmonary fibroblasts as well as their differentiation into myofibroblasts (Micera et al. 2001). Isolated lung fibroblasts constitutively express TrkA. Although NGF has the modulatory effects mentioned above on fibroblasts, it does not influence lung fibroblast proliferation, collagen production or metalloproteinase production or activation (Micera et al. 2001). These effects of NGF could be due to the induction and release of profibrotic factors like TFG-β and fibroblast growth factor (FGF)-2, and of cytokines involved in the remodeling mechanisms like IL-4 and IL13. Experiments in human conjunctival fibroblasts have shown an enhanced TGF-β1 production after long-term NGF exposure, and following specific TGF-β1 neutralization all the NGF-induced effects were ceased (Micera et al. 2005).

Neurotrophins

In NGF-treated PC12 cells, the activation of the smad signal transduction pathway, typical of TGF-β1, has also been reported (Lutz et al. 2004). Remodeling events in tissue repair, consequential of any inflammatory reaction or injury, are a complex process that can result in pathologic outcomes like fibrosis. Fibroblasts have been clearly shown to mediate most of the wound healing processes; one of the prime ways is by extracellular matrix (ECM) production. NGF stimulates fibroblast production of type I collagen (Nithya et al. 2003) and bronchial biopsy sections from patients with interstitial fibrosis show strong NGF staining (Micera et al. 2001). NGF also stimulates the proliferation of bronchial smooth muscle cells and is, therefore, perhaps a prime candidate involved in smooth muscle cell hypertrophy observed in asthma. Matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), also play an important role in the development of tissue fibrosis. In addition to ECM proteolysis, MMPs also process growth factors including the immature precursors pro-NGF and proBDNF (Lee et al. 2001) as well as the receptors, including TrkA, TrkC, and p75NTR. It has also been reported that NGF-induced activation of TrkA receptor induces MMP-9 expression in both primary cultured rat aortic smooth muscle cells and in smooth muscle cell lines genetically manipulated to express TrkA (Khan et al. 2002). This activation of MMP-9 by NGF was shown to occur by the Shc/mitogen-activated protein kinase pathway, indicating it to be a mediator in remodeling events occurring in the vascular wall in response to injury. After injury, MMP-9 mediates the migration of smooth muscle cells by releasing the attachment of the surrounding extracellular matrix. This allows the smooth muscle cells to migrate into the intimal space and is an essential step in formation of angiogenesis and increased vascularity. The microvasculature has been shown to contribute to airway wall remodeling in asthmatic patients and neurotrophin effects may support this process. TIMPs are secreted proteins that serve to inhibit MMPs and have relatively low selectivity. The relative proportion of the MMPs and their inhibitor TIMPs determines the proteolytic activity and is therefore critical for the turnover of extracellular matrix components within the tissues. Beside MMP expression, expression of TIMP-2 is also upregulated by NGF in various neuronal cell lines. However, so far the role of neurotrophins in regulation of extracellular matrix proteins, MMPs, and TIMPs is not well investigated in tissue remodeling in asthma. Further studies in this direction are required to better understand the airway remodeling observed in allergic asthma.

Allergic rhinitis Allergic rhinitis is a common chronic inflammatory disease of the upper airways with a pathophysiology similar to asthma, its counterpart of the lower airways. Asthma and allergic rhinitis are common comorbidities, suggesting the concept

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of “one airway, one disease.” Like asthma, allergic rhinitis is characterized by an inflammatory response including mucosal edema, increased vascular permeability, and leukocyte infiltration. The allergic responses are largely divided into two phases: systemic response in the induction phase and local allergic inflammation in the effector phase. The contribution of neurotrophins to the induction of the allergic response may be the same as in other allergic diseases, and the immunotrophic functions of neurotrophins for several immune cells involved in the initiation of the allergic Th2-response have been discussed above. The effector phase is characterized by sneezing, pruritus, rhinorrhea, and infiltration of the nasal mucosa with inflammatory cells, including eosinophils, neutrophils, lymphocytes, and mast cells. While direct studies of neurotrophins on these effector cells were not performed in the context of allergic rhinitis, the pathophysiologic events (e.g., epithelial–eosinophil or epithelial–mast cell interactions) are likely comparable to those in allergic asthma. Baseline levels of NGF protein are increased in the nasal fluid of rhinitis patients compared with control subjects (Sanico et al. 2000). Nasal provocation with allergen induces immediate symptoms of nasal irritation in allergic patients, together with a dose-dependent increase in NGF protein levels in nasal lavage fluid after allergen challenge. This increase in nasal fluid NGF concentrations is not attributable to plasma extravasation, as provocation with histamine increases concentrations of albumin but not NGF in nasal fluids. Therefore, enhanced NGF levels must originate from local sources of the allergic inflamed nose. Within the allergic inflamed nose tissue, NGF expression is found in infiltrated eosinophils but also in structural elements of the nasal mucosa such as the epithelium or the submucosal glands (Wu et al. 2006). The enhanced NGF expression in allergic rhinitis may provide a link between allergic inflammation and neural hyperresponsiveness to various inhalants. Allergic rhinitis is characterized by significantly increased reactivity against various inhalants, allergens, and environmental irritants such as tobacco smoke or cold dry air, resulting in sneezing and secretion of nasal fluid. Patients with allergic rhinitis show significantly greater nasal responses to stimulation with cold air compared with healthy volunteers (Sanico et al. 1999). In addition, rhinitis patients show a higher responsiveness of the neural apparatus of the nose as demonstrated by (i) significantly greater sneezing reflex after histamine provocation, (ii) significantly greater secretory responsiveness to capsaicin stimulation, and (iii) more intense plasma extravasation after capsaicin stimulation. These reflexes may be actively modulated by NT signaling, as has been shown in the lower respiratory tract. Stimulation of sensory neurons with capsaicin via activation of the vanilloid receptor-1 (TRPV1) results in an increased release of neuropeptides such as the tachykinins, which have multiple effects including the induction of vascular permeability, manifested by plasma protein extravasation in nasal fluids.

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Nasal epithelium Mucous glands RHINORRHEA

Blood vessels VASODILATATION PLASMA EXTRAVASTION

NGF Mast cell Histamine C5 Spinal cord

SNEEZING REFLEX Vidian nerve

Sensory nerve

CNS Fig. 22.9 In allergic rhinitis NGF modulates disease activity. Mast cells release histamine, which activates sensory nerves to produce pruritus and sneezing, as well as a nasal central reflex. This leads to activation of submucosal glands and rhinorrhea as well as vessel dilatation and plasma extravasation. Enhanced NGF expression by the nasal mucosa triggers mast cell and neuronal activity. (See CD-ROM for color version.)

Neurotrophins, in turn, increase membrane expression of TRPV1 nociceptors as well as modulating their signal transduction, resulting in an enhanced response to capsaicin (sensitization, hyperalgesia). Moreover, neurotrophins are target-derived growth factors and overexpression of NGF in the bronchial epithelium is associated with enhanced innervation (Hoyle et al. 1998). Therefore, increased NGF expression by the nasal mucosa may also alter the magnitude of innervation alongside elevated functional activity. However, enhanced sensory nerve activity results in increased pruritus and sneezing reflex as well as activation of submucosal glands leading to rhinorrhea (Fig. 22.9). Immunosuppressive therapy with local application of corticosteroids is a common treatment in allergic rhinitis. This is effective in inhibition of the allergic inflammation and also in the treatment of the clinical symptoms such as sneezing, pruritus, and rhinorrhea. The immunosuppressive action of corticosteroids results in a downmodulation of various biological functions of resident (e.g., mast cells) or infiltrating (e.g., eosinophils, immune cells). However, corticosteroids are also able to suppress neurotrophin synthesis in the airway epithelium (Hahn et al. 2006), and, thus, may block the described neuronal dysfunction triggered by enhanced local neurotrophin concentrations.

Atopic dermatitis Atopic dermatitis (AD) is a chronic multifactorial skin disease, which is the result of a complex network of genetic immunologic, and also environmental and psychologic factors. In atopic dermatitis various interactions between structural skin

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cells and the local nervous system as well as immune cells contribute to the pathology of the disease. The interplay between the local nervous system and infiltrating immune cells is particularly becoming a focus of clinical and basic research in AD. Elevated levels of NGF (Toyoda et al. 2002) and BDNF (Raap et al. 2005) were found in the plasma of AD patients and plasma NGF levels correlated positively with the disease activity. This finding indicates that neurotrophins are involved during the ongoing allergic inflammatory response in AD.

Neurotrophins

Mast cell Histamine

NGF Keratinocytes Substance P

Allergic skin inflammation Similar to other allergic diseases, e.g., those of the airways, neurotrophins are also produced in allergic skin disease by immune cells during the local inflammatory process. In AD many local as well as infiltrating immune cells (e.g., dendritic cells, Langerhans cells, mast cells, macrophages, lymphocytes, and eosinophils) are involved in the initiation as well as progression of the disease. Within the inflamed skin, a huge number of infiltrating cells show strong NGF expression while the normal skin areas of AD patients show weak NGF immunoreactivity (Dou et al. 2006). Moreover, the NGF content inside the granules of eosinophils obtained from AD patients was significantly elevated compared with cells from normal donors (Toyoda et al. 2003). Keratinocytes comprise an important source of neurotrophins in the diseased skin. Keratinocytes are recognized as a primary source of cutaneous neurotrophin production and keratinocyte neurotrophin release is enhanced during inflammatory conditions. Psoriatic keratinocytes release higher amounts of NGF than normal cells, and in AD skin an intense expression of NT-4 is found in pruritic lesions. IFN-γ is a strong inducer of keratinocyte NT4 as has been shown in culture as well as in INF-γ-injected skin. Recent findings indicate that NGF may also function as a signaling molecule between keratinocytes and mast cells. In the early phase of AD, mast cells are increased in the dermis and it is well known that NGF supports chemotaxis, maturation, and survival of mast cells in the body tissues. Thus, it is clear that enhanced NGF synthesis by keratinocytes may also promote dermal infiltration of mast cells as well as their activation, within the inflamed skin. Vice versa, mast cellderived histamine induces keratinocytes to upregulate NGF production (Kanda & Watanabe 2003), as do various other inflammatory cytokines. While NGF has been shown to degranulate mast cells in the skin, it stimulates its own synthesis via release of histamine (Fig. 22.10). Subsequently, plasma levels of histamine, as well as NGF, were significantly higher in patients with AD but also in serum and affected skin regions of DS-Nh mice, an animal model of AD. In vitro, the augmentation of keratinocyte NGF synthesis is inhibited by addition of loratadine, which is a strong histamine H1receptor antagonist (Yoshioka et al. 2006). The histamineinduced production of NGF, but also other cytokines such as GM-CSF, is mediated via H1 receptors on the keratinocyte

Sensory nerve Fig. 22.10 Nerve growth factor modulates interaction of keratinocytes with sensory nerves as well as mast cells in a vicious cycle within the allergic inflamed skin. (See CD-ROM for color version.)

cell surface and subsequent activation of intracellular protein kinase C. Thus, the NGF release after H1 histamine receptor activation contributes to a common inflammatory response of keratinocytes and is yet another example that neurotrophins play a role in the vicious cycle that amplifies allergic skin inflammation.

Pruritus In the epidermis of lesional skin of AD patients, cutaneous nerve fibers are present at higher densities. This increase in epidermal nerve fibers is at least partly responsible for the intense itch, one of the major clinical symptoms in AD. It is now accepted that NGF is one of the major mediators that determine skin innervation, corroborated in transgenic mice overexpressing NGF where hypertrophic sensory ganglia along with increased skin innervation is found. In a murine model of AD, treatment with a neutralizing anti-NGF antibody inhibited the proliferation of skin lesions and epidermal innervation, as well as intensity of itch–scratch cycles (Takano et al. 2005). However, the nature of relationship between NGF and pruritic conditions in AD is complex and involves multiple signaling between neurons, and structural and immune cells. Neuroimmune interactions are thought to be the most important trigger during the progression of this disease. In inflammatory skin disease, bidirectional pathways between the peripheral nervous system and immune system have been described. The interaction between cutaneous nerves and the immune cells is modulated by neuromediators such as neuropeptides and neurotrophins, which activate neurons and immune cells and also structural skin cells, such as keratinocytes, through specific receptors. Recent research indicates that the peripheral nervous system alters the course of AD through changes in cutaneous innervation and upregulated neuropeptide expression in lesional skin (Tobin et al.

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1995). AD lesions are characterized by high numbers of nerve fibers containing substance P, the most prominent member of the tachykinin neuropeptide family, while somatostatinimmunoreactive nerves were not found in the skin of AD patients in contrast with healthy subjects. Neuropeptides play an important role in immediate and delayed-type hypersensitivities and also mediate neurogenic inflammation in the skin. In particular, the release of substance P induces vascular responses, such as expression of adhesion molecules on endothelial cells, and also pruritus as a result of mast cell degranulation. Neuronal expression and release of tachykinins is mainly regulated by neurotrophins and the increased substance P may result from enhanced neurotrophin synthesis in AD skin. An increased epidermal expression of NGF and NT-4 in prurigo lesions of AD skin clearly supports this hypothesis (Grewe et al. 2000). Thus, neurotrophin expression in keratinocytes is upregulated in atopic dermatitis and may contribute to pruritus, which involves a complex signaling network between cutaneous nerves, mast cells, and also cells of the skin surface. While the primary function of keratinocytes is to provide the structural integrity of the skin and to protect against external injury, they are actively engaged in inflammatory skin responses by attracting immune cells and modulating their functions. However, these activities are also extended in chronic inflammatory skin disorders such as psoriasis or atopic dermatitis. Thereby, the expression of neurotrophins by activated keratinocytes is thought to regulate keratinocyte– nerve interactions during skin inflammation. Peptidergic sensory nerve fibers extending into the epidermis are in direct contact with keratinocytes. Enhanced synthesis of neurotrophins in the inflamed skin induces neuropeptide production and release in sensory skin innervation. Vice versa, substance P and neurokinin A are able to induce keratinocyte NGF (Burbach et al. 2001). While the direct effect of the neurosensory system on keratinocyte NGF production may be physiologically important for the maintenance and regeneration of cutaneous nerves in the normal skin, enhanced keratinocyte NGF synthesis may stimulate sensory innervation in a vicious cycle during allergic inflammation (Fig. 22.10).

Conclusions Studies of blood levels of neurotrophins revealed increased systemic concentrations of these factors during allergic diseases such as asthma or atopic dermatitis. While NGF is also elevated in autoimmune diseases such as systemic lupus erythematosus or systemic sclerosis, we might speculate whether upregulated neurotrophin expression is specifically linked to Th2-type allergic inflammation or more generally associated with an ongoing inflammatory response. There is growing evidence that NGF and also, potentially, the other members of the neurotrophin family are part of an integrated

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adaptive response to several offending stimuli, which connects cells of the immune and nervous system as well as structural cells. Initially discovered as growth factors supporting neuronal maintenance, it is now clear that their target cells are not limited to the nervous system. Cells of the immune system produce and respond to neurotrophins and studies of various allergic diseases reviewed over the last two decades indicate that neurotrophins behave as inflammatory cytokines delivering activating and survival signals to effector cells of the allergic response. In particular, the biology of mast cells and eosinophils is mainly affected by neurotrophins. However, a discussion on the potential mechanisms by which neurotrophins may participate in allergic inflammation will stray beyond what can be actually supported by experimental data. This also includes the functions of neurotrophins within structural tissue cells such as keratinocytes or epithelia which themselves contribute to the pathogenesis of allergic disease. On the other hand, it is indisputable from the results of many studies that enhanced local neurotrophin expression modulates activity of the peripheral nervous system and alterations in sensory innervation are frequently observed in allergic diseases. Moreover, they are responsible for typical clinical symptoms such as AHR in asthma or pruritus in atopic dermatitis. Based on these observations, a more intense study of the complex biological function of neurotrophins will become a potential tool for therapeutic intervention in the future.

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Neuropeptides David A. Groneberg and Axel Fischer

Central reflex

Summary Inflammatory events in allergic diseases such as allergic asthma or atopic dermatitis encompass the release of various neuropeptides from nerve fibers. This neurogenic response can be initiated not only by allergens but also by exogenous irritants, such as cigarette smoke or gases, and is characterized by a bidirectional linkage between neurons and inflammatory cells. The molecular mechanisms underlying neurogenic inflammation are orchestrated by a large number of neuropeptides including proinflammatory peptides such as tachykinins, neuropeptide tyrosine, or calcitonin gene-related peptide. Also, other biologically active molecules such as vasoactive intestinal polypeptide or pituitary adenylate cyclaseactivating peptide play a role in the modulation of inflammatory responses. Whereas numerous aspects of neuropeptide effects have been studied in laboratory animal models, little is known about precise neuroimmune events in human allergic diseases. However, different neuropeptide-modulating compounds may be used as targets for future therapeutic strategies.

Nodose ganglion

Vagus nerve

“Peripheral reflex“ Respiratory tract

Antidromic neuropeptide release Neurogenic inflammation

Allergen

Introduction In the event of allergic inflammation, neuropeptides such as the tachykinin substance P or calcitonin gene-related peptide (CGRP) are released into the periphery and can lead to inflammatory effects. The concepts of neurogenic inflammatory events in the airways and their potential importance for bronchial asthma have been established for more than a decade (Fig. 23.1). The initial activation of airway nerve fibers is based on the stimulation of receptors expressed on the terminal varicosities of airway nerves with exogenous irritants such as allergens, tobacco smoke or other air pollutants, or endogenous inflammatory mediators. This activation may


Central nervous system

Airway lumen

Fig. 23.1 Concept of neurogenic inflammation. After peripheral noxious stimulation by airborne substances such as allergens or tobacco smoke, orthodromic activation of sensory nerve fiber endings takes place. The classical pathway then leads via orthodromic transmission into the brainstem. Here, the signal is modulated via interneurons and then transmitted via efferent parasympathetic nerve fibers to induce parasympathetic effects after synaptic transmission in local intrinsic airway ganglia. The neural events orchestrating neurogenic inflammation start in the sensory ganglia perikarya with the induction of proinflammatory neuropeptide gene expression. Then the neuropeptides are transported antidromically via the sensory nerve fibers back to the peripheral endings in the airways. Here, they are locally released and propagate the events of neurogenic inflammation. (From Groneberg et al. 2004e, with permission.)

then lead via orthodromic central and antidromic local reflex pathways to symptoms of allergy including bronchoconstriction or mucus secretion (Vignola et al. 2000). Numerous effects are exerted after nerve stimulation and it is accepted that there is broad communication between

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neurons and inflammatory cells. These interactions are orchestrated by a large number of neuromediators such as tachykinins (Harrison & Geppetti 2001), CGRP, vasoactive intestinal polypeptide (VIP), gaseous molecules such as nitric oxide or carbon monoxide (Eynott et al. 2002, 2003), or endogenous opioids (Groneberg & Fischer 2001). The role of the innervation for inflammatory events is well established for experimental models of airway inflammatory and obstructive diseases such as bronchial asthma. In this respect, numerous mediators of inflammation are known to influence sensory and cholinergic nerve activity under conditions of allergic inflammation. It is also accepted that the airway innervation, and especially sensory nerve fibers, regulates all major features of human respiratory function. However, findings from animal experiments on the significance of neurogenic inflammation have not yet been fully extrapolated to the human situation. Since the different pharmacologic properties of airway nerve mediators orchestrate many aspects of airway inflammation and may be useful as targets for future therapeutic strategies, neurogenic inflammation is an important area of current research. The present chapter aims to give a comprehensive summary of neuropeptide actions in allergic inflammation.

neurons can be variable in their myelination, caliber, and central nervous input (Solway & Leff 1991). The largest portion of mammalian airway-innervating sensory nerve fibers originates from the vagal ganglia (Kummer et al. 1992; Fischer et al. 1996). A smaller number of airway sensory nerves originate from the dorsal root ganglia (Dalsgaard et al. 1984; Saria et al. 1985; Springall et al. 1987) (Fig. 23.2).

Brainstem

Vagus nerve Parasympathetic

Nodose ganglion

Sensory

Sympathetic ganglia

Local intrinsic ganglia

Symp.

Airway innervation Sensory

Alongside the two classical components of the autonomic nervous system, the sympathetic and parasympathetic innervation (Table 23.1), a third system termed “sensory innervation” projects to the airways and innervates all major respiratory effector cells. The sensory innervation of the mammalian airways, which has also been called the noncholinergic nonadrenergic innervation, constitutes a heterogeneous population of neurons. In this respect, sensory

DRG Fig. 23.2 Anatomy of airway innervation: schematic illustration of human airway innervation. The vagus nerve supplies all parasympathetic preganglionic neurons and the majority of sensory nerve fibers. Sympathetic nerve fibers originate from sympathetic cervical and thoracic ganglia. A minority of sensory nerve fibers originate from dorsal root ganglia (DRG). (From Groneberg et al. 2004e, with permission.)

Table 23.1 Neuromediators. Mediator

Receptor

Major origin

Major effects

Acetylcholine

Nicotinergic and cholinergic receptors

Parasympathetic fibers

Bronchoconstriction

Catecholamines

Adrenergic receptors

Sympathetic fibers

Bronchodilation

CGRP

CGRP receptors

Sensory fibers

Vasodilation, bronchial tone

NPY

NPY receptors

Sympathetic fibers

Inflammation

PACAP

VPAC1, VPAC2, PAC1

Parasympathetic, sympathetic, and sensory fibers

Bronchodilation, vasodilation, immunomodulation

Tachykinins

Tachykinin receptors (NK1, NK2)

Sensory fibers

Inflammation, bronchoconstriction, mucus secretion, plasma exudation

VIP

VPAC1, VPAC2

Parasympathetic, sympathetic, and sensory fibers

Bronchodilation, vasodilation, immunomodulation

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The majority of neurons that innervate the airways end in the commissural, ventrolateral, and medial areas of the nucleus tractus solitarius in the brainstem (Haxhiu et al. 1993; Perez Fontan et al. 2000a,b). In these areas, they form synapses with interneurons communicating with medullary networks (Haxhiu & Loewy 1996; Hadziefendic & Haxhiu 1999; Perez Fontan 2002). Even now, little is known about the resulting multineuronal circles. It is generally assumed that the circles represent reflex loops that are activated by the peripheral stimulation of sensory airway neurons leading to the enhancement or inhibition of cholinergic nerve fibers, which project to numerous target cells in the respiratory tract (Fig. 23.1). Apart from central multineuronal reflex circuits there is also a second mode of operation present in sensory neurons: peripheral stimulation in the respiratory tract can also lead to a local neuronal mechanism that is orchestrated by sensory neurons (Coleridge & Coleridge 1984; Coleridge et al. 1989) expressing proinflammatory neuropeptides, such as CGRP or tachykinins. As nerve fiber terminals, and also the receptors for these neuropeptides, are localized in the epithelium, vessel walls, bronchial smooth muscles, and around mucus glands (Lundberg et al. 1984a), the local stimulation of sensory nerve fibers projecting to these target cells and the subsequent neuropeptide release can lead to features of allergic inflammation such as hyperemia (Salonen et al. 1988), edema (Lundberg et al. 1984b), mucus hypersecretion (Coles et al. 1984), and contraction of the bronchial smooth muscle. It is also known that sensory nerve fibers project to local intrinsic airway ganglia (Lundberg et al. 1984a; Myers et al. 1996), which may depolarize in response to the presence of tachykinins (Myers & Undem 1993; Belvisi et al. 1994). Therefore, peripheral activation of sensory nerve fibers with consecutive neuropeptide release may also lead to modulation of centrally mediated medullary reflexes (Tanaka & Grunstein 1990).

Human respiratory sensory innervation There are major species-specific differences between human and animal respiratory tract innervation, as many of the findings on airway innervation or promising effects of compounds targeting sensory nerves were not replicated with human tissues. While guinea-pig innervation is the closest approach to the human situation among all common laboratory species, there are still differences present: in general, sensory nerve fibers containing substance P and CGRP are of lower number in the human respiratory tract as compared with guinea-pig airways. Whereas these fibers constitute a large portion of total epithelial nerve fibers in the guinea-pig airways, they have been estimated in preliminary studies at only about 1% in humans (Bowden & Gibbins 1992). However, these preliminary data need further morphologic

Neuropeptides

studies to clarify this issue. In contrast to the potential anatomic and functional discrepancies, airway neuronal plasticity is generally accepted to occur in human pulmonary diseases. To assess if alterations in neuronal subpopulations are disease-specific or an epiphenomenon of the inflammation, different subtypes of human chronic upper airway diseases including hyperreflectoric rhinitis (Heppt et al. 2002), aspirin-sensitive rhinitis (Groneberg et al. 2003a) and toxic rhinitis (Groneberg et al. 2003b) were examined. Hyperreflectoric rhinitis is a chronic upper airway inflammatory disease related to nonspecific hyperreactivity. Expression profiling for substance P, CGRP, VIP, and neuropeptide tyrosine (NPY) revealed an abundant presence of nerve fibers expressing these peptides in the airways. Neuropeptide expression in mucosal nerves was also quantitatively assessed and significant increases were found for substance P (3.00 ± 0.37 vs. 1.64 ± 0.34 staining intensity in the control group) and VIP (2.33 ± 0.42 vs. 0.82 ± 0.33) (Heppt et al. 2002). These results demonstrate differences in levels of neuropeptides in the innervation of human nasal mucosa between nonrhinitic and hyperreflectoric rhinitic subjects and point to a modulatory role of neuropeptide-specific subpopulations of nerve fibers in hyperreflectoric rhinitis. Irritative toxic rhinitis is induced by chemical compounds such as tobacco smoke, ozone, solvents, formaldehyde, nickel, or chrome, which are also known to be associated with the development and progression of chronic obstructive pulmonary disease (COPD) and asthma. Semiquantitative immunohistochemistry for substance P, CGRP, VIP and NPY demonstrated significant differences between rhinitis patients and control subjects: toxic rhinitis patients had significantly increased expression scores for VIP (2.83 ± 0.31 vs. 1.27 ± 0.47 control group) and NPY (3.17 ± 0.31 vs. 0.91 ± 0.37 control group) (Groneberg et al. 2003b). These results indicated a differential participation of subclasses of mucosal nerves in the pathophysiology of toxic rhinitis and suggested that the changes in nerve profiles found in toxic rhinitis and hyperreflectoric rhinitis are disease-specific and not an epiphenomenon of inflammation. Next to toxic rhinitis and hyperreflectoric rhinitis, aspirinsensitive rhinitis was investigated which represents the manifestation of aspirin intolerance in the upper airways (Groneberg et al. 2003a). The disease is a pseudoallergy against aspirin and related nonsteroidal antiinflammatory drugs. Immunohistochemical analysis demonstrated that aspirinsensitive rhinitis patients also had a significant increase in VIP-immunoreactive nerve fibers (Groneberg et al. 2003a). These changes observed for the different forms of chronic upper airway inflammation may be partly regulated via neurotrophins. Nerve growth factor (NGF) significantly increases the transcription of the preprotachykinin-A gene, which encodes for substance P and neurokinin A in vitro (Lindsay & Harmar 1989). NGF expression is increased in asthma (Olgart Hoglund & Frossard 2002), and might therefore account for the induction of substance P in airway

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nerves under inflammatory conditions. In this respect, earlier studies reported an increase in the number of substance P-immunoreactive nerve fibers in the airways of patients with fatal asthma (Ollerenshaw et al. 1991). Also, increased concentrations of substance P were documented in the bronchoalveolar lavage fluids of patients with asthma, with a further rise following allergen challenges (Nieber et al. 1992). Substance P has also been found in the sputum of asthmatic patients after inhalation of hypertonic saline (Tomaki et al. 1995). However, it was difficult to replicate these findings in other populations and other studies did not reveal any increases (Howarth et al. 1991; Lilly et al. 1995). At the receptor level, it was reported that NK1 receptor gene expression is increased in the airways of asthmatic patients (Adcock et al. 1993). Also, abnormal expression of NK2 receptors was documented in asthma (Bai & Bramley 1993).

Neuropeptides involved in neurogenic inflammation Among the different neuropeptides stored in and secreted from airway nerves, predominantly those expressed in sensory nerves have been shown to contribute to the events of neurogenic inflammation. They are secreted during airway inflammation in reaction to a multitude of inflammatory mediators. More than 100 different nonneuronal mediators of airway inflammation have been described so far (Barnes et al. 1998), which may all propagate directly or indirectly neuropeptide release from nerve fibers in diseases such as bronchial asthma or COPD (Chung 2001). These mediators may not only induce neuropeptide expression and secretion but also increase the transcriptional and translational expression of neuropeptide receptors on either neuronal, inflammatory, or respiratory target cells. They may also influence the degradation of neuropeptides in the periphery. Although neuropeptides usually originate from airway nerves, recent studies indicate nonneuronal cellular sources such as inflammatory or epithelial cells, especially in states of airway inflammation as found in bronchial asthma (Germonpre et al. 1999).

Tachykinins Tachykinins such as substance P or neurokinin A encompass a family of peptides released from airway nerves after noxious stimulation (Fig. 23.3). They influence numerous respiratory functions under both normal and pathologic conditions, including the regulation of airway smooth muscle tone, vascular tone, mucus secretion, and immune functions (Maggi et al. 1995). Apart from neuronal cells as the source of tachykinins, inflammatory and immune cells may also synthesize and release tachykinins under certain conditions. This second cellular source of tachykinins may play a role in inflammatory airway diseases. Dual tachykinin NK1 and

514

Fig. 23.3 Tachykinin ligand and receptor expression in airways. Doublefluorescence immunohistochemistry illustrating the abundant presence of tachykinergic nerve fibers and NK2 receptors in the airways. Tachykinergic fibers are identified with an anti-substance P antibody in the smooth muscle layer and in the subepithelial layer. The presence of the NK2 receptors in smooth muscle cells is visualized with an NK2 antibody in guinea-pig airways. Original magnification ×200. (See CD-ROM for color version.)

NK2 receptor antagonism has been shown to exert major bronchoprotective effects and may be of special interest for the development of novel therapeutic approaches. Also, NK3 receptors may participate in the mediation of effects but their precise role in the human respiratory tract still needs to be investigated. The family of tachykinins has recently been extended by the discovery of a third tachykinin gene that encodes previously unknown NK1 receptor-selective tachykinins such as hemokinin 1 or endokinin A and B. Together with other novel tachykinin peptides, such as C14TKL-1 and virokinin, they may also play a role in the respiratory tract and further research is required to define their respiratory biological role in health and disease. The release of the major tachykinins from sensory nerves is affected by numerous other mediators including opioids, dopamine, or histamine (Fig. 23.4). The neuronal induction of tachykinin expression has been demonstrated in animal models of allergic inflammation, where tachykinins act as potent regulators of neurogenic inflammation due to their proinflammatory effects on many airway effector cells (Fischer et al. 1996). Both substance P and neurokinin A derive from the preprotachykinin A gene. Both peptides share a common carboxy-terminus amino acid sequence containing the biologically active domain (Escher et al. 1982). In the upper and lower respiratory tract, tachykinin immunoreactivity has been shown to be present in nerve fibers localized to submucosal glands, airway smooth muscle, and vasculature (Lundberg et al. 1984a; Heppt et al. 2002). Retrograde neuronal tracing studies in rats and guinea pigs have demonstrated that sensory nerve fibers, which innervate the trachea, mainly originate from the jugular and nodose vagal sensory ganglia (Springall et al. 1987; Kummer et al. 1992). To the same

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Inhibition

Activation Sensory nerve +

Activation

CB2 −

B2 GABA-B H1 Capsaicin LTB-4 12-HPETE Proton Heat

H3

Inhibition TRPV1 −

a2

+

OP3(m) D2

Tachykinins, CGRP

NK1

NK2

CGRP-R

Respiratory target cells

Fig. 23.4 Modulation of sensory nerve activity. Sensory nerve-mediated airway effects are induced via antidromic release of proinflammatory neuropeptides such as tachykinins or CGRP and their receptors NK1, NK2, and CGRP-R. Sensory nerve activity is regulated via stimulation of the vanilloid receptor TRPV1, bradykinin (B2), or histamine (H1) receptor, and inhibited via activation of numerous receptors including adrenergic (a2), opioid (OP3), cannabinoid (CB2), dopaminergic (D2), or histamine (H3) receptors. (From Groneberg et al. 2004e, with permission.)

extent, tachykinin-expressing nerve fibers mainly originate from neurons localized in the jugular and dorsal root ganglia (Saria et al. 1985; Kummer et al. 1992). It was recently shown that a furin-mediated cleavage of the bovine respiratory syncytial virus fusion protein leads to the release of a peptide converted into a biologically active tachykinin termed “virokinin” (Zimmer et al. 2003). The cellular enzymes involved in the C-terminus maturation of virokinin are present in many established cell lines and, indeed, virokinin is secreted by virus-infected cells. In vitro experiments revealed that virokinin by itself is capable of inducing smooth muscle contraction (Zimmer et al. 2003). Therefore, the viral tachykinin-like peptide virokinin may be considered a novel form of molecular mimicry, by which a virus may benefit by affecting host immune responses (Zimmer et al. 2003; Patacchini et al. 2004). Tachykinins bind to three different G-protein-coupled receptors. These receptors can be distinguished by their molecular properties and different pharmacologic affinities (Nakanishi

Neuropeptides

et al. 1993). While substance P principally activates NK1 receptors, neurokinin A mainly acts via NK2, and neurokinin B via NK3 receptors (Frossard & Advenier 1991; Devillier et al. 1992; Joos et al. 2000a). A differential pattern of tachykinin receptor distribution is known to be present in the respiratory tract: while NK1 receptors are predominantly localized in the airway epithelium and around submucosal glands and vessels, NK2 receptors are mainly expressed in the airway smooth muscle layer (Martling et al. 1987; Komatsu et al. 1991; Laitinen et al. 1992). The majority of human data available is on the regulation and function of tachykinin ligands and their receptors in allergic inflammatory diseases (Brunelleschi 1999; Honore et al. 2000; Joos et al. 2000b; Collins 2001). The interactions of tachykinins and their receptors in diseases such as COPD are largely unknown. However, it has been reported that tachykinin gene expression is increased after chronic exposure to cigarette smoke in animals (Kwong et al. 2001) and this mechanism may also exist in humans.

Pulmonary effects of tachykinins Tachykinins such as substance P and neurokinin A exert a multitude of effects on respiratory target cells. Tachykinins, particularly NKA, potently constrict human airway smooth muscles in vitro via NK2 receptors, with significantly greater potency in smaller airways (Frossard & Barnes 1991). This suggests that tachykinins can play a role in regulating constrictory effects in more peripheral airways, whereas cholinergic fiber effects may dominate in larger airways. In asthma, neurokinin A leads to bronchoconstriction after both inhaled and intravenous administration (Crimi et al. 1993). In vitro, the removal of the epithelial layer significantly increases bronchoconstrictor responses to tachykinins (Frossard et al. 1989). This effect points to an increased importance of tachykinins in diseases with a distorted airway epithelium such as bronchial asthma or COPD. Tachykinins are also known to stimulate secretion from human submucosal glands in vitro. This effect is mediated via NK1 receptors. The exact molecular pathways leading to the induction of mucin genes such as the primary gel-forming mucins expressed in the upper and lower respiratory tract, MUC5AC or MUC5B (Groneberg et al. 2002a,b, 2003c, 2004a), have not been elucidated so far. Further effects mediated via NK1 receptor signaling are plasma vasodilatation and exudation, as well as acetylcholine release facilitation at cholinergic nerve terminals, thereby enhancing cholinergic neurotransmission (Watson et al. 1993).

Metabolism of tachykinins The predominant mode of airway enzymatic tachykinin cleavage is via two enzymes, neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) (Di Maria et al. 1998). As NEP is expressed in the airway mucosa and submucosa (Fig. 23.5), it plays a major role in the degradation of

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Fig. 23.5 Neuropeptide hydrolysis. Neutral endopeptidase (NEP) activity is abundantly present in the airways as demonstrated using an NEP activity assay in guinea-pig airways. (From Groneberg et al. 2006, with permission.) (See CD-ROM for color version.)

tachykinins in the airways. In contrast, ACE is predominantly expressed in vascular cells and therefore predominantly regulates the cleavage of intravascular peptides. The activity of NEP can affect the responsiveness to tachykinins in the airways (Fig. 23.5). NEP inhibition has been demonstrated to significantly increase tachykinin-induced bronchoconstriction. Removing the epithelium also decreases the activity of NEP and it is known that the removal of the epithelial layer significantly increases bronchoconstrictory responses to tachykinins. Also, tobacco smoke or exposure to viral infections can decrease NEP activity and thereby may diminish the cleavage and degradation of proinflammatory tachykinins (Di Maria et al. 1998).

Tachykinin antagonists: clinical applications Antagonism of the selective neuropeptide receptors represents a useful tool for discriminating the precise role of neuropeptides. This approach was used to identify the effect of tachykinins: the NK1 receptor antagonist CP96345 significantly blocked plasma exudation in response to vagal stimulation and to cigarette smoke (Lei et al. 1992). This receptor-specific antagonism did not have any impact on bronchoconstrictor responses, but was blocked by selective NK2 antagonists (Advenier et al. 1992). Antagonism of NK1 receptors also led to a decrease in bradykinin- and hyperpnea-induced plasma exudation, but did not alter acute allergen-induced plasma exudation in sensitized guinea pigs (Sakamoto et al. 1993). Although the precise role and extent of tachykinin-related neurogenic events is not completely understood for bronchial asthma, tachykinin receptor antagonism represents a potential target for novel therapeutic approaches. Initial clinical studies with asthmatic patients have not led to very promising results. The nonselective tachykinin antagonist FK-224 was found to have inhibitory effects on bradykinin-induced bronchoconstriction in asthmatic patients (Ichinose et al.

516

1992). A more potent NK1 receptor antagonist also revealed a reduction in the duration of exercise-induced bronchial asthma. However, there was no consistent effect on the maximal bronchoconstriction (Ichinose et al. 1996). The potent NK1 receptor antagonist CP99994 did not have effects on either hypertonic saline-induced bronchoconstriction or on cough (Fahy et al. 1995). In contrast to these early studies that were characterized by the use of single receptor antagonists, in a recently published study a dual tachykinin NK1/NK2 antagonist (DNK333) was shown to inhibit neurokinin A-induced bronchoconstriction in patients with bronchial asthma patients (Joos et al. 2004). A total of 19 male adults with mild asthma participated in the randomized, double-blind, placebo-controlled, crossover study protocol in which increasing concentrations of NKA (3.3 × 10–9 to 1.0 × 10–6 mol/mL) were inhaled at 1- and 10hour intervals. Either a single oral dose of DNK333 (100 mg) or a placebo was given. It was shown that DNK333 did not affect baseline lung functions but it protected against NKAinduced bronchoconstriction. The mean log10 provocative concentration leading to a 20% fall in forced expiratory volume in 1 s (FEV1) for NKA was –5.6 log10 mol/mL at 1 hour (DNK333) and –6.8 log10 mol/mL (placebo). The ratio is equivalent to a difference of 4.08 doubling doses. Ten hours after treatment, the ratio decreased to a difference of 0.9 doubling doses (Joos et al. 2004). The findings from this study demonstrate that the dual receptor antagonist DNK333 can be used to efficiently block neurokinin A-induced bronchoconstriction in bronchial asthma. It was also suggested that although NK2 receptors predominate in mediation of neurokinin A smooth muscle contraction, NK1 receptors might also be involved in these events. In view of the bronchoprotective effects of DNK333, further clinical studies need to be performed on the efficacy and safety of dual tachykinin receptor antagonists with regard to bronchial asthma.

Calcitonin gene-related peptide CGRP is a further major neuropeptide that belongs to the class of proinflammatory sensory neuropeptides in the airways (Springer et al. 2003). CGRP consists of 37 amino acids and is a product of calcitonin pre-mRNA alternative splicing. The neuropeptide is expressed and stored together with tachykinins in sensory nerve fibers (Palmer & Barnes 1987; Palmer et al. 1987; Groneberg et al. 2003b). In addition to its expression in airway sensory neurons, CGRP has also been detected in pulmonary neuroendocrine cells where it is expressed in both the solitary type and cluster type (neuroepithelial body) (Keith et al. 1991; Buvry et al. 1999). The molecular identity of CGRP receptors has not been identified in detail. Numerous receptors have been suggested to be associated with CGRP or related peptides but had to be reclassified as orphan receptors (Kapas & Clark 1995; Kapas et al. 1995; Hanze et al. 2002). However, it was recently shown that CGRP receptors belong to the family of G-protein-

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ADM receptor

CGRP-A receptor N

N N

N

N or C

C C

C

RAMP2 or 3

CL-R

C

CL-R C

RAMP1

CGRP-B receptor

C

N

C C N

N

CL-R

N

RAMP2/3

RAMP1

C

N

C RCP ER

??? Fig. 23.6 CGRP receptors. Illustration of the currently known and predicted receptors for CGRP and related peptides. The CL protein (formerly CRLR) in coexpression with RAMP1 leads to a functional CGRP-A receptor, whereas the coexpression of CL with RAMP2 or RAMP3 leads to a receptor with higher affinity for adrenomedullin (ADM). The CT protein functions alone as a calcitonin receptor (CT-R), but in coexpression with RAMP1 or RAMP3 it serves as an amylin receptor. A further receptor (CGRP-A receptor), which is related to a receptor component protein (RCP), has been suggested. ER, endoplasmic reticulum; RAMP, receptor affinity modifying protein. (From Springer et al. 2003, with permission.)

N N

N

N

CT-R

RCP C

C

Plasma membrane

C RAMP1 or 3

CT-R

CT-R C

C

C

C or

N

coupled seven transmembrane receptors, as do many other neuropeptide receptors (Fischer et al. 2001, 2002; Groneberg et al. 2001a). In contrast to other neuropeptide receptors, the activity of CGRP receptors is regulated by a family of receptor activity-modifying proteins leading to different type A or type B CGRP receptors (McLatchie et al. 1998) (Fig. 23.6). Binding sites for CGRP in human airways were identified first using autoradiography (Mak & Barnes 1988) with a wide distribution throughout the respiratory tract. Immunohistochemistry to precisely identify CGRP type A receptor expression in human bronchial blood vessels revealed receptor immunoreactivity in the endothelium of venules but not in the endothelium of arterioles (Springer et al. 2003).

Pulmonary effects of CGRP Numerous studies have provided evidence for a modulatory role of CGRP in multiple pulmonary functions. These include the regulation of airway smooth muscle tones and vessel tones (Springer et al. 2003). However, the exact mode of

N Amylin receptor

N

N CT-receptor

CGRP effects on the airway smooth muscle tone is still unclear as controversial bronchoconstrictor or bronchodilator effects have been reported in the past years in different species and preparations (Springer et al. 2003). Recently it was shown for human bronchi in vitro that CGRP causes a concentration-dependent contraction of epithelium-denuded human bronchi whereas no significant effects are found in epithelium-intact bronchi (Springer et al. 2004), indicating a potential altered effect of CGRP on airway tone in respiratory diseases with a damaged epithelial layer such as asthma or COPD. In human pulmonary arteries, CGRP causes a concentration-dependent relaxation of both endothelium-intact anddenuded arteries. Pretreatment with indomethacin prevents the CGRP-induced relaxation in pulmonary arteries suggesting that prostaglandins are involved in the intracellular signal transduction pathway. In contrast, nitric oxide (NO) is not involved in this mechanism as pretreatment with the NO synthase inhibitor L-NAME does not affect CGRP-induced

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vascular relaxation. CGRP-induced effects on both bronchi and vessels are prevented by application of the CGRPantagonist CGRP (8-37) (Springer et al. 2004). There are no published data about the effects of CGRP on human airway mucus secretion. However, although glandular areas only display a very low density of CGRP binding sites (Mak & Barnes 1988), CGRP may affect mucus secretion indirectly by increasing the blood flow to submucosal glands.

Metabolism of CGRP CGRP is subjected to proteolytic inactivation by several enzymes expressed in the human respiratory tract. The exact cleavage pathways have not been revealed so far but NEP (EC 3.4.24.11) inhibitors have been shown to increase some of the CGRP effects in the airways (Katayama et al. 1991).

sent in the airways. In the respiratory tract (Groneberg et al. 2002c) and in other organs (Groneberg et al. 2002d,e; Doring et al. 2005), short-chain peptide fragments of NPY may be rescavenged by peptide transporters such as PEPT2 (Groneberg et al. 2004c).

NPY: clinical applications Whereas the peptide has been focused for therapeutic options in the central nervous system, a potential use in the treatment of pulmonary inflammatory disorders has not been revealed so far due to the complex pulmonary effects of NPY. However, as selective antagonists and agonists and gene-depleted animals for the different receptors are now available, NPY may be of value for future strategies in airway nerve modulation.

Vasoactive intestinal polypeptide CGRP: clinical applications The complex effects of CGRP on the airway and vascular tone and the immune system have so far prevented the development of any therapeutic strategy based on CGRP targets (Springer et al. 2003).

Neuropeptide tyrosine NPY has long been proposed to play a role in the pathogenesis of inflammatory diseases (Groneberg et al. 2004b). NPY is a 36 amino acid neuropeptide that participates in the regulation of a large number of physiologic and pathophysiologic processes in the cardiorespiratory system, immune system, nervous system, and endocrine system.

Pulmonary effects of NPY Serum levels of NPY are increased during exacerbations of asthma, whereas the number of NPY-immunoreactive nerves in the airways remained constant in patients with inflammatory airway diseases such asthma or rhinitis. In addition to a role in the regulation of glandular activity, NPY exerts a major influence on humoral and cellular immune functions (Groneberg et al. 2004b). In this respect, NPY is known to modulate potent immunologic effects, such as immune cell distribution, T helper cell differentiation, mediator release, or natural killer (NK) cell activation. In addition to these direct effects, NPY also acts as an immunomodulator by influencing the effects of a variety of other neurotransmitters (Groneberg et al. 2004b).

Metabolism of NPY The active form of NPY can be degraded by specific enzymes, leading to the generation of NPY2–36 and NPY3–36. It is suggested that these truncated fragments may be ligands of particular NPY receptors. Dipeptidyl peptidase IV (EC 3.4.14.5) is a likely candidate responsible for the cleavage of NPY as this exopeptidase is a membrane-bound protease, which cleaves proline in the penultimate position, and is abundantly pre-

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One of the most important bronchodilatory peptides found in the human body is VIP (Groneberg et al. 2001a). It is one of the most abundant neuropeptides present in both the upper and lower human respiratory tract (Ghatei et al. 1982; Baraniuk et al. 1990; Groneberg et al. 2003a) and a likely neurotransmitter of the inhibitory nonadrenergic noncholinergic airway nervous system. Together with pituitary adenylate cyclase-activating peptide (PACAP), VIP influences numerous pulmonary functions (Said 1982). It is expressed in human airway nerve fibers innervating the pulmonary and bronchial vessels, the tracheobronchial airway smooth muscle layer, and submucosal glands (Dey et al. 1981; Lundberg et al. 1984c). Also, receptors for VIP have been identified in the airway epithelium, glands, and in inflammatory cells (Fischer et al. 2001, 2002; Groneberg et al. 2001b, 2003d).

Metabolism of VIP Similar to proinflammatory peptides, VIP is subjected to degradation by airway enzymes such as NEP (Goetzl et al. 1989), mast cell chymase, and mast cell tryptase (Caughey 1989). NEP hydrolysis fragments of VIP are physiologically inactive (Lilly et al. 1993). Also, chymase and tryptase VIP degradation products fail to relax vascular or airway smooth muscle (Caughey et al. 1988).

Pulmonary effects of VIP VIP has been shown to be a potent relaxant of airway smooth muscle. As the bronchodilatory effect of VIP in human bronchi is almost 100 times more potent than adrenergic dilation by isoproterenol, VIP is the most potent endogenous bronchodilator described so far (Palmer et al. 1986a). The predominant site of VIP-induced pulmonary dilatation appears to be the central airways. Vasodilation of pulmonary and systemic vessels is the second principal effect of VIP. VIP potently relaxes the vessels supplying the upper airways (Lundberg et al. 1981; Lung & Widdicombe 1987), trachea, bronchi (Laitinen et al. 1987),

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and pulmonary vessels (Hamasaki et al. 1983; Nandiwada et al. 1985; Obara et al. 1989). VIP-induced vasodilation is more potent in the tracheal circulation than in the bronchial circulation (Matran et al. 1989). The vasodilatory effect of VIP is 50 times more potent than prostacyclin (Saga & Said 1984) and is independent of endothelial cells (Barnes et al. 1986; Greenberg et al. 1987). There is a dense network of VIP-immunoreactive nerve fibers around airway submucosal glands (Dey et al. 1981), and VIP-binding sites have been localized to human submucosal glands, suggesting a regulatory role of VIPergic nerves in human airway mucus secretion. The effects of VIP on mucus secretion are very complex: VIP stimulates the secretion of mucus from tracheal submucosal glands of ferrets in vitro, being significantly more potent than isoproterenol (Peatfield et al. 1983). Furthermore, mucus secretion stimulated by cholinergic agonists is inhibited by VIP in ferret trachea but stimulated in cat trachea (Webber & Widdicombe, 1987; Shimura et al. 1988). In contrast to ferret trachea, VIP exhibits an inhibitory effect on methacholine-stimulated release of glycoproteins and lysozyme in human trachea (Coles et al. 1981), which is surprising because VIP increases cyclic AMP formation in submucosal gland cells (Frandsen et al. 1978), and therefore it would be expected to stimulate secretion.

VIP: clinical applications The precise role of VIP in the pathogenesis of asthma and COPD is still uncertain. Although therapy using the strong bronchodilator effects of VIP would offer potential benefits, the rapid inactivation of the peptide by airway peptidases has prevented a widespread use of effective VIP-based drugs so far. The potent bronchodilator properties of VIP gave rise to studies addressing the effectiveness of VIP-based therapy in bronchial asthma. Contrary to early studies (Morice et al. 1983) that reported a significant bronchodilation and protection against histamine-induced bronchoconstriction in asthmatic subjects, later studies failed to show protective effects of the peptide. In this respect, systemic administration was reported to have no effect on the airway tone in normal subjects (Palmer et al. 1986b). At higher doses, VIP even caused blood pressure decreases and tachycardia. Aerosol administration of VIP was shown to reduce the bronchial reactivity to histamine in mild asthma but did not significantly increase airway conductance (Barnes & Dixon 1984). Also, a further study using inhaled VIP as pretreatment did not significantly prevent exercise-induced asthma (Diamond et al. 1983). The explanation for the inability of VIP to cause protective effects is the rapid inactivation of the peptide by airway peptidases. To bypass the limited clinical effectiveness of the native peptide, which is subject to degradation by many enzymes, peptidase-resistant VIP analogs were generated. After the characterization of amino acid residues required for VIP receptor binding and activation (O’Donnell et al. 1991),

Neuropeptides

various compounds were synthesized and examined. The identification of major cleavage sites led to the synthesis of metabolically more stable analogs, namely cyclic peptides incorporating disulfide and lactam ring structures (Bolin et al. 1995). The most potent synthetic VIP analogs that proved to have the best metabolic stability and the longest duration of activity are the cyclic peptides Ro 25-1553, developed as an antiinflammatory bronchorelaxant agent (O’Donnell et al. 1994), and Ro 25-1392 (Xia et al. 1997). Both compounds have been characterized to be highly selective agonists of the VPAC2 receptor (Gourlet et al. 1997; Xia et al. 1997; Schmidt et al. 2001). Due to the extremely promising profiles of the respiratory effects of VIP, a double-blind, randomized, placebo-controlled, crossover study was recently performed to study the effects of Ro 25-1553 (Linden et al. 2003). The compound was administered by inhalation to patients with asthma and compared with the long acting β2-adrenoceptor agonist formoterol. Twenty-four patients with moderate stable asthma were included in a crossover protocol with the primary variable being the bronchodilatory effect assessed as increase in FEV1 after inhalation of Ro 25-1553 (100 or 600 μg) or formoterol (4.5 μg) (Linden et al. 2003). Side effects were identified by assessing blood pressure, serum potassium, electrocardiography, and echocardiography. In contrast to earlier unsuccessful studies with the native peptide VIP, it was shown that the inhalation of 600 μg Ro 25-1553 caused a rapid bronchodilatory effect (geometric mean increase in FEV1 compared with placebo) within 3 min of 6% (95% CI 4–9). The corresponding maximum bronchodilatory effect during 24 hours was similar for 600 μg Ro 25-1553 (7%; 95% CI 4–10) and the reference bronchodilator formoterol (10%; 95% CI 7–12). However, for both doses of the synthetic VIP agonist, the bronchodilatory effect was attenuated 5 hours after inhalation whereas formoterol had a bronchodilatory effect 12 hours after inhalation (Linden et al. 2003). Safety profiling of Ro 25-1553 and formoterol did not reveal any clinically relevant adverse reactions and side effects. Given the advantages of the strong topical effects of VIP, newly developed VIP agonists with a longer duration of bronchodilatory effects will be very attractive for potential clinical use.

Neuropeptides and cough Cough is an important defensive reflex of the airways and is also a very common symptom of respiratory diseases. Cough following a viral infection of the upper respiratory tract is usually transient, while persistent cough is associated with a whole range of conditions, including bronchial asthma or rhinosinusitis. A variety of studies focused on the involvement of neuropeptides in the modulation of the cough reflex. Direct functional effects were not found in pharmacologic

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studies, i.e., the potent NK1 receptor antagonist CP 99994 did not have effects on cough (Fahy et al. 1995). However, it was established that the vanilloid receptor TRPV1 plays a crucial role in the pathophysiology of the cough reflex (Groneberg et al. 2004d; Geppetti et al. 2006). Clinical trials using TRPV1 antagonists have not been carried out on cough so far but this receptor seems to be a principal target for the development of more effective antitussives devoid of side effects. Also, novel molecular targets for the delivery of drugs have been identified (Groneberg et al. 2004c) that offer promising pathways to deliver compounds directly to the airways via proton-coupled drug transport mechanisms (Groneberg et al. 2003e).

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Sakamoto, T., Barnes, P.J. & Fan Chung, K. (1993) Effect of CP96,345, a non-peptide NK1 receptor antagonist, against substance P-, bradykinin- and allergen-induced airway microvascular leakage and bronchoconstriction in the guinea pig. Eur J Pharmacol 231, 31–8. Salonen, R.O., Webber, S.E. & Widdicombe, J.G. (1988) Effects of neuropeptides and capsaicin on the canine tracheal vasculature in vivo. Br J Pharmacol 95, 1262–70. Saria, A., Martling, C.R., Dalsgaard, C.J. & Lundberg, J.M. (1985) Evidence for substance P-immunoreactive spinal afferents that mediate bronchoconstriction. Acta Physiol Scand 125, 407–14. Schmidt, D.T., Ruhlmann, E., Waldeck, B. et al. (2001) The effect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on induced tone in isolated human airways and pulmonary artery. Naunyn Schmiedebergs Arch Pharmacol 364, 314–20. Shimura, S., Sasaki, T., Ikeda, K., Sasaki, H. & Takishima, T. (1988) VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands. J Appl Physiol 65, 2537–44. Solway, J. & Leff, A.R. (1991) Sensory neuropeptides and airway function. J Appl Physiol 71, 2077–87. Springall, D.R., Cadieux, A., Oliveira, H., Su, H., Royston, D. & Polak, J.M. (1987) Retrograde tracing shows that CGRP-immunoreactive nerves of rat trachea and lung originate from vagal and dorsal root ganglia. J Auton Nerv Syst 20, 155–66. Springer, J., Geppetti, P., Fischer, A. & Groneberg, D.A. (2003) Calcitonin gene-related peptide as inflammatory mediator. Pulm Pharmacol Ther 16, 121–30. Springer, J., Amadesi, S., Trevisani, M. et al. (2004) Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery. Regul Pept 118, 127–34. Tanaka, D.T. & Grunstein, M.M. (1990) Maturation of neuromodulatory effect of substance P in rabbit airways. J Clin Invest 85, 345–50. Tomaki, M., Ichinose, M., Miura, M. et al. (1995) Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am J Respir Crit Care Med 151, 613–17. Vignola, A.M., Kips, J. & Bousquet, J. (2000) Tissue remodeling as a feature of persistent asthma. J Allergy Clin Immunol 105, 1041–53. Watson, N., Maclagan, J. & Barnes, P.J. (1993) Endogenous tachykinins facilitate transmission through parasympathetic ganglia in guinea-pig trachea. Br J Pharmacol 109, 751–9. Webber, S.E. & Widdicombe, J.G. (1987) The effect of vasoactive intestinal peptide on smooth muscle tone and mucus secretion from the ferret trachea. Br J Pharmacol 91, 139–48. Xia, M., Sreedharan, S.P., Bolin, D.R., Gaufo, G.O. & Goetzl, E.J. (1997) Novel cyclic peptide agonist of high potency and selectivity for the type II vasoactive intestinal peptide receptor. J Pharmacol Exp Ther 281, 629–33. Zimmer, G., Rohn, M., McGregor, G.P., Schemann, M., Conzelmann, K.K. & Herrler, G. (2003) Virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus. J Biol Chem 278, 46854–61.

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Late-phase Allergic Reactions in Humans Yee-Ean Ong and A. Barry Kay

Summary The late-phase reaction (LPR) in humans is an experimental model of allergic inflammation elicited usually in the nose, lower airways, or skin of atopic subjects following challenge with specific allergen. The LPR is a delayed-in-time reaction peaking between 3 and 9 hours after challenge, and is preceded by an early immediate response. A common feature of LPRs is vascular leakage with tissue swelling and edema. A wide range of cell types, pharmacologic mediators, cytokines, and chemokines have been identified in fluids and tissue sampled from LPRs. So far, no single agent has been shown to account for all the features of these tissue responses. Late asthmatic reactions can be provoked in isolation (i.e., without an immediate response) by allergen-derived T-cell peptide epitopes. Peptide challenge, like whole allergen provocation, leads to increased airway hyperresponsiveness in those subjects who experience a late asthmatic reaction. In the peptide model, the potent vasodilator calcitonin gene-related peptide was highly expressed in epithelial cells, infiltrating T cells, and smooth muscle at the peak of the late response, suggesting one mechanism that explains, at least in part, the events surrounding the formation of tissue swelling. However, the precise mechanisms of vascular leakage and edema are still not fully elucidated, although it is likely that agents derived from mast cells and T cells are crucial for the full expression of LPRs.

Introduction When the skin, nose, or airways of atopic subjects are provoked with a single dose of allergen this produces, respectively, and within minutes, an immediate cutaneous wheal-and-flare reaction, sneezing and runny nose, or wheezing. Depending on the dose of allergen, these immediate-type hypersensitivity


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responses are followed by a late-phase allergic reaction (LPR), which is slow to peak (6–9 hours) and slow to resolve. In the skin, LPRs are characterized by an edematous, red, and slightly indurated swelling, in the nose by sustained blockage, and in the lung by further wheezing. The LPR is extensively used as a model for studying the mechanisms of allergic inflammation and for screening drugs. Charles Blackley was the first to recognize, over 100 years ago, the association of allergen inhalation with symptoms of asthma several hours later, which often continued for several days (Blackley 1959). In 1934, Stevens noted that asthma symptoms could persist several days after allergen inhalation challenge (Stevens 1934). It was not until the 1950s that the response and time-course to allergen inhalation was more clearly described by Herxheimer (1952). Investigations into the pathogenesis of responses to inhaled allergens were well established by the 1970s (Booij-Noord et al. 1972; Pepys 1973). The development of a late asthmatic reaction (LAR) often results in increased airway responsiveness, which is reflected as an increase in morning dipping in peak expiratory flow rates for a few days following a single allergen challenge (Taylor et al. 1979). In 1977, Cockcroft made the classic observation that subjects who develop LARs following allergen provocation also develop an increase in airway responsiveness to histamine identified 24 hours after the challenge test, which persists for several days (Cockcroft et al. 1977a). The cutaneous late-phase reaction (CLPR) is characterized by a slowly developing, pink, edematous swelling at the challenge site. In large CLPR, there may be pruritus and systemic malaise, but these are unusual features. In most cases, the reaction is surprisingly indolent and the subject may be unaware of the reaction unless he or she is asked to look at the skin test site 4–6 hours after challenge. The reaction diameter can be measured accurately up to 8 hours after challenge, but becomes rather ill-defined thereafter. Following large CLPR, some diffuse swelling may persist for 48 hours, but this is not usually accompanied by erythema, and does not resemble the classical delayed-type hypersensitivity (tuberculin) reaction. The CLPR was originally considered to be an infrequent sequel to the immediate wheal-and-flare reaction. However, Frew and Kay (1988a) showed that all

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Methods of eliciting late-phase allergic reactions Airway challenge Late asthmatic responses can be achieved by experimental challenge in the laboratory. A number of methods have been employed, although perhaps the simplest and most reproducible is the tidal breathing method using a Wright nebulizer (Cockcroft et al. 1977b). Doubling or half-log incremental increasing concentrations of allergen are inhaled for 2 min from a Wright nebulizer at 15-min intervals. The starting allergen concentration is based on skin-prick tests of increasing concentrations of allergen extracts, with the starting concentration for inhalation being one half-log dilution lower than that which gave a 2-mm skin wheal. Baseline forced expiratory volume in 1 s (FEV1) (best of three attempts) is most commonly recorded initially after a 2-min inhalation of saline. Measurements are then repeated every 15 min after each allergen inhalation. If FEV1 has fallen by less than 10%, the next allergen concentration is inhaled. If FEV1 has fallen by 10–20%, it is repeated every 10 min until the maximal fall in FEV1 is achieved. If there is then a greater than 20% fall in FEV1, allergen inhalations are discontinued. If the fall in FEV1 is between 10 and 20%, the next concentration of allergen may be given. Once the end point has been achieved, the FEV1 is measured every 10 min

3.0

Normal saline control

Inhalational challenge 2.5 FEV1 (L)

atopic subjects are capable of developing a skin LPR if sufficient allergen is administered. The immediate response to nasal antigen challenge in sensitized patients comprises nasal congestion, rhinorrhea, sneezing, pruritus, and, often, conjunctival symptoms. These symptoms peak within a few minutes of the appropriate challenge and decrease over the next 1–2 hours (Naclerio et al. 1985). In 80% of subjects, the symptoms reach baseline levels within a couple of hours (Iliopoulos et al. 1990). Thereafter, in about 50% of these cases, recurrence of symptoms occurs 3–12 hours following the challenge. In some individuals, the decline in symptoms after the immediate reaction does not reach baseline levels; the clinical picture is that of continuing long-lasting reaction. Similarly, increased sneezing responsiveness to histamine has been observed after nasal allergen challenge, which dissipated after 12 days (Andersson et al. 1989a; Walden et al. 1991). In general, the molecular pathology of allergen-induced, late-phase lung, nasal, and skin reactions is similar. However, there are clear anatomic differences as regards permeability, content of smooth muscle, and autonomic nerve supply between the three tissues. The composition of mast cell granules between the different tissues (Irani et al. 1986) also varies, and may account for the different profile of response to various immunologic and nonimmunologic stimuli (Church et al. 1989).

Late-phase Allergic Reactions in Humans

Late asthmatic reaction

Early asthmatic reaction

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Time (h) Fig. 24.1 An example of an early and late asthmatic reaction provoked by inhalation of whole allergen (cat dander) in a cat-allergic subject. The early reaction is followed by a delayed-in-time late-phase response, which in this subject peaked at 8 hours. The response to allergen is compared with the control day in which the subject inhaled normal saline. (See CD-ROM for color version.)

until 1 hour and then hourly for up to 8 hours or longer depending on the protocol (Fig. 24.1). When performing repeated allergen inhalation challenges, identical conditions should be observed. These would include performing the challenges at the same time of day and adhering to the same technical and nontechnical factors. It is well recognized that an LAR may not be obtained in subjects challenged in the early morning, whereas this may be identified when challenged later in the day or evening. It has been postulated that this may be related to endogenous cortisol response, which might limit the development of a late response in some individuals. However, it has been shown that there are no changes in diurnal variation in serum cortisol concentrations in asthmatic subjects after allergen inhalation and no relationship to the size of the late response (Durham et al. 1989). Thus, the reason for the diurnal variation remains uncertain.

Nasal challenge Late nasal reactions can be produced by introduction of appropriate allergen into the nasal cavity either by means of a spray or presoaked disks. Allergen extracts are given in 10-fold increasing concentrations, with the starting concentration being the dose that caused a 2-mm wheal. Subjects are asked to inhale through the mouth to total lung capacity and hold their breath to minimize lower airway contamination by the test agent. Then, one squirt of the starting concentration is sprayed into one or both nostrils according to protocol from a metered-dose pump spray (Erin et al. 2005), or a disk is applied to the middle portion of the anterior nasal septum, posterior to the mucocutaneous junction for 1 min (Wagenmann et al. 2005). After 10–15 min, symptom scores derived from blockage, secretion, sneezes, itching eye, throat, conjunctivitis, cough, urticaria, or dyspnea are recorded

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Symptom score

Immediate (early) phase

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Time after challenge (hours) Fig. 24.2 Examples of immediate- and late-phase reactions in the nose assessed through symptom scores. (a) Example 1: the introduction of allergen in the nose leads to an immediate increase in nasal symptoms. These symptoms begin to dissipate soon after they peak and, within approximately 1 hour, they have almost returned to baseline. Later, 3– 4 hours after the allergen challenge, the symptoms return spontaneously and last for many hours and are still present 24 hours after allergen challenge. (b) Example 2: an identical immediate phase is followed by a partial reduction in symptoms, which remain elevated above baseline for many hours. A distinct late phase is not discernible but the magnitude of the symptomatology during the period that would be conventionally considered the “late phase” is similar to Example 1. (From Peebles & Togias 1997, with permission.)

(Fig. 24.2). The procedure is repeated until the highest concentration is reached or a positive response occurs (based on a predefined cumulative symptom score). The dose can be increased by increasing the number of squirts, until a response is obtained. Physiologic measurements can be made assessing resistance to air (which essentially reflects vascular engorgement), using rhinomanometry or measuring peak nasal inspiratory flow rate. Thickening of the paranasal sinus mucosa has been monitored (Pelikan & Pelikan-Filipek 1990). Nasal secretions can be collected, either spontaneously or by nasal lavage, and nasal biopsies taken. The glandular secretory apparatus can be assessed by local stimulation with a cholinergic agent such as methacholine (Raphael et al. 1988; Baroody et al. 1993). The activity of the neuronal apparatus can be monitored in the nose because stimulation of a mucosal irritant receptor inside one nasal cavity leads to the generation of a central reflex, the efferent arm of which produces a secretory response in the contralateral cavity (Konno & Togawa 1979; Baroody et al. 1993).

Cutaneous challenge Intradermal allergen challenge can be safely performed with predetermined concentrations of allergen using a 29G microfine syringe. The dose of allergen can be titrated according to the skin-prick test reaction (Charlesworth et al. 1989), or set using previous data (Frew & Kay 1988a). The immediate wheal

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(a)

(b)

Fig. 24.3 An early-phase and late-phase allergic skin reaction in an atopic subject challenged with house-dust mite extract by intradermal injection. (a) The early-phase reaction (15 min) is essentially a wheal-and-flare response, similar to that observed in a diagnostic skin-prick test. (b) The latephase reaction (6 h) is slightly raised, erythematous and with an edematous swelling, which is easy to palpate. (See CD-ROM for color version.)

and flare of the early reaction and the swollen erythematous skin characteristic of the CLPR is shown in Fig. 24.3. The late-phase skin reaction can be measured using a sharpened pencil point to detect the edge of the induration caused by the injections as previously described (Frew & Kay 1988a). An outline of the induration is drawn in ink and transferred using tape to form a permanent record. Alternatively, scanning laser Doppler imaging can be used to assess changes in skin blood flow (Clough et al. 1998). Samples can be obtained by means of skin biopsy to assess cellularity, while the skin chamber/skin window technique and cutaneous microdialysis are particularly useful to examine mediators. Recent alterations have been made to the traditional skin window technique in order to obtain sufficient cells to study by flow cytometry, while also collecting supernatant for study of mediators (Fernvik et al. 1999; Ong & Kay 2006).

Progression of early- and late-phase reactions Initially, atopic individuals were thought to be either single early responders or to have both early and late reactions (dual responders) to intradermal allergen challenge (Slavin et al. 1964). It has now been shown that there is a dose– response relationship between allergen dose and continuation of the skin reaction to the formation of an LPR (Frew & Kay 1988a). Formation of a CLPR after allergen challenge required a higher concentration threshold of allergen than the early-phase reaction (EPR), but all atopics were capable of mounting a LPR if challenged with sufficient allergen. It is

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possible that late asthmatic and late nasal reactions would also be ubiquitous if the airway effects of the EPR did not limit the dose of allergen that can safely be given. Isolated LARs have been shown to be induced by occupational agents (Paggiaro & Chan 1987), allergen-derived peptides (Haselden et al. 1999; Ali et al. 2004), and low-dose allergen (Ihre et al. 1988). Similarly, late nasal reactions have also been reported in the absence of early responses (Pelikan 1978). However, isolated late cutaneous reactions have not been reported, even when intradermal challenge with allergen-derived peptides has resulted in distant LARs (see below) (Haselden et al. 1999; Oldfield et al. 2001). The situation is more complex in patients with urticaria (see Chapter 90). Delayed-pressure urticaria is an example of a skin reaction that resembles an LPR but has no preceding immediate phase. In addition, a large immediate hive can be provoked with an ice cube in cold urticaria, but this is not followed by an LPR even with very large wheals. Thus, in this situation, the magnitude of the EPR does not seem related to an LPR.

Priming Following nasal or inhaled allergen challenge, the response to repeated local stimulation with the same allergen is increased. Priming was first described in the 1960s and its occurrence has since been confirmed by several investigators in both experimental and natural allergen exposure (Connell 1968, 1969; Bacon et al. 1981; Iliopoulos et al. 1990; Koh et al. 1997). In contrast, repeated cutaneous allergen challenges have shown both a reduction in the size of response (so-called refractory period) (Shaikh et al. 1977) or priming (Weller et al. 1996), depending on the time between challenges. The mechanism by which priming occurs is unclear. Although nonspecific target-organ responsiveness to inflammatory mediators released during allergen challenge can be implicated as a possible contributor to priming, there is evidence that the amount of inflammatory mediators recovered after allergen challenge in the primed state is significantly elevated as compared with baseline provocation (Pipkorn et al. 1987a; Wachs et al. 1989; Iliopoulos et al. 1990).


Mast cells There is incontrovertible evidence that the allergen-induced EPR is due to allergen cross-linking IgE antibodies on mast cells. The number of mast cells in bronchoalveolar lavage (BAL) (Diaz et al. 1989), skin (Macfarlane et al. 2000), and nasal epithelium (Fokkens et al. 1992) has been shown to decrease after allergen challenge. However, mast cell numbers were raised in bronchial mucosal biopsies 24 hours after allergen challenge (Crimi et al. 1991), and nasal lamina propria also shows increased levels of perivascular mast cells 10 hours after challenge (Juliusson et al. 1992). It is possible that this apparent discrepancy occurs as a result of continuous migration of mast cells into the epithelial surface fluid (Fokkens et al. 1992). The cells show an increased capacity to degranulate as they approach the airway surface (Djukanovic et al. 1990). Mapping techniques have shown that mast cells are present in normal skin with the greatest density occurring just below the dermal–epidermal junction (Cowen et al. 1979), particularly concentrated around blood vessels, nerves, and appendages (Eady et al. 1979). Application of inhalant allergen to the abraded skin of sensitive individuals with atopic dermatitis has been shown to induce an increase in the number of cutaneous mast cells (Mitchell et al. 1986). Studies using immunohistochemistry have shown a decrease in tryptase-containing mast cells after allergen provocation suggesting mast cell degranulation had occurred, and these remained reduced for up to 48 hours (Macfarlane et al. 2000). In nasal studies, some authors have found a correlation between mast cell numbers and the severity of early phase symptoms reported (Juliusson et al. 1992); however, others have not (Fokkens et al. 1992). In seasonal allergen exposure, a positive correlation has been reported between local mast cell density at the start of the allergy season and the severity of symptoms (Pipkorn et al. 1988). Circulating basophils infiltrate tissues at sites of allergen challenge in the skin (Solley et al. 1976; Mitchell et al. 1986; Charlesworth et al. 1989), nose (Iliopoulos et al. 1992), and lung (Guo et al. 1994). However, their numbers are small and influx after allergen challenge tends to occur later than the peak of the LPR itself, suggesting that this cell type is not directly responsible for tissue edema.

Immunology of late-phase reactions Eosinophils (see Chapter 12) Pepys, studying patients with allergic bronchopulmonary aspergillosis, initially proposed that the LAR was a type III reaction initiated by IgG antibodies (Pepys et al. 1968). However, later studies identified the IgE dependency of late responses occurring in the skin and the lung (Dolovich et al. 1973; Solley et al. 1976; Kirby et al. 1986). Mast cells, basophils, and dendritic cells possess high-affinity IgE receptors (FcεRI), and other cell types including monocytes, alveolar macrophages, and eosinophils recognize IgE through the low-affinity receptor (FcεRII, CD23).

In recent years, eosinophils have been regarded as proinflammatory cells in allergic disease that show selective enrichment in involved tissues (Wardlaw 1999). Allergen-induced LARs are accompanied by changes in blood eosinophils (Booij-Noord et al. 1972; Durham & Kay 1985; Cookson et al. 1989), which have both a temporal and quantitative relationship with the size of the late response and increase in airway responsiveness (Frick et al. 1989). Late-phase responses are associated with eosinophilia in the lung (de Monchy et al. 1985), nose (Bascom et al. 1988), and skin (Frew & Kay 1988b),

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and the appearance of hypodense eosinophils suggests they are in a state of activation (Frick et al. 1989). There is evidence of eosinophil degranulation in asthmatics (Wardlaw et al. 1988; Adelroth et al. 1990), after nasal allergen challenge (Andersson et al. 1989b) and in skin blister fluid after cutaneous challenge (Zweiman et al. 1991). Disease severity in asthmatics correlates with the number and state of activation of eosinophils in the bronchial mucosa and BAL fluid (Wardlaw et al. 1988). In contrast, there was no correlation between eosinophil numbers and the size of the allergen-induced LPR at 6 hours in the skin model (Frew & Kay 1988b). It seems unlikely that eosinophils are essential for the expression of LPRs. Anti-interleukin (IL)-5 had no effect on the LAR (Leckie et al. 2000) or the CLPR (Phipps et al. 2004). It is uncertain whether, in the former study, IL-5 depleted bronchial mucosal eosinophils; in the skin the eosinophildepleting effect was far more effective. Also, as described below, isolated LAR induced by inhalation of T-cell peptides was not associated with a significant increase in airway eosinophils (Ali et al. 2007).

Neutrophils (see Chapter 13) Neutrophils are residents of the airways in normal individuals as well as subjects with asthma. They accumulate in large numbers after allergen challenge in the lungs (Nocker et al. 1999; Macfarlane et al. 2000), nose (Bascom et al. 1988), and skin (Ying et al. 1999). The CXC chemokine IL-8, initially described as neutrophil chemotactic factor, was first noted after allergen challenge (Lee & Kay 1982). Although allergic asthma is traditionally thought to be due to eosinophilic inflammation, several studies have described the presence of neutrophils and IL-8 particularly in severe asthma (Jatakanon et al. 1999; Wenzel et al. 1999). In addition, peripheral blood neutrophils become activated in association with allergen-induced early and late-phase asthmatic reactions (Papageorgiou et al. 1983; Durham et al. 1984; Carroll et al. 1985). Although neutrophils are identified after allergen challenge in rhinitis (Lim et al. 1995) and asthma, their functional role in the development of late responses has yet to be determined. Neutrophils have the potential to release a mixture of lysozomal enzymes, oxygen metabolites, and leukotriene (LT)B4, which may be of relevance to the LPR. For example, vascular endothelial growth factor (VEGF) has been found in several cell types, including neutrophils and eosinophils, and is localized to the secretory vesicles (Gaudry et al. 1997). Tumor necrosis factor (TNF)-α stimulation has been shown to induce VEGF release by isolated human neutrophils (Gaudry et al. 1997). VEGF has an important role in angiogenesis but also induces endothelial hyperpermeability via the action of endothelial VEGF-R2 and thus may play a role the development of edema (Bates & Harper 2002; Byrne et al. 2005). The role of vascular permeability is considered later.

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T cells (see Chapter 3) Most of the work examining the role of T cells in allergic disease has been in the field of asthma. In the 1980s the concept arose that chronically activated T helper cells perpetuated the inflammatory response in allergic diseases and an association between T cells, their products, and asthma severity was noted (Walker et al. 1991). The T-cell hypothesis of asthma developed from studies of allergen-induced LARs (Gonzalez et al. 1987) and acute severe asthma (Corrigan et al. 1988), and was supported by the observation that there was a Th2 cytokine profile in asthma (Robinson et al. 1992; Bentley et al. 1993). Interest in the role of T cells in asthma arose from the concept that, in addition to participating in IgE synthesis, T-cell products may also have effects on the airways either through direct action or via the recruitment of inflammatory cells. Sampling of both peripheral blood of asthmatics during exacerbations as well as the airways of asthmatic patients revealed T cells with features of activation (Gonzalez et al. 1987; Azzawi et al. 1990; Robinson et al. 1993a). After description of the Th1/Th2 dichotomy, cells bearing mRNA for IL-4 and IL-5 (signature Th2 cytokines) were detected in the airways of atopic asthmatic patients (Ying et al. 1997). This linked Th2 cells with IgE synthesis through the actions of IL-4, and also eosinophil airway inflammation through IL-5, together with IL-3 and granulocyte–macrophage colonystimulating factor (GM-CSF). Over recent years it has been suggested that Th2 cytokines contribute to asthma pathology in a variety of ways. In addition to stimulating IgE production (IL-4, IL-13), the effects include maturation of eosinophils (IL-5, IL-9), upregulation of the eosinophil/basophil selective adhesion molecule vascular cell adhesion molecule (VCAM)-1 (IL-4, IL-13), mast cell development (IL-3, IL-9, stem-cell factor), and, in the context of asthma, airway hyperreactivity (IL-9, IL-13) and mucus hypersecretion (IL-4, IL-9, IL-13) (Kay 2001). It is also suggested that there may be direct interaction between T cells and airway smooth muscle, and IL-5 has been shown to increase smooth muscle contractility in vitro (Hakonarson et al. 1999). In humans, LARs were provoked by intradermal challenge with T-cell peptide epitopes. However, as mentioned above, the expected infiltration of eosinophils and Th2 cells into bronchial mucosa was not observed and there was no measurable release of histamine or eicosanoids in BAL, raising the possibility that there may be purely T cell-dependent pathways to airway narrowing in asthma (Haselden et al. 2001). Although some studies also showed the presence of Th1 cytokines in serum and BAL fluid from asthmatics (Krug et al. 1996), most studies confirm a Th2 predominance. An increase in nasal mucosal CD4+ T lymphocytes is also seen after nasal allergen challenge, and there is a correlation between expression of the IL-2 receptor (CD25+) and CD4+ cells, suggesting that CD4+ cells may be activated after allergen challenge (Varney et al. 1992a; Lim et al. 1995). Interest-

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ingly, in some studies, mononuclear cells (which presumably include lymphocytes) do not rise significantly, suggesting that the allergen-induced inflammatory response may vary between individuals (Lim et al. 1995). As with the lung, the majority of the lymphocytes in the nose (Durham et al. 1992) and skin (Barata et al. 1998) after allergen challenge were Th2 type.

Antigen-presenting cells (see Chapter 8) Increased numbers of Langerhans cells have been found in epithelium and lamina propria of the nose after sequential nasal allergen challenge (Godthelp et al. 1996). Similar results have been observed during grass pollen season in patients with seasonal allergic rhinitis and these increases were inhibited by the use of topical corticosteroids (Till et al. 2001).


It is not possible to determine the cellular sources of histamine, PGD2, and LTC4 recovered in these studies. Mast cells have been shown to produce all three substances, eosinophils the lipid-derived mediators, while basophils release only histamine and LTC4. Therefore, it is possible that mast cells are responsible for the early release of histamine and, in conjunction with eosinophils, for PGD2 and LTC4 levels in the first 6–8 hours. The absence of tryptase and PGD2 from the LPRs suggests that basophils rather than mast cells may be responsible for the second wave of mediators in the airways. There is also evidence that kinins may be involved in the late-phase skin response, as activation of the kallikrein–kinin cascade (see Chapter 20) paralleled the magnitude of the LPR (Atkins et al. 1987).

Cytokines (see Chapter 3) Pharmacologic mediators Early release of the mast cell products histamine, tryptase, prostaglandin (PG)D2, and LTC4 has been detected in nasal lavage fluid (Naclerio et al. 1985; Juliusson et al. 1991), and also in BAL (Sedgwick et al. 1991; Wenzel et al. 1991) after local allergen challenge. Skin blister studies have also shown histamine release within 5 min of allergen challenge (Ting et al. 1980), as well as tryptase and PGD2 (Shalit et al. 1988; Charlesworth et al. 1989; Reshef et al. 1989). Dermal microdialysis techniques have detected histamine release in the wheal area after allergen challenge, but not in the flare which is thought to occur by a neurogenic reflex mechanism (Petersen et al. 1997). The quantity of mediators produced by mast cells correlates significantly with the clinical intensity of the immediate-phase nasal reaction (Naclerio et al. 1985). There is a second increase in histamine, LTC4, and kinins in those patients who exhibit a late nasal response (Naclerio et al. 1985; Pipkorn et al. 1987b). Although the late increase in LTC4 is small compared with levels found in the early phase (Pipkorn et al. 1987b), a metabolite of LTC4, LTE4, is also detected in the late phase in higher quantities than in the early phase, perhaps as a result of metabolism. Interestingly, PGD2 and tryptase do not reappear in increased concentrations in the late-phase nasal response (Naclerio et al. 1985; Gosset et al. 1993). Similarly, increases have been seen in histamine and LTC4 levels in BAL 6 hours after challenge (Diaz et al. 1989), and the terminal metabolite LTE4 has been recovered in increased amounts in the urine (Taylor et al. 1989). In the skin after allergen challenge, histamine continues to be detected at a lower level for up to 12 hours, although some investigators have noted a second peak at 12 hours (Charlesworth et al. 1989). PGD2 levels peak 1–4 hours after allergen challenge before decreasing over the next 8 hours (Charlesworth et al. 1989; Reshef et al. 1989). In contrast, LTC4 is elevated for 7 hours with significantly raised levels only between 4 and 6 hours (Reshef et al. 1989).

After antigen challenge, elevated levels of several cytokines have been detected in nasal lavage, BAL, and skin blister fluid during the LPR. These include IL-1β (Bochner et al. 1990; Sim et al. 1994; Virchow et al. 1995) and IL-6 (Lee et al. 1992; Sim et al. 1994; Virchow et al. 1995). IL-4, IL-5, IL-13, GM-CSF, and TNF-α have all been detected in lavage fluid after nasal and bronchial challenges (Sim et al. 1994; Bradding et al. 1995; Huang et al. 1995; Virchow et al. 1995; Batra et al. 2004; Erin et al. 2005; Ong & Kay 2006). The source of cytokines that are found in lavage or skin blister fluid is yet to be fully determined. Bradding et al. (1993) found, in immunohistochemical staining of inferior turbinates of patients with perennial rhinitis, that 90% of the IL-4 and IL-6 immunoreactive cells were mast cells, while 50% of the IL-5 immunoreactive cells were mast cells. IL-5 immunoreactivity was also found in eosinophils. Studies have also shown that the majority of the cells expressing IL4, IL-5 in the nose and lungs after allergen challenge are CD3+ T cells, with the remainder being mast cells and some eosinophils (Ying et al. 1993, 1995, 1997). Although allergic disease has traditionally been viewed as a Th2-driven disease, there is some work that has suggested inbuilt regulatory mechanisms with Th1 or regulatory cytokines playing a role. Previous work using in situ hybridization to assess mRNA for Th1 and Th2 cytokines did not show a significant increase in interferon (IFN)-γ mRNA (Kay et al. 1991). However, some studies have shown elevated IFN-γ in acute and chronic asthma (Corrigan & Kay 1990; Cembrzynska-Nowak et al. 1993), and correlated this with disease activity in subjects with asthma (ten Hacken et al. 1998). Furthermore, T cells obtained from BAL are able to produce IFN-γ on stimulation (Cho et al. 2005). There is also growing evidence to suggest that Th1 cytokines counteract the activity of Th2 lymphocytes, with IFN-γ being the most potent in suppressing Th2-type allergic responses (Gavett et al. 1995; Lack et al. 1996; Tang et al. 2001). Therefore, Th1 cytokines within areas of allergic inflammation may be acting to “switch off” the allergic response.

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In addition, there were significant increases in the level of the regulatory cytokine IL-10 in allergen-challenged blisters. IL-10 is produced by a variety of cells, including regulatory T cells (Tr1) as well as macrophages (Moore et al. 1993; Groux et al. 1997). It is thought to play a possible role in the regulation of allergic responses through a number of different pathways, including its suppressive effects on Th1 and Th2 cells (Romagnani 1995), and cytokines (Fiorentino et al. 1991), as well as by inhibiting eosinophil survival (Takanaski et al. 1994). It is also a potent suppressor of IgE while it simultaneously increases IgG4 production (Akdis et al. 1998). Given its antiinflammatory effects, it has been suggested that a deficiency in this cytokine may result in a more intense inflammatory reaction in asthmatic subjects. Although some investigators have reported reduced levels of IL-10 in asthmatics (Borish et al. 1996), others have reported higher levels of both IL-10 mRNA (Robinson et al. 1996; Magnan et al. 1998), as well as IL-10 protein in the BAL of asthmatics compared with normal individuals (Colavita et al. 2000). Similarly, elevated levels of transforming growth factor (TGF)-β have been detected in the BAL of asthmatics (Redington et al. 1997). Previous work has suggested that TGF-β may have antiinflammatory properties (Haneda et al. 1997, 1999), as well as a role in the remodeling seen in chronic asthma (Kumar et al. 2004). It is possible that these regulatory cytokines attempt to act in conjunction with Th1 cytokines to downregulate the allergic inflammatory response that has been elicited.

Chemokines Following allergen challenge in asthmatics, both macrophagederived chemokine (MDC) and thymus and activationregulated chemokine (TARC) were strongly upregulated on airway epithelial cells, and Th2 cells in the bronchial mucosa expressed CCR4, and some of them CCR8, but no CCR3 (Panina-Bordignon et al. 2001). Macrophage inflammatory protein (MIP)-1β, the ligand for CCR5 and interferon-inducible protein (IP)-10, the CXCR3 ligand, preferentially attract Th1 cells in vitro. In sarcoidosis, a typical Th1 cell-mediated lung disease, the T cells isolated from BAL expressed high levels of CXCR3 (Agostini et al. 1998). However, Campbell et al. (2001) examined BAL T cells comparing asthmatic subjects and nonasthmatic control subjects and showed no difference in the expression of CCR5 and CXCR3, with only low expression of CCR4. Both Th1 (IP-10) and Th2 (MDC and TARC) chemokines have been recovered from BAL after allergen challenge (Liu et al. 2004). Furthermore, some work suggests that chemokines may induce tissuespecific (rather than disease-specific) migration of T cells. Memory for skin-homing circulating lymphocytes is found in a population of circulating lymphocytes expressing cutaneous lymphocyte antigen (CLA) (Berg et al. 1991). In addition, TARC (CCL17) has been shown to be expressed by cutaneous endothelium and binds to CCR4 expressed at high levels on CLA-positive skin memory lymphocytes (Campbell et al.

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1999). More recently, keratinocytes have been shown to express cutaneous T cell-attracting chemokine (CTACK, CCL27), which binds to the receptor CCR10, also specifically expressed on circulating skin-homing CLA-positive T lymphocytes (Morales et al. 1999). Increased levels of CCR4 have been found in the skin after allergen challenge (Nouri-Aria et al. 2002), and elevated levels of CCR4 and CCR10 have also been demonstrated in an animal model of delayed-type hypersensitivity (Reiss et al. 2001) suggesting that, in the skin, the TARC/CCR4 axis may act as a specific skin-homing mechanism rather than having a Th2 disease bias. Increased levels of RANTES and MIP-1α have been identified in nasal secretions after allergen challenge (Weido et al. 1996; Kramer et al. 2001). Similarly, increased levels of eotaxin, eotaxin-2, RANTES, MIP-1α, and MCP-4 have been found after intradermal allergen challenge (Zweiman et al. 1997; Ying et al. 1999, 2001).

Neural inflammation LPRs in the lungs, nose, and skin provide three different methods of assessing the role of neurogenic inflammation. The lower airways allow us to study the interaction of nerves, neuropeptides, and smooth muscle; the nose allows investigators to stimulate one nasal passage and study the effects of neurogenic inflammation in the other; and the skin model can be used to study the disruption of nerve supply whether by use of local anesthetics or interruption of nerve supply. Initial interest in the concept of neurogenic inflammation arose when stimulation of dorsal root ganglion (a sensory nerve) led to vasodilation via an antidromic (conduction opposite to normal) pathway (Bayliss 1901). The axon reflex model was proposed whereby local injury could spread by orthodromic and then antidromic conduction of impulses (Langley 1900). Bruce (1913) demonstrated that acute inflammation caused by the application of mustard oil to the skin required an intact sensory system. Lewis and Grant (1924) then showed that sensory innervation provides the pathway for a local reflex which is required for the flare of the wheal-and-flare response after allergen challenge.

Airways Substance P is the predominant neuropeptide found in BAL of asthmatics (Nieber et al. 1991, 1992). Substance P and neurokinin A increase in BAL after allergen challenge (Nieber et al. 1992; Tomaki et al. 1995; Heaney et al. 1998). It appears that substance P exerts its proinflammatory effects mainly by stimulation of the neurokinin (NK)1 receptors, whereas neurokinin A (NKA) mainly acts on NK2 receptors. Increased levels of NK1 and NK2 receptor mRNA expression have been found in asthmatics as compared with nonasthmatics (Adcock et al. 1993; Bai et al. 1995). On inhalation, substance P induces airway hyperresponsiveness (AHR)

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mainly characterized by microvascular leakage, mucus secretion, and inflammatory cell responses (Joos et al. 2000), whereas inhaled NKA mainly causes bronchoconstriction (Joos et al. 1987; Cheung et al. 1993). Calcitonin gene-related peptide (CGRP), which acts predominantly as a vasodilator, has also been localized in airway nerves (Palmer et al. 1987; Martling et al. 1988). Although neuropeptides were traditionally thought to derive from nerves, there is considerable evidence that there are cellular sources that produce neuropeptides and also bear NK receptors (Ho et al. 1997; Kay et al. 2007). Several studies have been conducted assessing tachykinin NK1/NK2 receptor antagonists. In one study, the NK1/NK2 receptor antagonist FK224 failed to protect against NKAinduced bronchoconstriction (Joos et al. 1996). In contrast, other tachykinin receptor antagonists, DNK333 and CS-003, have been shown to produce a significant rightward shift of the dose–response curve to NKA (Joos et al. 2004; Schelfhout et al. 2006). More recently, a dual NK1/NK2 receptor antagonist, AVE5883, conferred protection against NKA-induced bronchoconstriction but had no beneficial effects against allergen-induced early or late-phase asthmatic reactions, methacholine PC20, or sputum differential cell counts, raising a question as to the role of neurogenic inflammation in this model (Boot et al. 2007).


Skin Nerve fibers containing a variety of neuropeptides, including substance P and CGRP, have been demonstrated in human skin (Schulze et al. 1997). Intradermal injection of substance P results in an immediate wheal-and-flare reaction (Foreman et al. 1983), whereas the main effect of CGRP is a long-lasting (4–6 hours) intense erythema (Brain et al. 1985). Additionally, CGRP potentiates edema formation evoked by mediators such as substance P (Brain & Williams 1985). This work suggested that neuropeptides may have an important role to play in both the allergen-induced EPR and LPR. Previous work on two subjects with impaired sensory function (one with sensory denervation due to trauma, the second after administration of local anesthetic) showed that stimulation of mast cells with anaphylactogenic anti-IgE resulted in a wheal, but the complete absence of a flare, corroborating evidence that the flare in the EPR is due to a neurogenic reflex mechanism. However, in both of these subjects, there was no apparent reduction in the LPR on the sensorydeprived site as compared with a control site (Umemoto et al. 1976), suggesting that while neurogenic inflammation was responsible for the flare of the EPR, it had a limited role in the LPR.

Action of drugs Nose After nasal allergen challenge, substance P and CGRP are released into nasal secretions of atopic patients (Mosimann et al. 1993; Nieber et al. 1993). In allergic rhinitis patients, but not in normal control subjects, exogenous substance P induces nasal obstruction (Devillier et al. 1988). In contrast, CGRP instillation in the nasal cavity increased nasal blood flow and caused the sensation of nasal obstruction (Rangi et al. 1990), but had no immediate effects on albumin leakage or glandular secretion (Guarnaccia et al. 1994). Nerve growth factor has been found in nasal lavage fluid and also in cells, particularly mast cells, in allergic rhinitis (Sanico et al. 2000; Wu et al. 2006). There is evidence for the participation of nasal nerves in aspects of the LPR. Most studies have reported that the number of sneezes during the LPR, although much less than the EPR, is elevated compared with appropriate controls (Naclerio et al. 1985). When a localized, unilateral nasal allergen challenge is performed, ipsilateral and contralateral elevations in airway resistance, as well as in levels of histamine, IL-4, IL-8, and airways secretions are seen (Baroody et al. 1993; Wagenmann et al. 2005). Although it is not possible to refute the hypothesis that local allergen application may lead to systemic contralateral effects unrelated to neurogenic inflammation, there is evidence that stimulation of local sensory nerves with capsaicin can lead to dose-dependent leukocyte influx, albumin leakage, and glandular secretion (Sanico et al. 1997).

The b agonists The β agonists, when given prior to allergen, will generally inhibit the immediate asthmatic response but not the late response (Cockcroft & Murdock 1987). In nebulized form, salbutamol has been shown to partially inhibit the late response when given prior to allergen, and similarly inhaled β agonists may have a partial effect when given during the late response (Twentyman et al. 1991). Their effects may be more a reflection of prolonged functional antagonism rather than a specific effect on the underlying mechanism of the LAR (Twentyman et al. 1990). The cutaneous allergeninduced EPR can also be attenuated by the administration of β2 agonists by inhibition of allergen-induced mast cell degranulation both in vitro and in vivo. Intradermal terbutaline and salbutamol administration resulted in inhibition of the allergen-induced early and late-phase skin reactions (Petersen & Skov 1999) and also an immediate reduction in the release of histamine and PGD2 synthesis (Petersen & Skov 2003).

Antihistamines Antihistamines have been an integral part of the treatment of allergic rhinitis for many years and predominantly reduce symptoms of sneezing and pruritis, but have limited effects on nasal blockage (Hilberg 1995). Antihistamines have been shown to predominantly affect the early phase nasal reaction, with limited effect in the late-phase reaction (Baroody &

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Naclerio 2000; Saengpanich et al. 2002). Although antihistamines are not used in the treatment of asthma, they have been shown to reduce the size of both the early and late asthmatic reactions (Hamid et al. 1990; Wasserfallen et al. 1993), as well as reduce eosinophil accumulation 24 hours after allergen challenge (Redier et al. 1992). Similarly, histamine H1 receptor antagonists such as cetirizine have been shown to inhibit both the early and, to a lesser extent, late-phase allergen-induced skin reactions (Varney et al. 1992b; Nielsen et al. 2001). Although cetirizine has been shown to reduce early total protein extravasation, there was no evidence of a reduction in the release of inflammatory mediators, such as histamine, tryptase, or LTB4, or a reduction in the numbers of inflammatory cells after allergen challenge.

Mast cell stabilizers Sodium cromoglycate has been shown to inhibit both the early and late asthmatic response to allergen (Crimi et al. 1986; Cockcroft & Murdock 1987). Cromolyn has been shown to be effective in protecting the nasal mucosa from both the early and late nasal responses to allergen (Pelikan 1982); however, the therapeutic role of cromolyn in allergic rhinitis is rather limited (Naclerio 1991). There have been contradictory results as to the effect of mast cell stabilizers such as sodium cromoglycate on allergen-induced skin reactions. Locally applied cromolyn does not appear to cause a reduction in the allergeninduced EPR when administered at the same time as allergen or anaphylactogenic anti-IgE (Ting et al. 1983; Kimata & Igarashi 1990). However, when pretreatment with cromolyn was given, there was a reduction in the EPR (Kimata & Igarashi 1990). One other study has shown that although local cromolyn administered at the same time as anti-human IgE did not alter the EPR, there was a significant reduction in the LPR (Gronneberg & Zetterstrom 1985). Differences in dosage, as well as the time of administration, may have played a role in the differences between these studies.

Leukotriene receptor antagonists Early trials of leukotriene receptor antagonists (LTRAs) showed benefit only on the early asthmatic response (Fuller et al. 1989). However, later LTRAs have been shown to inhibit both the early and late asthmatic response to inhaled allergen as well as the development of AHR (Taylor et al. 1991; Hamilton et al. 1998). There is general agreement that LTRAs do not inhibit allergen-induced cutaneous EPRs (Hill & Krouse 2003; White et al. 2005). However, there has been contradictory work as regards their effects on the cutaneous LPR, with some authors finding a reduction in size of these reactions after administration of LTRAs while others have not (Simons et al. 2001; Sekerel & Akpinarli 2003).

inhaled corticosteroids inhibit the late response almost completely and some authors have also shown partial inhibition of the early response (Burge et al. 1982; Cockcroft & Murdock 1987). In addition, there is a reduction in bronchial hyperreactivity after inhaled allergen challenge (Kraan et al. 1985; Cockcroft & Murdock 1987). Even when inhaled beclomethasone dipropionate was administered after the early asthmatic response caused by inhaled allergen challenge, the LAR was attenuated (Cockcroft et al. 1993; Paggiaro et al. 1994). Inhaled beclomethasone dipropionate given for a week prior to allergen challenge was able to reduce the allergen-induced eosinophil accumulation and activation (Gauvreau et al. 1996; Kelly et al. 2000; Parameswaran et al. 2000), and certain inhaled steroid formulations have been shown to reduce sputum expression of IL-4 and IL-5 after allergen challenge (Hauber et al. 2006). Similarly, short-term local steroid application has a greater effect on the allergen-induced late nasal reaction as compared with the early reaction (Pelikan 1982). However, with pretreatment for 7 days, the early as well as the late nasal reaction is reduced, with a reduction in histamine and kinins in nasal lavage fluid (Pipkorn et al. 1987a). Six weeks treatment with topical steroids prior to allergen challenge showed a reduction in T cell as well as eosinophil accumulation (Rak et al. 1994). Cytokine and chemokine levels in nasal mucosa are also reduced after treatment with topical steroids (Kleinjan et al. 2000; Erin et al. 2005). Systemic treatment with steroids prior to allergen challenge inhibited eosinophil influx and the release of eosinophil granule proteins, but had no effect on T cells (Bascom et al. 1988, 1989). In contrast, a single dose of oral corticosteroid has been shown to significantly reduce the size of the allergen-induced CLPR, and also the infiltration of inflammatory cells such as T cells and eosinophils in the absence of any effect on the EPR (Charlesworth et al. 1991; Varney et al. 1992b). Prednisolone has also been found to ablate the secondary rise of histamine seen during allergen-induced late-phase reactions, and reduce levels of LTC4 (Charlesworth et al. 1989). Prolonged corticosteroid use led to a reduction in allergen-induced early phase skin reactions and also a reduction in the number of skin mast cells (Pipkorn et al. 1989).

Cyclosporin A Evidence for a T-cell component to the LAR was provided by Sihra et al. (1997), who showed that a single dose of cyclosporin A inhibited the late, but not the early, reaction in mild asthmatics after allergen challenge. This supported the view that T-cell products may be acting independently of mast cell mediators in at least some aspects of the late phase mechanism(s).

Corticosteroids

Anti-IgE

Inhaled corticosteroids are the mainstay of treatment in all but the mildest of asthmatics. Studies have shown that

Two studies have shown that anti-IgE (omalizumab) has a greater inhibiting effect on the LPR than the EPR. Fahy et al.

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Anti-IgE (Omalizumab)

100%

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(1997) showed that anti-IgE, given twice weekly for 3 weeks, suppressed the early and late asthmatic reaction, as well as the number of circulating eosinophils. Inhibition of the LAR was more marked than that of the early asthmatic response, suggesting either mast cell activation leading to downstream inflammatory effects, or nonmast-cell IgE-dependent events involving IgE (Fc) receptors on antigen-presenting cells (Fig. 24.4). Ong et al. (2005) studied 24 atopic allergic volunteers who received omalizumab or placebo for 12 weeks. Paired intradermal challenges of allergen (30 biological units) and diluent control were administered on nine occasions at 2-week intervals. Compared with placebo, omalizumab-treated patients had a progressive reduction in the LPR, which was significantly greater than the effect on the EPR. In addition, significant reduction of the LPR was reached within 2 weeks of commencing treatment, compared with 8 weeks for the EPR. The study raised the possibility that a major effect of IgE in asthma may be related to its role in antigen presentation to T cells.

Immunotherapy Allergen-specific immunotherapy has been shown to reduce LARs in children after 1 year of therapy, without a significant change in the EPR (Warner et al. 1978). Later studies have shown that immunotherapy reduced both the allergeninduced early and late asthmatic reactions, as well as airway hyperreactivity, and abrogated eosinophil influx and eosinophil cationic protein release after inhaled allergen challenge (Arvidsson et al. 2004). The majority of studies have been conducted to assess the effect of immunotherapy with respect to seasonal allergic rhinitis, rather than using models of allergic disease. Successful immunotherapy has been associated with a reduction of eosinophil accumulation into tissues after local allergen provocation. This has been demonstrated in the nose (Furin et al. 1991), together with a decrease in CD4+ T lymphocytes and an increase in IFN-γ (Durham et al. 1996). Allergen-specific immunotherapy has also been shown to cause a reduction in the size of cutaneous allergen-induced LPRs (Pienkowski et al. 1985; Varney et al. 1993). Although

3 4 Time (h)

5

6

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Before treatment

Per cent of baseline FEV1

Placebo Per cent of baseline FEV1

Fig. 24.4 Effect of a humanized murine monoclonal antibody directed to the FceR1binding domain of human IgE (omalizumab) on allergic airway responses. The effects of 56 days of treatment with anti-IgE in a parallel group, randomized, double-blind, placebocontrolled study of 19 allergic asthmatic subjects was studied. There was partial inhibition of the early reaction and almost total abrogation of the late asthmatic reaction. (From Fahy et al. 1997, with permission.) (See CD-ROM for color version.)


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the mechanisms by which immunotherapy exerts its beneficial effects have still not been fully elucidated, skin biopsies taken during the LPR have shown a reduction in the number of CD4+ T cells and eosinophils. In addition, although the Th2 pattern of cytokines persisted, there was an enhanced expression of Th1 cytokine mRNA (Varney et al. 1993). Similarly, studies that examined the effect of Fel d1-derived peptide immunotherapy have also shown a reduction in the size of the cutaneous and bronchial LPR after local allergen challenge (Oldfield et al. 2002; Alexander et al. 2005a), and this has been associated with an increase in cells with a Th1 cytokine profile (Alexander et al. 2005b), although other studies have shown an increase in serum IL-10 (Oldfield et al. 2002).

Late-phase reaction provoked by allergenderived T-cell peptides A novel method of antigen challenge has been developed that involves administration of allergen-derived T-cell peptide epitopes. It was originally shown that direct T-cell activation through intradermal injection of short peptides derived from the cat allergen Fel d1 could induce an isolated LAR in asthmatic patients with cat allergy (Haselden et al. 1999; Oldfield et al. 2001). These LPRs peaked between 3 and 9 hours after peptide inhalation and had a similar time-course of onset and resolution to LARs induced by whole allergen extract. They are termed “isolated” late reactions as there was no early (immediate) asthmatic reaction, presumably because the peptides were too short to cross-link IgE on mast cells (Fig. 24.5). Like whole antigen, peptides administered by either intradermal injection (Haselden et al. 1999) or by inhalation (Ali et al. 2004), induce LARs in a proportion of atopic asthmatics (responders) but not in others (nonresponders) (Fig. 24.6). Peptides were shown to bind to major histocompatibility complex (MHC) class II molecules, but did not cross-link IgE in a basophil histamine-release assay. The peptide-induced LAR was MHC-restricted in that it only occurred in those individuals with MHC class II able to bind the injected peptides, further supporting a role for T cells in the observed response. Thus, the model has the advantage of

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Early asthmatic reaction

Mast cell

FEV1 (% baseline)

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Allergen

0 –10

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Fig. 24.6 Characteristics of T-cell peptide responders and nonresponders. Changes in FEV1 after peptide challenge (closed circles) in 12 responders (a) and 12 nonresponders (b), as well as the effect of diluent (open triangles).

MHC class II

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2

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APC

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Fig. 24.5 Diagrammatic representation of mechanisms of late asthmatic reactions elicited by (a) whole allergen and (b) allergen-derived peptides. APC, antigen-presenting cell. (See CD-ROM for color version.)

providing information on the T-cell component of allergic airway inflammation, independently of initial mast cell activation. Examples of the different patterns of airway response after challenge with whole allergen or T-cell peptides are shown in Fig. 24.7. T-cell peptide challenge, either by the intradermal route or by inhalation, was not associated with increases in BAL, in a wide range of pharmacologic mediators including histamine, leukotrienes, prostaglandins, and neurotrophins. Also, there were no increases in the numbers of eosinophils, neutrophils, and basophils in the airway wall (Haselden et al. 2001). On the other hand, responders, but not nonresponders, had high expression of the potent vasodilator CGRP (but not neurokinin-A or substance P) in bronchial biopsies and BAL fluid recovered at 6 hours, i.e., when the LAR was well established.

Calcitonin gene-related peptide CGRP is a 37-amino acid peptide resulting from alternative splicing of mRNA from the calcitonin gene (Springer et al. 2003), and is part of the adrenomedullin, calcitonin, amylin family of polypeptides and a potent arterial and venous vasodilator (Brain & Grant 2004). Brain et al. (1985) showed, in experimental animals and humans, that CGRP had a pro-

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longed mode of action when injected into the skin (5–6 hours with as little as 15 pmol). CGRP does not induce permeability per se, but appears to act synergistically with several mediators of inflammation, including histamine, to produce marked and prolonged edema (Brain & Williams 1985). In the airways, in health, CGRP is contained within small sensory nerves, epithelial neuroendocrine cells (NEC), and aggregates of NEC, neuroendocrine bodies. Aoki-Nagase et al. (2002) showed CGRP immunoreactivity throughout the airway epithelium and in the submucosa of mice sensitized and challenged with specific antigen. Furthermore, sensitized CGRP gene-disrupted mice had significant attenuation of both airway hyperreactivity and CGRP expression after antigen challenge. CGRP has also been shown to be synthesized and secreted in vitro by the type II alveolar cell line A549 (Li et al. 2004). There are also many studies showing that the related neuropeptide, substance P, is expressed by several inflammatory cells, including neutrophils and eosinophils (O’Connor et al. 2004). There is controversy regarding the effects of CGRP on bronchial smooth muscle. Earlier claims that it is a potent bronchoconstrictor (Palmer et al. 1987) have not been confirmed and, in any event, this would have been surprising as CGRP increases levels of cAMP in airway smooth muscle cells indicating a relaxing effect. On the other hand, the peptide has been shown to have constrictor effects on damaged (epithelium-denuded) human airways (Springer et al. 2004). To date, there is no convincing evidence that CGRP is overtly expressed in asthma or allergic disease, although previous studies were performed on biopsies from asthmatics obtained at baseline (Howarth et al. 1995; Chanez et al. 1998). Dakhama et al. (2002) suggested that CGRP may have a regulatory role,

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as its administration to sensitized and challenged mice resulted in the normalization of AHR. Nevertheless, these clinical data indicated that changes in the vasculature may be critical in the pathogenesis of late-phase allergic reactions and, if confirmed, will have important implications for understanding mechanisms in bronchial, nasal, and cutaneous late-phase allergic reactions. CGRP immunoreactivity in bronchial biopsies from peptide-challenged asthmatics with LARs were similar to the findings in wild-type mice with positive cells being observed throughout the airway wall, i.e., in epithelial cells, infiltrating inflammatory cells, and airway smooth muscle (Fig. 24.8). Colocalization experiments showed that these were largely CD3+/CD4+ and CD68+ cells (Fig. 24.9). The concept that T cells and macrophages can be recruited in allergic airway disease for the release of the vasoactive peptide CGRP is novel and

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–60 (c)

supports the view that vascular leakage may be a component of subacute (i.e., LAR) as well as acute airway narrowing.

T cells and airway hyperresponsiveness It has also been shown that, in asthmatic subjects, selective activation of allergen-specific T cells by inhalation of allergenderived peptides is sufficient to induce increases in nonspecific AHR, a cardinal feature of asthma, which in turn is accompanied by a predominantly T cell, bronchial mucosal inflammatory response. Thus, in responders but not nonresponders, inhalation of peptides produced increased AHR (measured 7 days after peptide provocation), as well as an LAR that peaked at 6 hours after challenge. The elevated numbers of CD3+, CD4+, and TARC+ cells in biopsies supports the view that increased AHR is linked to T-cell activation and was in keeping with previous animal studies. For example, adoptive transfer experiments in Brown Norway rats have shown that AHR can be transferred by allergen-specific CD4+ T cells (Haczku et al. 1977; Mishima et al. 1998). Moreover, in mice, using a combination of antiT-cell monoclonal antibody, T-cell transfer, and bone marrow transplantation, it was shown that T cells enhanced genetically determined AHR (De Sanctis et al. 1977). Also, depletion of murine CD4+ T lymphocytes prevented antigen-induced AHR and pulmonary eosinophilia (Gavett et al. 1995; Hogan et al. 1998).

Peptide-specific T cells

–60 (a)


Time (h)

It was also found that at baseline, i.e., before challenge, the responders had significantly raised serum cat-specific IgE compared with nonresponders. It was previously shown Fig. 24.7 (left) Different patterns of airway response after challenge with whole allergen or T-cell peptides. In approximately 50% of allergic asthmatic patients, inhalation of whole-allergen extract results in a biphasic reduction in FEV1. The early asthmatic reaction occurs within the first hour. The late asthmatic reaction (LAR) is initiated between 2 and 4 hours and peaks at 6–9 hours. Challenge of allergic asthmatic patients with T-cell peptides through either the intradermal route or the inhaled route results in an isolated LAR. (a) Whole cat dander. An asthmatic patient with cat allergy was challenged with either nebulized saline (open circles) or nebulized whole cat dander allergen extract (filled circles) through the inhaled route. In contrast to saline, challenge with allergen resulted in both early asthmatic response and LAR. A reduction of 20% in FEV1 was arbitrarily considered significant to allow for normal variation in airway caliber in an asthmatic subject (dotted line). (b) Intradermal peptides. An asthmatic patient with cat allergy was challenged with either saline or a mixture of overlapping peptides spanning the majority of the Fel d 1 molecule. Administration of saline or peptide was through intradermal injection in the volar aspect of the forearm. In contrast to the saline control, injection of peptides resulted in an isolated LAR. (c) Peptides by inhalation. An asthmatic patient with cat allergy was challenged with either saline or a mixture of overlapping peptides spanning the majority of the Fel d 1 molecule. Administration of saline or peptide was through inhalation of nebulized material (particle size approximately 5 mm). In contrast to challenge with saline, peptides induced an isolated LAR. (From Larche et al. 2003, with permission.)

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

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CD3


CD68

Fig. 24.9 Bronchial biopsies from T-cell peptide challenge showing colocalization of CGRP immunoreactivity to CD3+ and CD4+ T lymphocytes, neutrophils, eosinophils, and CD68+ macrophages. The left panel shows colocalization of CGRP to CD3 cells (indicated by arrows). Single CGRP cells are green (indicated by white arrows) and single CD3+ cells are red (indicated by red arrows). Double-positive (yellow) CGRP/CD68+ cells are shown in the right panel. (From Kay et al. 2007, with permission.) (See CD-ROM for color version.)

that dual responders, i.e., those who develop early and late asthmatic reactions and subsequent increased AHR after whole allergen challenge, tended to have raised allergenspecific IgE compared with single early responders (Cockcroft et al. 1979). This observation may help to predict why some cat-sensitive individuals, and not others, develop an LAR after the Fel d1 peptide challenge. Thus, elevated allergenspecific serum IgE may reflect high IgE density on airway dendritic cells and/or the frequency of allergen-specific T cells in the airway mucosa. This study supported the view that increased AHR occurs after an LAR, irrespective of whether this is induced by whole allergen (and therefore preceded by a mast cell-dependent early response) or is an isolated peptide-induced LAR as in the present study. In both situations, i.e., whole allergen- or peptide-challenge, the subjects who develop an LAR have elevated serum allergen-specific IgE. Thus, T-cell activation, rather than mast cell activation, may Fig. 24.8 (opposite) Photomicrographs of CGRP immunoreactivity in mucosal bronchial biopsies from peptide-induced late asthmatic reactions. (a) Responder challenged with peptides showing CGRP-immunoreactive cells in basal cells along a length of basement membrane. The epithelium is denuded. (b) Responder challenged with diluent showing basal cells and denuded epithelium but no immunoreactivity. (c) Responder challenged with peptide showing numerous immunoreactive cells within a disrupted epithelium together with positive infiltrating cells below the basement membrane, as well as in association with small blood vessels. (d) A nonresponder after peptide challenge. There are occasional CGRPimmunoreactive cells below the basement membrane. The epithelium

be more crucial for the development of increased AHR as previously observed in animal studies (De Sanctis et al. 1977; Haczku et al. 1977; Mishima et al. 1998).

Downstream events The following scenario is proposed to explain the events occurring after peptide challenge. Inhaled T-cell peptide epitopes bind to MHC class II molecules expressed in the airways, leading to activation of peptide-specific resident effector memory T cells. Activation of these cells enhances local production of TARC from antigen-presenting cells, structural cells, and accessory cells. Elevated TARC expression leads to recruitment and activation of CD3+/CD4+ T cells, production of inflammatory cytokines and, ultimately, increased AHR. As described above, the mechanism of the LPR itself may be related to elevated expression of CGRP in epithelial cells, infiltrating submucosal CD3+/CD4+ cells, and in smooth is intact. (e) Responder challenged with peptide showing numerous infiltrating CGRP-positive inflammatory cells below the basement membrane and also immunoreactivity in association with airway smooth muscle. (f) A responder challenged with diluent showing no immunoreactive submucosal inflammatory cells or CGRP staining in association with airway smooth muscle. (g) A confocal micrograph of a responder challenged with peptide showing CGRP-positive epithelial cells and immunoreactive smooth muscle. (h) A nonresponder challenged with peptide showing no smooth muscle or epithelial immunoreactivity. (See CD-ROM for color version.)

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muscle in responders (but not nonresponders) after peptide challenge.

Concluding comments The concept that mast cell products are largely responsible for EPRs in the lung, nose, and skin is uncontroversial. In addition, mast cell degranulation may contribute to the LPR by means of histamine-induced eotaxin upregulation contributing to eosinophil accumulation (Menzies-Gow et al. 2004). In the skin model, stimulation of mast cells with anaphylactogenic IgE is sufficient to cause both early- and late-phase skin reactions (Dolovich et al. 1973), suggesting that mast cells may cause the cutaneous LPR through as yet undetermined mediators. Antigen-presenting cells take up allergen, process it, and present peptides in the context of MHC class II to CD4+ Th2 cells. This is presumed to cause release of the Th2 cytokines IL-4, IL-5, and IL-13. In humans, IL-5 acts selectively on eosinophils and basophils. By associating with its receptor, IL-5 effects eosinophil growth and differentiation (in particular the terminal differentiation of committed eosinophil precursors) (Yamaguchi et al. 1988; Clutterbuck et al. 1989; Lopez et al. 1992), migration (Warringa et al. 1992), activation and effector function (Kita et al. 1992; Carlson et al. 1993), and survival (Yamaguchi et al. 1991). In allergic disease, eosinophils migrate to sites in a relatively select fashion with up to 100-fold enrichment of eosinophils over neutrophils in tissues. This is a multistep process directed by Th2-cytokine producing T cells (Wardlaw 1999). The first step involves increased production and release of eosinophils from the bone marrow under the influence of IL-5, acting synergistically with specific chemoattractants such as eotaxin (Clutterbuck et al. 1989; Palframan et al. 1998). Next, the target organ vasculature displays increased adhesiveness for eosinophils. The Th2 cytokines IL-4 and IL-13 induce expression of the adhesion molecule VCAM-1 on endothelium during allergic inflammation, which binds eosinophils through very late antigen (VLA)-4, a receptor not expressed on neutrophils, and P-selectin to which eosinophils bind with greater avidity than neutrophils (Symon et al. 1996; Edwards et al. 2000; Woltmann et al. 2000). In addition, IL-4 and IL-13 contribute towards perpetuating IgE production, working in conjunction with CD40 and CD40 ligand. IL-13, in addition, contributes to bronchial hyperresponsiveness. The association between eosinophils and allergy is well established. Local tissue eosinophilia has been described in asthma, atopic rhinitis, and atopic dermatitis. Leukotrienes, which can be produced by eosinophils as well as eosinophil granule products, are found during LPRs in all three tissues after allergen challenge, suggesting that eosinophils actively secrete mediators (Naclerio et al. 1985; Andersson et al. 1989b; Diaz et al. 1989; Reshef et al. 1989; Wardlaw et al. 1989;

538

Zweiman et al. 1991). Disease severity in asthmatics correlated with the number and state of activation of eosinophils in the bronchial mucosa and BAL fluid (Wardlaw et al. 1988). Thus, the general belief over the last 10–15 years has been that the LPR is largely due to the release of eosinophil products. However, as stated above, studies with mepolizumab, a humanized monoclonal anti-IL-5 antibody, produced no appreciable effects on the LAR, AHR, or other clinical outcomes including lung function (Leckie et al. 2000; Flood-Page et al. 2003a). Anti-IL-5 also had no effect on the late-phase skin reaction (Phipps et al. 2004). There is currently an interest in the concept that eosinophils play a role in repair and remodeling processes (Levi-Schaffer et al. 1999) and, indeed, in both the lung and skin models, reduction of eosinophils led to a decrease in markers of airway and skin remodeling (FloodPage et al. 2003b; Phipps et al. 2004). Moreover, Kariyawasam et al. (2007) found that in dual responders with asthma the 24-hour increase in airway wall cellular inflammation, including tissue eosinophilia, resolved by 7 days, whereas the increases in AHR and markers of remodeling persisted. Thus, persistent inflammation does not appear to be necessary for ongoing remodeling and AHR. A further difference between the tissues may lie in T-cell activation and function. Again, as described above, studies using allergen-derived peptides have shown that inhalation of Fel d1-derived peptides by cat allergic asthmatics resulted in isolated LARs (Ali et al. 2004). It has also been shown that intradermal administration of these peptides cause distant LARs in the absence of cutaneous LPRs (Haselden et al. 1999), suggesting that either the T cells in the skin are not able to respond to the peptides or that the mechanisms that cause the LAR are not able to manifest themselves in skin. For example, if the peptide-induced LAR was due to smooth muscle contraction, there would be a lack of response in the skin even if mediators responsible for the LAR were released in the skin. Furthermore, Frew and Kay (1988b) had previously suggested that the skin might lack “activated” T cells in contrast to the airways of asthmatics (Robinson et al. 1993b). It is possible that “inactive” T cells in the skin are unable to generate a response when exposed to the Fel d1-derived peptides. Furthermore, repeat allergen challenges result in increased T-cell accumulation without causing an increase in the size of the allergen-induced LPR (Ong et al. 2005). Taken together with previous Fel d1 peptide studies, this suggests that LARs may have a predominant T-cell component, whereas, in the skin, late reactions are largely mast cellmediated. The common denominator is vascular permeability and vascular leakage, which presumably involve several mediators. This hypothesis is diagrammatically represented in Fig. 24.10. These speculations are in keeping with a number of studies on the role of the vasculature in asthma. These include the demonstration of increases in angiogenic factors, such as VEGF and angiogenin (Hoshino et al. 2001), as well as stromal cell-derived factor 1 in asthmatic airway epithelium

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Ag

Early-phase reaction

MC

(Prominent in late skin reactions) Vascular permeability factors APC

Late-phase reaction

(Prominent in late asthmatic reactions) CD4+ Th2 Eos

Remodeling

IL-5 Bone marrow Eotaxin Fig. 24.10 Hypothesis for mechanisms of late-phase allergic reactions in the skin and airways. It is proposed that eosinophils (Eos), although mobilized by antigen, play a role in downstream events (i.e., remodeling and repair) rather than contribute directly to tissue swelling. Mast cell (MC) permeability factors are prominent in skin late-phase reactions, whereas T-cell permeability factors play a major role in late asthmatic reactions. The role of airway smooth muscle in late asthmatic reactions is unclear. Airway wall edema may be sufficient to account for narrowing. APC, antigen-presenting cell. (See CD-ROM for color version.)

(Hoshino et al. 2003). Furthermore, increases in subepithelial vessels of the airways were observed in newly diagnosed asthmatics, but not in COPD patients or normal control subjects using a high magnification bronchovideoscope (Tanaka et al. 2003). The role of airway smooth muscle in LARs is unclear. Airway wall edema may be sufficient to account for narrowing. Of interest was the observation that inhalation of the loop diuretic furosemide had a protective effect on the allergen-induced early and late asthmatic reaction (Bianco et al. 1989). Therefore, there may be differences in emphasis of the prominent cell type involved in the formation of the LPR in the airways as compared with the skin. Finally, it is important to emphasize that the common feature of late-phase allergic reactions, whatever the organ, is tissue swelling and edema. Increases in our knowledge of mechanisms of vascular permeability and leakage in the context of allergic tissue reactions may well hold the key to understanding the final common pathways involved in this useful model of allergic inflammation.

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Antihistamines F. Estelle R. Simons and Keith J. Simons

Summary In this chapter, the important role played by histamine in human health, in immunomodulation, and in allergic inflammation is reviewed. The four currently known types of histamine receptors are compared and contrasted. The main focus of the chapter is on H1-antihistamines, which are among the most commonly used medications in the world. The molecular basis for H1-antihistamine action as inverse agonists, rather than antagonists or blockers, is described. The first-generation, potentially sedating H1-antihistamines, which have never been optimally investigated in humans, are discussed briefly. The therapeutically more important and well-investigated secondgeneration, nonsedating H1-antihistamines are discussed more extensively. Clinically relevant differences in the pharmacologic profiles of H1-antihistamines are reviewed, with emphasis on their onset of action, potency, and duration of action. H1-antihistamines play an important role in the treatment of allergic rhinitis, allergic conjunctivitis, and urticaria, and their efficacy as well as their limitations in these disorders is described. In contrast to the first-generation H1-antihistamines, the second-generation H1-antihistamines are relatively free from adverse effects, including central nervous system adverse effects and cardiac toxicity, not only when administered in usual doses but also if taken in overdose. The second-generation H1-antihistamines are therefore medications of choice. Use of H1-antihistamines in special populations such as infants, young children, pregnant women, and the elderly is discussed. Clinically advantageous H1-antihistamines developed with the aid of molecular techniques might be available in the future.

Histamine and histamine receptors Histamine is an important natural body constituent that is expressed in central nervous system (CNS) neurons, gastric mucosa parietal cells, mast cells, and basophils, and exerts its


diverse biological effects through four or more types of histamine receptors. H1, H2, H3, and H4 receptors differ in their expression, signal transduction, and function (Table 25.1). All types of histamine receptors are heptahelical transmembrane molecules that transduce extracellular signals through G proteins to intracellular second-messenger systems. The four different types of histamine receptors have distinct intracellular signals. In addition, differences in the affinities of these receptors for histamine are highly decisive for the biological effects of histamine and for drugs that target histamine receptors (Leurs et al. 2002; de Esch et al. 2005; Jongejan et al. 2005; Bongers et al. 2007). All known types of histamine receptors have constitutive activity, which is defined as the ability to trigger downstream events in the absence of ligand binding (Fig. 25.1). The active and inactive states of the receptors exist in equilibrium. Agonists stimulate the active conformation, and inverse agonists (historically termed histamine antagonists) stimulate the inactive conformation. H1 and H2 receptors are widely expressed in the body, in contrast to H3 and H4 receptors. H1-receptor polymorphisms have been described, but not yet optimally studied. In humans, H1 receptors have approximately 45% homology with muscarinic receptors (Leurs et al. 2002). Through the H1 receptor, histamine is involved in cell proliferation and differentiation, hematopoiesis, embryonic development, regeneration, and wound healing. In the CNS, histamine is produced in neurons with cell bodies in the tuberomamillary nucleus of the posterior hypothalamus, from which axons project to the frontal and temporal cortices and other regions of the brain. In this phlyogenetically old neurotransmitter system, histamine is involved in the regulation of basic body functions such as energy and endocrine homeostasis, sleeping, waking, cognition, memory, and anticonvulsant activity (Schneider et al. 2002; Haas & Panula 2003). Targeted disruption of H1 receptors in a murine model results primarily in the impairment of neurologic function such as learning, memory, and locomotion, and in aggressive behavior, although some immunologic abnormalities, including impaired antigen-specific responses of T cells and B cells also occur (Toyota et al. 2002).

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Table 25.1(a) Histamine receptors.

H1 receptor

Year described/

Receptor

Chromosomal

year human

proteins,

localization,

gene cloned

humans

humans

Receptor expression

coupling

Activated intracellular signals

1966/1993

487 amino

3p25, 3p14–p21

Nerve cells, airway and vascular smooth

Gaq/11

Ca2+, protein kinase C, PLC, cGMP,

G protein

PLD, PLA2, NF-kB

muscle, endothelial cells, epithelial cells,

acids, 56 kDa

neutrophils, eosinophils, monocytes/ macrophages, DCs, T and B cells, hepatocytes, chondrocytes H2 receptor

1972/1991

359 amino

5q35.3

Gas

Nerve cells, airway and vascular smooth

Adenylate cyclase, cAMP, c-Fos, cJun, protein kinase C, P70S6K

muscle, endothelial cells, epithelial cells,

acids, 40 kDa

neutrophils, eosinophils, monocytes/ macrophages, DCs, T and B cells, hepatocytes, chondrocytes H3 receptor

1983/1999

445 amino

20q13.33

High expression in histaminergic neurons,

Gi/o

Inhibition of adenocyclase, activation

acids, 70 kDa;

eosinophils, DCs, monocytes, low expression

of PLA2, modulation of the MAPK

splice variants

in peripheral tissues

pathway, activation of the Akt/GSK3b axis, modulation of intracellular Ca2+, and inhibition of Na+/H+ exchanger activity

H4 receptor

1994/2000

390 amino

18q11.2

acids, kDa n/a

Gi/os

High expression on bone marrow and

Enhanced Ca2+, inhibition of cAMP

peripheral hematopoietic cells, neutrophils, eosinophils, DCs, T cells, basophils, mast cells

Table 25.1(b) Histamine receptors (contd). H1 receptor

H2 receptor

H3 receptor

H4 receptor

Histamine

↑ pruritus, ↑ pain, ↑ vasodilation

↑ gastric acid secretion

Prevents excessive

Differentiation of

function,

↑ vascular permeability

↑ vascular permeability

bronchoconstriction

myeloblasts and

general

↑ hypotension, flushing, headache, tachycardia,

↑ hypotension, flushing, headache,

Mediates pruritus (no mast

promyelocytes

bronchoconstriction, stimulation of airway vagal

tachycardia

cell involvement)

Mediates pruritus (no

afferent nerves and cough receptors

↑ chronotropic and ↑ inotropic activity,

Nasal congestion

mast cell involvement)

↓ atrioventricular node conduction time

bronchodilation

Nasal congestion

↑ mucus production (airway) Histamine

↑ release of histamine and other mediators

↓ eosinophil and neutrophil chemotaxis

Probably involved in control

↑ calcium flux in human

function

↑ cell adhesion molecule expression and

↓ IL-12 by dendritic cells

of neurogenic inflammation

eosinophils

in allergic

chemotaxis of eosinophils and neutrophils

↑ IL-10 and development of Th2 or

through local neuron–mast

↑ eosinophil chemotaxis

inflammation

↑ APC capacity, costimulatory activity on B cells

tolerance-inducing DCs

cell feedback loops

↑ IL-16 production

and immune

↑ cellular immunity (Th1)

↑ humoral immunity

↑ proinflammatory activity

(H2 receptor also involved)

modulation

↑ IFN-g, autoimmunity

↓ cellular immunity

↑ APC capacity

↓ humoral immunity and IgE production

suppresses Th2 cells and cytokines indirect role in allergy, autoimmunity, malignancy, graft rejection Presynaptic heteroreceptor

Histamine

Sleep/wakefulness, food intake, thermal

function

regulation

↓ histamine, dopamine,

in the CNS

Emotions/aggressive behavior, locomotion,

serotonin, norepinephrine,

memory, learning

and acetylcholine release

Neuroendocrine

To be defined

Inverse agonists

> 40, including cetirizine, desloratadine,

Cimetidine, famotidine, nizatidine, and

Medications in development

Medications in

(formerly called

fexofenadine, levocetirizine, and loratadine for

ranitidine for peptic ulcer and related

for treatment of narcolepsy,

development for allergic

antagonists)

allergic rhinoconjunctivitis and urticaria

disorders

dementia, schizophrenia,

rhinitis treatment

and other CNS disorders APC, antigen-presenting cell; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DCs, dendritic cells; IFN, interferon; IL, interleukin; MAPK, mitogen-activated protein kinase; n/a, information not available; NF-kB, nuclear factor kB; NOS, nitric oxide synthase; PL, phospholipase. The information about histamine receptors and their inverse agonists is based on in vitro studies, studies in animal models (H1, H2, H3, and H4 receptor-deficient mice, H1/H2receptor-deficient mice and histidine decarboxylase-deficient mice), and on studies in humans. Peripheral and central H1 receptors do not differ, although isoforms of H1 receptors, including species isoforms, exist. Receptor binding varies depending on the system used to study it and the inverse agonist being investigated; for example, in the Chinese hamster ovary cell membrane model, the Ki (nmol/L) of second-generation H1-antihistamines in current use ranges from 0.87 (desloratadine) to 175 (fexofenadine).

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Extracellular

Extracellular

Extracellular

Effect of agonist (histamine)

Inactive

Effect of inverse agonist (antihistamine)

Inactive

Inactive

Active (a)

Intracellular

Active (b)

Antihistamines

Intracellular

Fig. 25.1 The histamine H1 receptor: a simplified two-state model. (a) The inactive state of the histamine H1 receptor is in equilibrium with the active state. (b) An agonist has a preferential affinity for the active state and stabilizes the H1 receptor in this conformation, consequently causing a shift in the equilibrium toward the active state. (c) An inverse agonist such as an H1-antihistamine has a preferential affinity for the inactive state,

Active (c)

stabilizes the receptor in this conformation, and causes a shift in the equilibrium toward the inactive state. All H1-antihistamines described to date function as inverse agonists. Intracellular and extracellular are defined in relation to the cell membrane. (From Leurs et al. 2002, with permission.) (See CD-ROM for color version.)

Histamine and immunomodulation Histamine affects chronic inflammation through at least four distinct receptor types on macrophages, dendritic cells, T lymphocytes, B lymphocytes, epithelial cells, endothelial cells, and other cells such as eosinophils, basophils, fibroblasts, and keratinocytes (Traidl-Hoffmann et al. 2006). In this complex system, the expression of the receptor types is influenced by the microenvironment, and differs according to the stage of cell differentiation (Triggiani et al. 2007). Histamine regulates a number of essential events in the immune response, including cell proliferation, cytokine production, and expression of cell adhesion molecules and major histocompatibility complex (MHC) class II antigens (Jutel et al. 2006). Through the H1 receptor and the H3 receptor, histamine induces proinflammatory activity and increased antigenpresenting cell capacity. Through the H2 receptor, it plays a suppressive role on monocytes and monocyte-derived dendritic cells. Th1 cells show predominant, but not exclusive, expression of the histamine H1 receptor, and histamine induces increased proliferation of Th1 cells, as well as interferon-γ production. On Th2 cells, the expression of the histamine H2 receptor predominates and, through it, histamine acts as a negative regulator of proliferation, and of interleukin (IL)-4 and IL-13 production. Histamine enhances Th1-type responses through the histamine H1 receptor, whereas both Th1- and Th2-type responses are negatively regulated through the histamine H2 receptor. These distinct effects suggest roles for the histamine H1 receptor and the histamine H2 receptor on T cells for autoimmunity and for peripheral tolerance, respectively (Akdis & Simons 2006) (Fig. 25.2).

Intracellular

HR2 Induction of IL-10 Suppression of IL-12 Th2 or tolerance Inducing DC

High HR2 Low HR1 Suppressed Th2 cytokines tolerance

Th2

HR2: induction of humoral immunity and suppression of cellular immunity. HR2-deficient mice show suppressed specific IgE

Monocyte dendritic cell HR1-4 DC

HR1/HR3 proinflammatory activity, increased APC capacity

Histamine

Th1

High HR1 Low HR2 Increased IFN-g autoimmunity

B cell

HR1: blocking of humoral immunity and induction of cellular immunity. HR1-deficient mice show increased specific IgE

Fig. 25.2 Histamine plays an important role in allergic inflammation. In lymphatic organs and subepithelial tissues, it regulates monocytes and dendritic cells that express all four known subtypes of histamine receptors, and T cells which express histamine H1 receptors and histamine H2 receptors, as well as B cells and other cells. APC, anti-presenting cell; DC, dendritic cell. (From Akdis & Simons 2006, with permission.) (See CD-ROM for color version.)

Histamine also modulates antibody production, as a costimulatory receptor on B cells. Histamine H1 receptors predominantly expressed on Th1 cells may block humoral immune responses by enhancing the Th1 cytokine response. In contrast, histamine enhances humoral immune responses through the histamine H2 receptor. Allergen-specific IgE production

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is differentially regulated in histamine H1 receptor and histamine H2 receptor-deficient mice. Histamine H1 receptordeleted mice show increased allergen-specific IgE production, in contrast to histamine H2 receptor-deleted mice, which show suppressed IgE production. Bphs, a non-MHC-linked gene involved in the susceptibility to many autoimmune diseases, has been identified as the histamine H1-receptor gene in mice (Jutel et al. 2006).

Histamine in allergic inflammation Histamine plays a key role in the intricate network of cellular events described as allergic inflammation (MacGlashan 2003). Along with preformed mediators of inflammation such as tryptase, as well as newly generated leukotrienes, prostaglandins, and other mediators, it is released from the granules of mast cells and basophils after cross-linking of surface high-affinity IgE receptors by allergen. Less commonly, it is released from these cells through mechanisms that are independent of IgE and the IgE receptor. Although histamine is less potent than some other mediators such as leukotrienes, after allergen challenge it is found locally in relatively large microgram quantities per one million cells. In contrast, leukotrienes are found in picogram quantities per one million cells. In the early allergic response, most of the effects of histamine occur through H1 receptors; these include itching and sneezing due to neural reflexes, edema due to vasodilation and increased permeability of the vascular endothelium, and bronchoconstriction due to smooth muscle contraction. In addition to its role in the early allergic response, histamine plays an important role in the late allergic response by activating various signal cascades, including NF-κB, followed by the production of proinflammatory cytokines such as IL-4 and IL-13, the expression of cell adhesion molecules and class II antigens, and chemotaxis of eosinophils and other cells. The clinical significance of the complex interrelationships of histamine, its receptors, and other G-protein coupled receptors remains to be elucidated. In anaphylaxis, some symptoms such as hypotension, tachycardia, flushing, and headache occur through both H1 and H2 receptors in the vasculature. Cutaneous itching in atopic dermatitis and nasal congestion in allergic rhinitis may occur through H3 or H4 receptors, as well as through H1 receptors. There are also some hints that H1-antihistamines subtly modulate the allergic response by affecting the balance between Th1, Th2, and Treg cells when suppressing the inflammatory cell accumulation. For example, in humans, H1-antihistamine premedication during the initial dose escalation phase of allergen-specific immunotherapy may improve the longterm efficacy of treatment (Muller et al. 2001; Golightly & Greos 2005; Bryce et al. 2006; Caproni et al. 2006; Petecchia et al. 2006).

554

Clinical pharmacology During the past decade, understanding of the molecular mechanisms by which H1-antihistamines interact with the histamine H1 receptor has increased greatly. It is now known that they do not “block” these receptors. Rather, all H1antihistamines in use function as inverse agonists that have a preferential affinity for the inactive state of histamine H1 receptors. They stabilize the receptors in this conformation, with a shift in equilibrium toward the inactive state and consequent downregulation of acute and chronic inflammation (Leurs et al. 2002). Traditionally, H1-antihistamines have been classified into six chemical groups: the ethanolamines, ethylenediamines, alkylamines, piperazines, piperidines, and phenothiazines. Now, the more commonly used classification is a functional one in which H1-antihistamines are divided into firstgeneration, potentially sedating agents and second-generation, relatively nonsedating H1-antihistamines (Simons 2003). The term third-generation has been used to market some newer H1-antihistamines; however, this designation should be reserved for clinically advantageous H1-antihistamines designed with the use of molecular techniques, and no such H1-antihistamines are currently available (Holgate et al. 2003). Many second-generation H1-antihistamines have been identified by screening and structural modification of preexisting medications in the class. For example, acrivastine is structurally similar to triprolidine; cetirizine is a metabolite of hydroxyzine; levocetirizine is the active R-enantiomer of cetirizine; desloratadine is a metabolite of loratadine; and fexofenadine is a metabolite of terfenadine (Simons 2003; Hair & Scott 2006). New H1-antihistamines continue to be developed and introduced for clinical use (Corcostegui et al. 2006; Keam & Plosker 2007).

Pharmacokinetics Pharmacokinetics is defined as the study of the absorption, distribution, metabolism, and elimination of medications. The pharmacokinetics of the first-generation H1-antihistamines have never been optimally investigated, even in healthy adults or adults with allergic diseases, let alone in special groups such as children, the elderly, or patients with hepatic or renal dysfunction. Clearance rates and terminal elimination half-life values are known for a few of these medications, but not for all of them. Although all the first-generation H1antihistamines are metabolized by the hepatic cytochrome P450 (CYP450) system, and interactions with other drugs, foods, or herbal products potentially occur, few interaction studies of these medications have been performed (Table 25.2). In contrast, the pharmacokinetics of most of the secondgeneration H1-antihistamines have been extensively studied. After oral administration, peak plasma concentrations of

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Antihistamines

Table 25.2(a) Pharmacokinetics and pharmacodynamics of oral H1-antihistamines differ in healthy young adults.

H1-antihistamine (metabolite)

tmax (hours) after single dose§

Terminal elimination half-life (hours)

Elimination unchanged in the urine/ feces (%)

Clinically relevant drug/drug interactions*

Onset/ duration of action† (hours)

First-generation Chlorpheniramine‡

2.8 ± 0.8

27.9 ± 8.7

n/a

Possible

3/24

Diphenhydramine‡

1.7 ± 1.0

9.2 ± 2.5

n/a

Possible

2/12

Doxepin‡ Hydroxyzine‡

2 2.1 ± 0.4

13 20.0 ± 4.1

n/a n/a

Possible Possible

n/a 2/24

Second-generation Acrivastine Cetirizine Desloratadine Ebastine (carebastine) Fexofenadine*

1.4 ± 0.4 1.0 ± 0.5 1–3 (2.6–5.7) 2.6

1.4–3.1 6.5–10 27 (10.3–19.3) 14.4

59/0 60/10 0 (75–95)/0 12/80

Unlikely Unlikely Unlikely n/a Unlikely

1/8 1/≥ 24 2/≥ 24 2/≥ 24 2/24

Levocetirizine Loratadine (descarboethoxyloratadine) Mizolastine Rupatadine

0.8 ± 0.5 1.2 ± 0.3 (1.5 ± 0.7) 1.5 0.75–1.0

7 ± 1.5 7.8 ± 4.2 (24 ± 9.8) 12.9 6 (4.3–13.0)

86/13 trace

Unlikely Unlikely

0.5/0 n/a

n/a Proven

Usual adult dose

4 mg t.i.d.–q.i.d. 12 mg (SR) b.i.d.† 25–50 mg t.i.d.–q.i.d. or h.s.† 25–50 mg t.i.d. or h.s.† 25–50 mg t.i.d. or h.s.†

Population in which dose adjustment may be required

G G, H H H

n/a G, R, H R, H R, H R

1/> 24 2/24

8 mg t.i.d. 5–10 mg daily 5 mg daily 10–20 mg daily 60 mg b.i.d. or 120 mg or 180 mg daily 5 mg daily 10 mg daily

1/24 2/24

10 mg daily 10 mg daily

n/a G, R, H

R, H H

The legend for Table 25.2(a) and Table 25.2(b) appears on p. 556.

these medications are reached within 1–2 hours. Terminal elimination half-life values range from about 2 hours for acrivastine to 27 hours for desloratadine. Protein binding of the second-generation H1-antihistamines ranges from 60% (fexofenadine) to 99% (levocetirizine). Cetirizine and levocetirizine are eliminated largely unchanged in the urine, and fexofenadine is eliminated mostly unchanged in the feces (Simons 2003, 2004; Molimard et al. 2004; Golightly & Greos 2005; Hair & Scott 2006; Keam & Plosker 2007). The pharmacokinetics of many of the second-generation H1-antihistamines have been studied in special groups such as children, the elderly, and patients with hepatic or renal dysfunction. For example, in very young children, traditional pharmacokinetic studies and population pharmacokinetic studies have been performed for cetirizine, desloratadine, levocetirizine, and loratadine (Salmun et al. 2000; Simons 2003; Cranswick et al. 2005; Simons & Simons 2005; Simons et al. 2005; Gupta 2007; Gupta et al. 2007). Clinically relevant pharmacokinetic interactions between second-generation H1-antihistamines and other drugs, foods, or herbal products, are few (Zhou et al. 2004; Bressler 2006; Prenner et al. 2006). For example, although some individuals have decreased ability to convert desloratadine to 3-hydroxy-desloratadine, the major metabolite, clinically relevant drug accumulation does not occur. It is recommended, however, that fexofen-

adine not be administered within 15 min after the ingestion of antacids that contain aluminum and magnesium, which potentially decrease its absorption (Golightly & Greos 2005). Rupatadine is metabolized by the hepatic cytochrome P450 system, and significant drug interactions between rupatadine and agents such as ketoconazole, erythromycin, and grapefruit juice that inhibit CYP3A4 activity have been reported (Picado 2006; Keam & Plosker 2007).

Pharmacodynamics Pharmacodynamics involves the study of the onset, amount, and duration of action of medications in relationship to their plasma concentrations. The pharmacodynamics of H1-antihistamines are readily studied using inhibition of the histamine-induced wheal and flare (erythema), a standardized biological assay of peripheral H1-activity. H1-antihistamines decrease wheal size by decreasing the vascular permeability and leakage of plasma proteins. They decrease flare (erythema) size by decreasing vasodilation caused by the histamineinduced axon reflex. Less commonly, suppression of the allergen-, compound 48/80-, or codeine-induced wheals and flares (erythema) is used in this bioassay. There are few pharmacodynamic studies of the firstgeneration H1-antihistamines. In contrast, the pharmacodynamics of most of the second-generation H1-antihistamines

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Table 25.2(b) Pharmacokinetics and pharmacodynamics of H1-antihistamines for intranasal/ophthalmic use.

H1-antihistamine (metabolite)

tmax (hours) after single dose§

Topical intranasal/ophthalmic Azelastine** 5.3 ± 1.6 (desmethylazelastine) (20.5)

Terminal elimination half-life (hours)

Elimination unchanged in the urine/ feces (%)

Clinically relevant drug/drug interactions

Onset/ duration of action¶ (hours)

22–27.6 (54 ± 15)

2(3)/0

No

0.5/12

0.137 mg/nasal spray, 1–2 sprays b.i.d.; 0.5% ophthalmic solution, 1 drop b.i.d.

n/a

Usual adult dose§

Population in which dose adjustment may be required

Emedastine**

1.4 ± 0.5

7

6.7/n/a

No

0.25/12

0.05% ophthalmic solution, 1 drop q.i.d.

n/a

Epinastine**

2–3

6.5

25/70

No

0.1/12

0.05% ophthalmic solution, 1 drop b.i.d.

n/a

Ketotifen**

2–4

20–22

60–70/ 30–40

No

0.25/12

0.025% ophthalmic solution, 1 drop b.i.d. (q8–12h)

n/a

Levocabastine**

1–2

35–40

65–70/20

No

0.25/12

50 mg/nasal spray 2 sprays b.i.d.; or 0.05% ophthalmic solution, 1 drop t.i.d.–q.i.d.

n/a

Olopatadine**

0.5–2

7.1–9.4

59–73/n/a

No

0.25/12

0.1% ophthalmic solution, 1 drop b.i.d. (q6–8h); 0.2% ophthalmic solution, 1 drop once daily

n/a

Results are expressed as mean ± standard deviation, unless otherwise indicated. b.i.d. twice daily; G, geriatric; H, hepatic impairment; n/a, information not available or incomplete; q.i.d., four times daily; R, renal impairment; t.i.d., three times daily. * Drug interactions: fexofenadine should not be administered within 15 min of ingestion of aluminum- and magnesium-containing antacids, which decrease its absorption; although grapefruit juice and rifampin may also decrease its absorption, and erythromycin, ketoconazole, and verapamil may increase its absorption, fexofenadine has a very wide benefit-to-risk ratio and these effects are unlikely to have clinical significance; nevertheless, effectiveness should be monitored. † Onset/duration of action is based on wheal and flare studies. ‡ Five or six decades ago when many of the first-generation H1-antihistamines were introduced, pharmacokinetic and pharmacodynamic studies were not required by regulatory agencies. They have subsequently been performed for some of these drugs. Empirical dosage regimens persist; for example, the manufacturers’ recommended diphenhydramine dose for allergic rhinitis is 25–50 mg q4–6h and the diphenhydramine dose for insomnia is 25–50 mg at bedtime. The use of sustained-action formulations persists despite the long terminal elimination half-life values identified for medications such as chlorpheniramine. § Time from oral intake to peak plasma concentration. ¶ Intranasal and ophthalmic H1-antihistamines: onset and duration of action, and usual adult dose refers to topical application; for nasal use, the doses refer to sprays in each nostril, and for ophthalmic use the doses refer to one drop in each eye. ** Intranasal and ophthalmic H1-antihistamines: pharmacokinetic parameters after oral administration.

have been well studied (Murdoch et al. 2003; Simons 2003, 2004; Curran et al. 2004; Golightly & Greos 2005; Hair & Scott 2006; Keam & Plosker 2007). In healthy and allergic individuals, all H1-antihistamines inhibit the histamine-induced wheal and flare to some extent. The magnitude of the effect, the time to peak effect, and the duration of the effect are medication and dose-related (Popov et al. 2006). For a few

556

H1-antihistamines such as cetirizine and fexofenadine, wheal and flare (erythema) suppression has been shown to correlate with H1-antihistamine concentrations in the skin (Simons et al. 2002). For other H1 receptors such as levocetirizine, suppression has been shown to correlate with H1-receptor occupancy by free (unbound) H1-antihistamine (Simons KJ et al. 2007).

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The onset of action ranges from about 1 hour for cetirizine and levocetirizine to about 2 hours for fexofenadine, desloratadine, and loratadine. The duration of action for most of the orally administered second-generation H1-antihistamines is at least 24 hours, facilitating once-daily dosing. Tolerance to the suppressive effect on skin test reactivity to allergens does not occur, as documented in rigorously controlled, double-blind studies of several months duration. After discontinuing a second-generation H1-antihistamine that has been taken regularly for 1 week or longer, residual suppression of skin test reactivity to histamine and to allergens varies with the H1-antihistamine (Simons 2003, 2004). Available evidence suggests that residual effects of a short course of loratadine or desloratadine last for 1 day, those of fexofenadine last for 2 days, and those of cetirizine and levocetirizine last for 3– 4 days. Some systemic absorption of intranasal and ophthalmic H1-antihistamines such as azelastine, emedastine, epinastine, levocabastine, and olopatadine occurs, and may be associated with transient suppression of skin-test reactivity. Regardless of the terminal elimination half-life of these medications, which ranges from about 7 hours (emedastine, epinastine, olopatadine) to 40 (levocabastine) hours, they are administered at 6–12 hour intervals because of washout from the nasal mucosa or conjunctivae. No dose adjustments are required in special populations (Bielory et al. 2005). In individuals with allergic rhinoconjunctivitis, pharmacodynamic studies of orally or topically active H1-antihistamines, with a focus on onset of action, can be conducted using nasal or ophthalmic challenge tests. Challenges with histamine or allergen can be administered on an individual basis, or groups of individuals can be challenged with allergen, either in a purpose-built chamber or room (Day et al. 2006; Horak et al. 2006; Stuebner et al. 2006), or in an outdoor setting when ambient seasonal pollen counts are high. Doses and dose intervals for the second-generation H1antihistamines are therefore based on objective information obtained from pharmacokinetic and pharmacodynamic studies. Moreover, modified dose regimens have been defined, where required, in infants, children, elderly persons, patients with hepatic or renal dysfunction, or those taking other medications concurrently (Table 25.2) (Simons 2003; Golightly & Greos 2005; Hair & Scott 2006). For some H1-antihistamines, such as cetirizine and levocetirizine, wheal and flare inhibition correlates with relief of symptoms of allergic rhinitis, beginning at 1 hour and peaking at 5–7 hours after the dose, several hours after the peak plasma concentration. H1-antihistamine effects persist even when plasma concentrations become undetectable, probably due to high plasma/tissue concentration ratios, as determined by measurement of H1-antihistamine concentrations in skin biopsy specimens, and for some medications, the presence of active metabolites (Simons et al. 2002). For other H1antihistamines, such as loratadine and desloratadine, the

Antihistamines

pharmacodynamics of the wheal and flare (erythema) response do not seem to predict efficacy in allergic rhinitis. In this model, desloratadine 10 mg and levocetirizine 1.25 mg provide equivalent suppression 4 hours after dosing, although both of these medications are administered in a dose of 5 mg daily for allergic rhinoconjunctivitis or urticaria treatment (Popov et al. 2006).

Efficacy H1-antihistamines prevent and relieve symptoms in seasonal/ intermittent and perennial/persistent allergic rhinitis, allergic conjunctivitis, and acute and chronic urticaria. The rationale for their use in these and other allergic disorders is based on the following: (i) local challenge with histamine reproduces many of the acute symptoms of these diseases; (ii) challenge with allergen or other relevant stimulus can result in local or systemic increases in histamine concentrations; (iii) during disease activity, histamine concentrations may increase locally or even systemically; (iv) pretreatment with an H1antihistamine prevents or relieves symptoms after challenge with histamine or with allergen (Simons 2003). H1-antihistamines sometimes relieve symptoms incompletely in allergic disorders because, in addition to histamine, leukotrienes and other mast cell and basophil products also play a role in allergic inflammation. The dose–response curve for efficacy of H1-antihistamines in allergic rhinoconjunctivitis and urticaria is relatively flat, i.e., doubling or trebling of the manufacturers’ recommended dose does not necessarily result in a corresponding two- or three-fold increase in efficacy. In chronic/perennial/persistent allergic diseases, H1antihistamines are best taken on a regular basis rather than as needed, in order to prevent allergic inflammation and associated symptoms. Tolerance to clinical efficacy does not develop. First-generation H1-antihistamines have not been optimally studied in any allergic disease, and the evidence base for their use is largely empirical. In contrast, the use of second-generation H1-antihistamines for relief of symptoms in seasonal/intermittent, and perennial/persistent allergic rhinoconjunctivitis, and in chronic urticaria is supported by a strong evidence base consisting of randomized, doublemasked, placebo-controlled clinical trials lasting weeks or months. In these trials, there are adequate numbers of participants enrolled, and data on attrition and adherence are reported. Second-generation H1-antihistamines are therefore medications of choice in these diseases (Murdoch et al. 2003; Curran et al. 2004; Simons 2004; van Cauwenberge et al. 2004; Golightly & Greos 2005; Hair & Scott 2006; Keam & Plosker 2007).

Allergic rhinoconjunctivitis In allergic rhinoconjunctivitis, H1-antihistamines prevent and relieve the sneezing, nasal and conjunctival itching, rhinorrhea, tearing, and conjunctival erythema of the early response to allergen (Fig. 25.3). They also have a small beneficial effect

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Calcium-ion channels

H1-receptor Nuclear factor-kB Decreased allergic inflammation, itching, sneezing, rhinorrhea, and whealing

Decreased antigen presentation, expression of cell-adhesion molecules, chemotaxis, and proinflammatory cytokines

Decreased mediator release

Potential adverse effects of H1-antihistamines

H1-receptor

Muscarinic receptor

a-adrenergic receptor

Serotonin receptor

IKr and other cardiac-ion channels

Decreased neurotransmission in the central nervous system, increased sedation, decreased cognitive and psychomotor performance, and increased appetite

Increased dry mouth, urinary retention, and sinus tachycardia

Hypotension, dizziness, and reflex tachycardia

Increased appetite

Prolonged QT intervals, sometimes resulting in ventricular arrhythmias

Fig. 25.3 Benefits and potential adverse effects of H1-antihistamines. H1-antihistamines downregulate allergic inflammation mainly through the H1 receptor. First-generation H1-antihistamines potentially cause adverse effects not only through H1 receptors in the central nervous system, but also through the muscarinic, a-adrenergic, serotonin receptors, and cardiac ion channels. (From Simons 2004, with permission.) (See CD-ROM for color version.)

on the nasal blockage that characterizes the late allergic response (Plaut & Valentine 2005). They improve quality of life (Juniper et al. 2005). Second-generation H1-antihistamines such as cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, mizolastine, and rupatadine have greater efficacy than placebo, as documented in well-designed clinical trials (Simons et al. 2003a; Golightly & Greos 2005; Pasquali et al. 2006; Canonica et al. 2007). There are relatively few published clinical trials in which their efficacy relative to each other, or to first-generation H1-antihistamines has been investigated; however, when such trials have been conducted, no clear overall superior efficacy of one H1antihistamine over another has been documented consistently (van Cauwenberge & Juniper 2000; Berger et al. 2006). When orally administered H1-antihistamines are compared with intranasal or ophthalmic H1-antihistamines, the latter are found to have a more rapid onset of action; for example, 15 min for topical azelastine versus 150 min for orally administered desloratadine (Horak et al. 2006); however, they require administration several times daily, in contrast to once-daily dosing for the orally administered second-generation H1antihistamines (Bielory et al. 2005). Few H1-antihistamines have been optimally studied in children with allergic rhinoconjunctivitis (Wahn et al. 2003).

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Selection of an H1-antihistamine for an individual should be based on his/her preference for a particular H1-antihistamine formulation, route of administration, or dose regimen, and above all, on considerations of benefits versus risk of adverse effects (Blaiss 2006; Raphael et al. 2006). More head-to-head comparisons among the H1-antihistamines are needed. In allergic rhinitis, second-generation H1-antihistamines have comparable efficacy to intranasal cromolyn or nedocromil, and to the leukotriene modifier montelukast, although in seasonal and persistent allergic rhinitis the combination of desloratadine or levocetirizine with montelukast may be more effective than monotherapy with any of these agents (Ciebiada et al. 2006). In order to provide increased relief of nasal congestion, H1-antihistamines are sometimes marketed in fixed-dose combinations with pseudoephedrine or other decongestants. H1-antihistamines are less effective than intranasal glucocorticoids, especially for relief of nasal congestion (Saengpanich et al. 2003; Barnes et al. 2006). In individuals with allergic rhinoconjunctivitis whose symptoms are primarily conjunctival, H1-antihistamines administered topically to the affected areas are the medications of choice, not only for their antihistaminic effects, but also for their antiallergic and antiinflammatory effects, and their rapid onset of action (3–15 min) (Bielory et al. 2005).

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H1-antihistamines, whether topically or orally administered, have a more favorable therapeutic index than any other class of medications used in the treatment of allergic conjunctivitis (Treatment Guidelines from The Medical Letter 2007).

Other airway disorders H1-antihistamines are widely used to relieve symptoms of upper respiratory tract infections, otitis media, sinusitis, and nonspecific cough; however, the published evidence does not support their use in these disorders (De Sutter et al. 2003; Simons 2003; Chang et al. 2006; Griffin et al. 2006). Pretreatment with an H1-antihistamine provides some protection against bronchospasm induced in the clinical laboratory by histamine, exercise, hyperventilation, cold, dry air, hypertonic or hypotonic saline, distilled water, adenosine-5 monophosphate, or allergen (Wilson 2006). Although H1antihistamines do no harm in asthma, they are not recommended, as such, for the treatment of asthma. While they are reported to decrease concurrent symptoms of allergic rhinitis and asthma significantly in individuals who have allergic inflammation throughout the upper and lower airways, their effect on allergic rhinitis is greater than their effect on asthma (Bousquet et al. 2001; Baena-Cagnani et al. 2003). In an 18month study in very young children with atopic dermatitis and house-dust mite or grass sensitization who were at risk for development of asthma, regular administration of cetirizine was reported to delay asthma onset (Warner et al. 2001); however, in a population of more highly atopic young children, this observation was not confirmed with levocetirizine.

Urticaria H1-antihistamines are efficacious in acute urticaria, defined as lasting < 6 weeks, and chronic urticaria, defined as lasting ≥ 6 weeks, including physical urticarias such as cholinergic, cold, and pressure-induced urticarias. They are not effective in urticarial vasculitis or non-allergic angioedema. They decrease itching and reduce the number, size, and duration of wheals and erythema (flares), and significantly improve quality of life. In acute urticaria, both first- and secondgeneration H1-antihistamines are widely used, although there is not much published evidence in support of their efficacy in this disorder (Zuberbier et al. 2006). In two different large, randomized, double-masked, placebo-controlled studies in young atopic children in which efficacy in acute urticaria was a planned secondary outcome, cetirizine and levocetirizine effectively prevented and treated the hives (Simons, F.E.R. et al. 2001, 2007a). The first-generation H1-antihistamines remain in widespread use for chronic urticaria, despite a paucity of efficacy studies, and concerns about potential adverse effects, which may or may not be clinically apparent. In contrast, the secondgeneration H1-antihistamines cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine, mizolastine, and rupatadine have been well studied in chronic urticaria

Antihistamines

(Simons 2003, 2004; Kozel & Sabroe 2004; Golightly & Greos 2005; Hair & Scott 2006; Kapp & Pichler 2006; Keam & Plosker 2007; Nettis et al. 2006; Zuberbier et al. 2006; Dubertret et al. 2007; Gimenez-Arnau et al. 2007; Ortonne et al. 2007). In chronic urticaria, although use of two different H1antihistamines on the same day (specifically, a nonsedating medication in the morning and a sedating medication at bedtime) has been recommended (Kaplan 2002), this strategy has not been prospectively tested in randomized, doublemasked, placebo-controlled trials. In individuals with chronic urticaria that is not relieved by an H1-antihistamine alone, an H2-antihistamine such as cimetidine administered concurrently with an H1-antihistamine may give added relief. This treatment strategy is worth a 3–4 week trial (Kaplan 2002). Simultaneous administration of the H1-antihistamine desloratadine with the leukotriene D4-antagonist montelukast does not appear to offer any consistent therapeutic advantage over desloratadine alone (Di Lorenzo et al. 2004). In some individuals, chronic urticaria and associated pruritus can be difficult to treat. If H1-antihistamine treatment fails, they may require an immunomodulator such as an oral corticosteroid, cyclosporin, sulfasalazine, hydroxychloroquine, mycophenolate, or tacrolimus; however, all these medications have a more limited evidence base for efficacy than H1-antihistamines in chronic urticaria, and most of them potentially have severe adverse effects (Kozel & Sabroe 2004; Treatment Guidelines from The Medical Letter 2007).

Atopic dermatitis and other skin disorders The evidence that H1-antihistamines relieve itch in atopic dermatitis, and provide a glucocorticoid-sparing effect in atopic dermatitis is not convincing (Klein & Clark 1999). In this disorder, histamine may act as a pruritogen through H4 receptors on peripheral neurons (Dunford et al. 2007); in addition, cytokines such as IL-31, and other agents, may play an important pruritogenic role (Boguniewicz et al. 2006). The use of H1-antihistamines to relieve symptoms in individuals with mastocytosis, or to prevent itchy local allergic reactions to bites of insects such as mosquitoes, is supported by small clinical trials (Karppinen et al. 2006).

Anaphylaxis A recent Cochrane collaboration review of 2070 studies of H1-antihistamines in anaphylaxis did not reveal even one study that provided high-quality evidence for or against the use of H1-antihistamines in this disease (Sheikh et al. 2007). Individuals who experience anaphylaxis in the community should not depend on an oral H1-antihistamine for first-aid treatment, because H1-antihistamines are not life-saving, and do not relieve respiratory, cardiovascular, or gastrointestinal symptoms, although they decrease itch and hives. After administration by mouth, absorption and onset of action of H1-antihistamines takes at least 1–2 hours. First-generation H1-antihistamines, such as diphenhydramine, potentially

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cause somnolence and impaired recognition of anaphylaxis symptoms. In many individuals, improvement in anaphylaxis attributed to H1-antihistamine treatment likely reflects spontaneous improvement due to endogenous epinephrine, endothelin, or angiotensin II (Simons 2006).

CNS and vestibular system disorders: the unfavorable therapeutic index of first-generation H1-antihistamines Although not medications of choice, the first-generation H1-antihistamines diphenhydramine, doxylamine, and pyrilamine are the most widely used medications in the world for insomnia relief. They are also still used for treatment of akathisia, serotonin syndrome, anxiety, and other conditions affecting the CNS. Diphenhydramine, hydroxyzine, cyproheptadine, and promethazine remain in use for perioperative sedation and for analgesia. Concerns have been raised about their use in conscious sedation, not only because of their potential CNS adverse effects, but also because they may cause respiratory depression, and fatality. Promethazine has received a black-box warning from the United States Food and Drug Administration regarding use in young children, due to its association with a high rate of occurrence of CNS adverse effects, respiratory depression, and death in this age group (Starke et al. 2005). Dimenhydrinate, diphenhydramine, meclizine, and promethazine block the histaminergic signal from the vestibular nucleus to the vomiting centre in the medulla, and are used for antiemetic effects, and for prevention and treatment of motion sickness, vertigo, and related disorders. The firstgeneration H1-antihistamines have an unfavorable benefitto-risk ratio in the treatment of CNS and vestibular disorders. Military and commercial aviation authorities do not permit pilots to use these medications. Other public transportation workers such as ships’ captains, bus, truck, and taxi drivers, and members of the general public, must decide for themselves whether or not they are willing to take the risk of using a first-generation H1-antihistamine while operating a vehicle.

Adverse effects First-generation H1-antihistamines First-generation H1-antihistamines, administered in usual doses, potentially cause a wide variety of adverse effects in many body systems (Simons 2003, 2004). In contrast to the flat dose–response curve for efficacy, the dose–response curve for adverse effects is steep; that is, the adverse effects potentially increase with increasing doses. Through muscarinic receptors, these older H1-antihistamines may cause pupillary dilation, dry eyes, dry mouth, urinary retention and hesitancy, decreased gastrointestinal motility and constipation. Through α-adrenergic receptors, they potentially

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cause peripheral vasodilation, postural hypotension, and dizziness. Through serotonin receptors, they potentially cause appetite stimulation and weight gain (Simons 2004) (Fig. 25.3). The main concern, however, is that all first-generation H1-antihistamines administered in usual doses interfere with histamine’s neurotransmitter effects through H1 receptors in the CNS, and potentially cause CNS adverse effects (Shamsi & Hindmarch 2000; Tashiro et al. 2005). These include drowsiness, sedation, somnolence, fatigue, and impairment of memory, cognitive function, and psychomotor performance, and, less commonly, headache, dystonia, dyskinesia, agitation, confusion, and hallucinations. Positron emission tomography (PET) with 11C-doxepin as the positron-emitting ligand reveals that these medications occupy 70–90% of the H1 receptors in the frontal cortex, temporal cortex, hippocampus, and pons (Tashiro et al. 2002, 2006). Blood–brain barrier penetration is related to their lipophilicity, relatively low molecular weights, and lack of substrate recognition by the P-glycoprotein efflux pump that is expressed on the luminal surfaces of nonfenestrated endothelial cells in the CNS vasculature (Chen et al. 2003). Impairment of CNS function by first-generation H1antihistamines in usual doses has been documented in the absence of CNS symptoms. After taking one of these older medications at bedtime, some individuals have residual CNS adverse effects the next morning, the so-called antihistamine “hangover.” Tolerance to the CNS effects of the firstgeneration H1-antihistamines does not necessarily occur. The CNS effects of a first-generation H1-antihistamine are similar to, and exacerbate, those produced by alcohol or by a major tranquilizer. After overdose with first-generation H1-antihistamines, CNS symptoms predominate. In adults, these symptoms may culminate in delirium and coma. In children, paradoxical excitation, irritability, hyperactivity, insomnia, hallucinations, and seizures may occur. After overdose, first-generation H1antihistamines, such as diphenhydramine and hydroxyzine, also potentially cause dose-related cardiac adverse effects, including sinus tachycardia, reflex tachycardia, supraventricular arrhythmias, and prolongation of the QT interval and ventricular arrhythmias. These effects occur through interaction with muscarinic receptors, α-adrenergic receptors, and blockade of cardiac ion currents, especially the rapid component of the delayed rectifier potassium current (Yap & Camm 2002; Liu et al. 2007). Deaths attributed to first-generation H1-antihistamines due to accidental overdose, suicide, and homicide (in infants) have been reported in the literature for more than half a century (Wyngaarden & Seevers 1951). The plasma concentrations associated with fatality have been identified for medications such as diphenhydramine and brompheniramine (Simons 2003; Nine & Rund 2006). Up to 30 000 diphenhy-

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dramine overdoses are reported to poison control centers in the USA every year. Triage guidelines have been published in order to help identify individuals with these overdoses who urgently require transportation to a hospital emergency department for supportive treatment, and those who can be safely managed in other healthcare facilities, or at home (Scharman et al. 2006).

Second-generation H1-antihistamines In contrast to all of the foregoing, the second-generation H1antihistamines are unlikely to cause CNS effects when taken in manufacturers’ recommended doses, although in higher doses some of them, such as cetirizine, may cause sedation (Simons 2004; Golightly & Greos 2005). They are therefore medications of choice for pilots and others in safety-critical jobs, elite and professional athletes, and indeed in anyone who needs to remain alert. Relative lack of penetration into the CNS (Chen et al. 2003) has been documented by PET scan studies, which demonstrate that 0 to ≤ 30% of H1 receptors in the CNS are occupied by these newer medications, and by electroencephalographic monitoring, sleep latency studies, and standardized performance tests ranging from simple reaction time tests to complex sensorimotor tasks such as computermonitored driving (Shamsi & Hindmarch 2000; Hindmarch et al. 2002; Nicholson et al. 2003; Tashiro et al. 2005). Lack of CNS effects, confirmed objectively for the most part in healthy volunteers, may be of even greater significance in individuals with allergic rhinoconjunctivitis or urticaria, in whom symptoms such as congestion and extreme itching may reduce the duration and quality of nocturnal sleep, and increase daytime somnolence. In “real world,” prescription-event monitoring studies conducted in thousands of individuals with allergic rhinitis during the first 30 days after introduction of a new H1antihistamine in the UK, a low risk of sedation has been reported for medications such as fexofenadine, cetirizine, and loratadine (Mann et al. 2000), and more recently, for desloratadine and levocetirizine (Layton et al. 2006). The second-generation H1-antihistamines do not exacerbate the CNS effects of alcohol, diazepam, or other CNS-active substances when coadministered with these substances (Weiler et al. 2000; Ridout et al. 2003; Barbanoj et al. 2006; Scharf & Berkowitz 2007). Since withdrawal of regulatory approval for astemizole and terfenadine in most countries more than a decade ago (Woosley 1996), the second-generation H1-antihistamines remaining in use are free from potential cardiac adverse effects (Yap & Camm 2002; Hove-Madsen et al. 2006; Liu et al. 2007). According to published reports, accidental massive (e.g., up to 20-fold) overdoses of second-generation H1antihistamines such as cetirizine, fexofenadine, and loratadine have not resulted in any serious CNS or cardiovascular adverse effects, or in any deaths (Golightly & Greos 2005).

Antihistamines

Individuals who suffer from one or more allergic diseases often use H1-antihistamines intermittently or regularly for months, years, or even decades, yet there are only a few published randomized, controlled studies documenting the long-term safety of these medications. These include 6–12 month-long studies of fexofenadine and of levocetirizine in adults with allergic rhinitis, and three studies in very young children: a 12-month study of loratadine, an 18-month study of cetirizine, and an 18-month study of levocetirizine (Simons et al. 1999; Bachert et al. 2004; Grimfeld et al. 2004; Simons, F.E.R. et al. 2007b).

Other considerations Uncommonly, after usual doses, euphoria and “getting high” are reported for diphenhydramine, dimenhydrinate, and other first-generation H1-antihistamines. Rare adverse effects of both first- and second-generation H1-antihistamines include contact dermatitis, fixed-drug eruptions, photosensitivity, fever, elevation of liver enzymes and hepatitis, cytopenias, urticaria, and even anaphylaxis. First-generation H1-antihistamines, particularly those in the phenothiazine class, have been associated with sudden infant death syndrome, although causality has never been proven. First-generation H1-antihistamines applied topically to excoriated or abraded skin may cause systemic and local adverse effects. Intranasal or ophthalmic H1-antihistamines may cause stinging or burning of the nasal mucosa or the conjunctivae, and some such as azelastine potentially cause dysgeusia (bitter taste). No H1antihistamines currently approved for use are considered to have carcinogenic or tumor-promoting effects in humans (Nadalin et al. 2003; Simons 2003; Golightly & Greos 2005; Vythoulka et al. 2006).

Use of H1-antihistamines in infants and very young children H1-antihistamines are commonly given to infants and young children, not only for treatment of allergic rhinoconjunctivitis and urticaria, but also for colds, otitis media, and other disorders in which their efficacy remains unproven. Firstgeneration H1-antihistamines are available without prescription and are extensively marketed in a wide variety of tasty liquid formulations, often in combination with other medications. There are no prospective safety studies of these older H1-antihistamines in infants or very young children. The second-generation H1-antihistamines cetirizine and desloratadine have been prospectively studied in infants aged 6–11 months (Simons et al. 2003b; Gupta 2007). The long-term safety of cetirizine, levocetirizine, and loratadine has been confirmed in children aged 12–36 months, as noted previously. Studies of all three medications involved monitoring of adverse event reports, body mass and height measurements, and blood hematology and chemistry tests; in addition, electrocardiograms were performed in the cetirizine

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and loratadine studies. The 18-month duration cetirizine and levocetirizine studies included documentation of developmental milestones, and testing behavior and intellectual performance (Stevenson et al. 2002). For these second-generation H1-antihistamines, safety profiles were similar to safety profiles of children being treated with placebo.

Use of H1-antihistamines in pregnancy and lactation With any medication, and any class of medications, potential teratogenicity is a concern. Few medications, and no H1antihistamines, have been designated as FDA Category A, denoting negative studies in animals and negative human data. Several H1-antihistamines, such as the first-generation medications chlorpheniramine and diphenhydramine, and the orally administered second-generation medications cetirizine, levocetirizine and loratadine, as well as emedastine for ophthalmic use, have been designated as FDA Category B, denoting that studies in animals have shown no adverse effects and data in humans are not available; or that studies in animals have shown adverse effects but studies in humans have not shown adverse effects. These six medications are therefore considered to be relatively safe for use if needed in pregnancy. All other H1-antihistamines are designated as Category C, denoting drugs for which studies in animals have shown adverse effects, and data in humans are not available, or, drugs for which neither studies in animals nor studies in humans have been performed (Simons 2004). H1-antihistamines that are not approved for use in the USA (ebastine, mizolastine, and rupatadine) have not been categorized by the FDA. H1-antihistamines are secreted into breast milk. Nursing infants receive approximately 0.1% of an orally administered maternal dose, and first-generation H1-antihistamines potentially cause sedation and other adverse effects in these infants.

Use of first-generation H1-antihistamines in the elderly The vulnerability of the elderly to adverse effects from any CNS-active chemical is an important issue, because 25% of individuals older than age 65 years have some baseline cognitive impairment, often with no obvious sign of dysfunction. Use of first-generation H1-antihistamines for allergic rhinoconjunctivitis and urticaria in elderly individuals is a concern because of potential drug–drug and drug–herbal product interactions, potential antimuscarinic effects, and potential CNS effects through the H1 receptor. Diphenhydramine administration for insomnia in institutionalized elderly individuals has been associated with an increased cognitive impairment, delirium, inattention, disorganized speech, altered consciousness, and need for urinary catheter placement (Simons 2003; Hansen et al. 2005).

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Future directions H1-antihistamines are first-line medications in the treatment of allergic rhinitis, allergic conjunctivitis, and urticaria. Secondgeneration H1-antihistamines, which are relatively free from adverse effects, should be used in preference to the firstgeneration medications in the treatment of these disorders. The latter remain in use, however, and, in many countries, regulatory agencies appear to be less concerned about the potential CNS toxicity of these older H1-antihistamines than they are about the potential cardiac toxicity of H1antihistamines. Most of the new H1-antihistamines developed in recent years are structurally related to existing medications in the class. To date, no second-generation H1-antihistamine appears to be superior to the others, and none of them appears to be a true third- or next- or new-generation H1-antihistamine (Holgate et al. 2003). Nevertheless, in the future, clinically advantageous H1-antihistamines may be designed with the use of molecular techniques and will merit the designation “third-generation.” Some of these medications may possess intrinsic H2, H3, or H4 antihistamine properties, or antileukotriene properties.

References Akdis, C.A. & Simons, F.E.R. (2006) Histamine receptors are hot in immunopharmacology. Eur J Pharmacol 533, 69–76. Bachert, C., Bousquet, J., Canonica, G.W. et al. (2004) Levocetirizine improves quality of life and reduces costs in long-term management of persistent allergic rhinitis. J Allergy Clin Immunol 114, 838–44. Baena-Cagnani, C.E., Berger, W.E., DuBuske, L.M. et al. (2003) Comparative effects of desloratadine versus montelukast on asthma symptoms and use of beta 2-agonists in patients with seasonal allergic rhinitis and asthma. Int Arch Allergy Immunol 130, 307–13. Barbanoj, M.J., Garcia-Gea, C., Antonijoan, R. et al. (2006) Evaluation of the cognitive, psychomotor and pharmacokinetic profiles of rupatadine, hydroxyzine and cetirizine, in combination with alcohol, in healthy volunteers. Human Psychopharmacol 21, 13–26. Barnes, M.L., Ward, J.H., Fardon, T.C. & Lipworth, B.J. (2006) Effects of levocetirizine as add-on therapy to fluticasone in seasonal allergic rhinitis. Clin Exp Allergy 36, 676–84. Berger, W.E., Lumry, W.R., Meltzer, E.O. & Pearlman, D.S. (2006) Efficacy of desloratadine, 5 mg, compared with fexofenadine, 180 mg, in patients with symptomatic seasonal allergic rhinitis. Allergy Asthma Proc 27, 214–23. Bielory, L., Lien, K.W. & Bigelsen, S. (2005) Efficacy and tolerability of newer antihistamines in the treatment of allergic conjunctivitis. Drugs 65, 215–28. Blaiss, M.S. (2006) Diphenhydramine vs desloratadine comparisons must consider risk-benefit ratio. Ann Allergy Asthma Immunol 97, 121–2.

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Boguniewicz, M., Schmid-Grendelmeier, P. & Leung, D.Y.M. (2006) Atopic dermatitis. J Allergy Clin Immunol 118, 40–3. Bongers, G., Bakker, R.A. & Leurs, R. (2007) Molecular aspects of the histamine H3 receptor. Biochem Pharmacol 73, 1195–204. Bousquet, J., van Cauwenberge, P.B., Khaltaev, N. in collaboration with the World Health Organization (2001) Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol 108, 1A–14A, S147–S334. Bressler, R. (2006) Grapefruit juice and drug interactions. Exploring mechanisms of this interaction and potential toxicity for certain drugs. Geriatrics 61, 12–18. Bryce, P.J., Mathias, C.B., Harrison, K.L., Watanabe, T., Geha, R.S. & Oettgen, H.C. (2006) The H1 histamine receptor regulates allergic lung responses. J Clin Invest 116, 1624–32. Canonica, G.W., Tarantini, F., Compalati, E. & Penagos, M. (2007) Efficacy of desloratadine in the treatment of allergic rhinitis: a meta-analysis of randomized, double-blind, controlled trials. Allergy 62, 359– 66. Caproni, M., Volpi, W., Giomi, B., Torchia, D., Del Bianco, E. & Fabbri, P. (2006) Cellular adhesion molecules in chronic urticaria: modulation of serum levels occurs during levocetirizine treatment. Br J Dermatol 155, 1270– 4. Chang, A.B., Peake, J. & McElrea, M.S. (2006) Antihistamines for prolonged non-specific cough in children. Cochrane Database Syst Rev 3, CD005604. Chen, C., Hanson, E., Watson, J.W. & Lee, J.S. (2003) P-glycoprotein limits the brain penetration of nonsedating but not sedating H1antagonists. Drug Metabol Dispos 31, 312–18. Ciebiada, M., Gorska-Ciebiada, M., DuBuske, L.M. & Gorski, P. (2006) Montelukast with desloratadine or levocetirizine for the treatment of persistent allergic rhinitis. Ann Allergy Asthma Immunol 97, 664–71. Corcostegui, R., Labeaga, L., Innerarity, A., Berisa, A. & Orjales, A. (2006) In vivo pharmacological characterisation of bilastine, a potent and selective histamine H1 receptor antagonist. Drugs Res Dev 7, 219–31. Cranswick, N., Turzikova, J., Fuchs, M. & Hulhoven, R. (2005) Levocetirizine in 1–2 year old children: pharmacokinetic and pharmacodynamic profile. Int J Clin Pharmacol Ther 43, 172–7. Curran, M.P., Scott, L.J. & Perry, C.M. (2004) Cetirizine: a review of its use in allergic disorders. Drugs 64, 523–61. Day, J.H., Ellis, A.K., Rafeiro, E., Ratz, J.D. & Briscoe, M.P. (2006) Experimental models for the evaluation of treatment of allergic rhinitis. Ann Allergy Asthma Immunol 96, 263–77. de Esch, I.J.P., Thurmond, R.L., Jongejan, A. & Leurs, R. (2005) The histamine H4 receptor as a new therapeutic target for inflammation. Trends Pharmacol Sci 26, 462–9. De Sutter, A.I.M., Lemiengre, M., Campbell, H. & Mackinnon, H.F. (2003) Antihistamines for the common cold. Cochrane Database Syst Rev 3, CD001267. Di Lorenzo, G., Pacor, M.L., Mansueto, P. et al. (2004) Randomized placebo-controlled trial comparing desloratadine and montelukast in monotherapy and desloratadine plus montelukast in combined therapy for chronic idiopathic urticaria. J Allergy Clin Immunol 114, 619–25. Dubertret, L., Zalupca, L., Cristodoulo, T. et al. (2007) Once-daily rupatadine improves the symptoms of chronic idiopathic urticaria: a randomised, double-blind, placebo-controlled study. Eur J Dermatol 17, 223– 8.

Antihistamines

Dunford, P.J., Williams, K.N., Desai, P.J., Karlsson, L., McQueen, D. & Thurmond, R.L. (2007) Histamine H4 receptor antagonists are superior to traditional antihistamines in the attenuation of experimental pruritus. J Allergy Clin Immunol 119, 176–83. Gimenez-Arnau, A., Pujol, R.M., Ianosi, S. et al. (2007) Rupatadine in the treatment of chronic idiopathic urticaria: a double-blind, randomized, placebo-controlled multicentre study. Allergy 62, 539– 46. Golightly, L.K. & Greos, L.S. (2005) Second-generation antihistamines: actions and efficacy in the management of allergic disorders. Drugs 65, 341–84. Griffin, G.H., Flynn, C., Bailey, R.E. & Schultz, J.K. (2006) Antihistamines and/or decongestants for otitis media with effusion (OME) in children. Cochrane Database Syst Rev 4, CD003423. Grimfeld, A., Holgate, S.T., Canonica, G.W. et al. (2004) Prophylactic management of children at risk for recurrent upper respiratory infections: the Preventia I Study. Clin Exp Allergy 34, 1665–72. Gupta, S., Khalilieh, S., Kantesaria, B. & Banfield, C. (2007) Pharmacokinetics of desloratadine in children between 2 and 11 years of age. Br J Clin Pharmacol 63, 534–40. Gupta S.K., Kantesaria, B., Banfield, C., Wang, Z. (2007) Desloratadine dose selection in children aged 6 months to 2 years: comparison of population pharmacokinetics between children and adults. Br J Clin Pharmacol 64, 174–84. Haas, H. & Panula, P. (2003) The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci 4, 121–30. Hair, P.I. & Scott, L.J. (2006) Levocetirizine: a review of its use in the management of allergic rhinitis and skin allergies. Drugs 66, 973–96. Hansen, J., Klimek, L. & Hormann, K. (2005) Pharmacological management of allergic rhinitis in the elderly: safety issues with oral antihistamines. Drugs Aging 22, 289–96. Hindmarch, I., Shamsi, Z. & Kimber, S. (2002) An evaluation of the effects of high-dose fexofenadine on the central nervous system: a double-blind, placebo-controlled study in healthy volunteers. Clin Exp Allergy 32, 133–9. Holgate, S.T., Canonica, G.W., Simons, F.E.R. et al. (2003) Consensus Group on New-Generation Antihistamines (CONGA): present status and recommendations. Clin Exp Allergy 33, 1305–24. Horak, F., Zieglmayer, U.P., Zieglmayer, R. et al. (2006) Azelastine nasal spray and desloratadine tablets in pollen-induced seasonal allergic rhinitis: a pharmacodynamic study of onset of action and efficacy. Curr Med Res Opin 22, 151–7. Hove-Madsen, L., Llach, A., Molina, C.E. et al. (2006) The proarrhythmic antihistaminic drug terfenadine increases spontaneous calcium release in human atrial myocytes. Eur J Pharmacol 553, 215–21. Jongejan, A., Bruysters, M., Ballesteros, J.A. et al. (2005) Linking agonist binding to histamine H1 receptor activation. Nat Chem Biol 1, 98–103. Juniper, E.F., Stahl, E., Doty, R.L., Simons, F.E.R., Allen, D.B. & Howarth, P.H. (2005) Clinical outcomes and adverse effect monitoring in allergic rhinitis. J Allergy Clin Immunol 115 (suppl. 1), S390–S413. Jutel, M., Blaser, K. & Akdis, C.A. (2006) Histamine receptors in immune regulation and allergen-specific immunotherapy. Immunol Allergy Clin North Am 26, 249–59.

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Pharmacology

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Pasquali, M., Baiardini, I., Rogkakou, A. et al. (2006) Levocetirizine in persistent allergic rhinitis and asthma: effects on symptoms, quality of life and inflammatory parameters. Clin Exp Allergy 36, 1161–7. Petecchia, L., Serpero, L., Silvestri, M., Sabatini, F., Scarso, L. & Rossi, G.A. (2006) The histamine-induced enhanced expression of vascular cell adhesion molecule-1 by nasal polyp-derived fibroblasts is inhibited by levocetirizine. Am J Rhinol 20, 445–9. Picado, C. (2006) Rupatadine: pharmacological profile and its use in the treatment of allergic disorders. Exp Opin Pharmacother 7, 1989–2001. Plaut, M. & Valentine, M.D. (2005) Clinical practice. Allergic rhinitis. N Engl J Med 353, 1934–44. Popov, T.A., Dumitrascu, D., Bachvarova, A., Bocsan, C., Dimitrov, V. & Church, M.K. (2006) A comparison of levocetirizine and desloratadine in the histamine-induced wheal and flare response in human skin in vivo. Inflamm Res 55, 241–4. Prenner, B., Kim, K., Gupta, S. et al. (2006) Adult and paediatric poor metabolisers of desloratadine: an assessment of pharmacokinetics and safety. Exp Opin Drug Safety 5, 211–23. Raphael, G.D., Angello, J.T., Wu, Mei-M. & Druce, H.M. (2006) Efficacy of diphenhydramine vs desloratadine and placebo in patients with moderate-to-severe seasonal allergic rhinitis. Ann Allergy Asthma Immunol 96, 606–14. Ridout, F., Shamsi, Z., Meadows, R., Johnson, S. & Hindmarch, I. (2003) A single-center, randomized, double-blind, placebocontrolled, crossover investigation of the effects of fexofenadine hydrochloride 180 mg alone and with alcohol, with hydroxyzine hydrochloride 50 mg as a positive internal control, on aspects of cognitive and psychomotor function related to driving a car. Clin Ther 25, 1518–38. Saengpanich, S., deTineo, M., Naclerio, R.M. & Baroody, F.M. (2003) Fluticasone nasal spray and the combination of loratadine and montelukast in seasonal allergic rhinitis. Arch Otolaryngol Head Neck Surg 129, 557–62. Salmun, L.M., Herron, J.M., Banfield, C., Padhi, D., Lorber, R. & Affrime, M.B. (2000) The pharmacokinetics, electrocardiographic effects, and tolerability of loratadine syrup in children aged 2 to 5 years. Clin Ther 22, 613–21. Scharf, M. & Berkowitz, D. (2007) Effects of desloratadine and alcohol coadministration on psychomotor performance. Curr Med Res Opin 23, 313–21. Scharman, E.J., Erdman, A.R., Wax, P.M. et al. (2006) Diphenhydramine and dimenhydrinate poisoning: an evidence-based consensus guideline for out-of-hospital management. Clin Toxicol 44, 205–23. Schneider, E., Rolli-Derkinderen, M., Arock, M. & Dy, M. (2002) Trends in histamine research: new functions during immune responses and hematopoiesis. Trends Immunol 23, 255–63. Shamsi, Z. & Hindmarch, I. (2000) Sedation and antihistamines: a review of inter-drug differences using proportional impairment ratios. Human Psychopharmacol Clin Exp 15 (suppl. 1), S3–S30. Sheikh, A., ten Broek, V.M., Brown, S.G.A. & Simons, F.E.R. (2007) H1-antihistamines for the treatment of anaphylaxis with and without shock. Cochrane Database Syst Rev 1, CD006160. Simons, F.E.R. (2003) Antihistamines. In: Adkinson, N.F. Jr, Yunginger, J.W., Busse, W.W., Bochner, B.S., Holgate, S.T. & Simons, F.E.R., eds. Middleton’s Allergy: Principles and Practice, 6th edn, vol. I. Mosby, St Louis, pp. 834–69.

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Simons, F.E.R. (2004) Advances in H1-antihistamines. N Engl J Med 351, 2203–17. Simons, F.E.R. (2006) Anaphylaxis, killer allergy: long-term management in the community. J Allergy Clin Immunol 117, 367–77. Simons, F.E.R. & Simons, K.J. (2005) Levocetirizine: pharmacokinetics and pharmacodynamics in children age 6 to 11 years. J Allergy Clin Immunol 116, 355– 61. Simons, F.E.R., on behalf of the ETAC Study Group (1999) Prospective, long-term safety evaluation of the H1-receptor antagonist cetirizine in very young children with atopic dermatitis. J Allergy Clin Immunol 104, 433– 40. Simons, F.E.R., on behalf of the ETAC Study Group (2001) Prevention of acute urticaria in young children with atopic dermatitis. J Allergy Clin Immunol 107, 703– 6. Simons, F.E.R., Silver, N.A., Gu, X. & Simons, K.J. (2002) Clinical pharmacology of H1-antihistamines in the skin. J Allergy Clin Immunol 110, 777– 83. Simons, F.E.R., Prenner, B.M., Finn, A., for the Desloratadine Study Group (2003a) Efficacy and safety of desloratadine in the treatment of perennial allergic rhinitis. J Allergy Clin Immunol 111, 617–22. Simons, F.E.R., Silas, P., Portnoy, J.M., Catuogno, J., Chapman, D. & Olufade, A.O. (2003b) Safety of cetirizine in infants 6 to 11 months of age: a randomized, double-blind, placebo-controlled trial. J Allergy Clin Immunol 111, 1244– 8. Simons, F.E.R., on behalf of the ETAC study group (2005) Population pharmacokinetics of levocetirizine in very young children: the pediatricians’ perspective. Pediatr Allergy Immunol 16, 97–103. Simons, F.E.R., on behalf of the Early Prevention of Asthma in Atopic Children (EPAAC) Study Group (2007a) H1-antihistamine treatment in young atopic children: effect on urticaria. Ann Allergy, Asthma, Immunol 99, 261– 6. Simons, F.E.R., on behalf of the Early Prevention of Asthma in Atopic Children (EPAAC) Study Group (2007b) Safety of levocetirizine treatment in young atopic children: an 18-month study. Pediatr Allergy Immunol 18, 535– 42. Simons, K.J., Strolin-Benedetti, M., Simons, F.E.R., Gillard, M. & Baltes, E. (2007) Relevance of H1-receptor occupancy to antihistamine dosing in children. J Allergy Clin Immunol 119, 1551–4. Starke, P.R., Weaver, J. & Chowdhury, B.A. (2005) Boxed warning added to promethazine labeling for pediatric use. N Engl J Med 352, 2653. Stevenson, J., Cornah, D., Evrard, P. et al. (2002) Long-term evaluation of the impact of the H1-receptor antagonist cetirizine on the behavioral, cognitive and psychomotor development of very young children with atopic dermatitis. Pediatr Res 52, 251–7. Stuebner, P., Horak, F., Zieglmayer, R. et al. (2006) Effects of rupatadine vs placebo on allergen-induced symptoms in patients exposed to aeroallergens in the Vienna Challenge Chamber. Ann Allergy Asthma Immunol 96, 37– 44. Tashiro, M., Mochizuki, H., Iwabuchi, K. et al. (2002) Roles of histamine in regulation of arousal and cognition: functional neuroimaging of histamine H1 receptors in human brain. Life Sci 72, 409–14. Tashiro, M., Horikawa, E., Mochizuki, H. et al. (2005) Effects of fexofenadine and hydroxyzine on brake reaction time during cardriving with cellular phone use. Hum Psychopharmacol 20, 501–9.

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Tashiro, M., Mochizuki, H., Sakurada, Y. et al. (2006) Brain histamine H1 receptor occupancy of orally administered antihistamines measured by positron emission tomography with 11C-doxepin in a placebo-controlled crossover study design in healthy subjects: a comparison of olopatadine and ketotifen. Br J Clin Pharmacol 61, 16–26. Toyota, H., Dugovic, C., Koehl, M. et al. (2002) Behavioral characterization of mice lacking histamine H(3) receptors. Mol Pharmacol 62, 389–97. Traidl-Hoffmann, C., Munster, I., Ring, J. & Behrendt, H. (2006) Impact of desloratadine and loratadine on the crosstalk between human keratinocytes and leukocytes: Implications for anti-inflammatory activity of antihistamines. Int Arch Allergy Immunol 140, 315–20. Treatment Guidelines from The Medical Letter (2007). Drugs for allergic diseases 5, 71–80. Triggiani, M., Petraroli, A., Loffredo, S. et al. (2007) Differentiation of monocytes into macrophages induces the upregulation of histamine H1 receptor. J Allergy Clin Immunol 119, 472–81. van Cauwenberge, P. & Juniper, E.F. (2000) Comparison of the efficacy, safety and quality of life provided by fexofenadine hydrochloride 120 mg, loratadine 10 mg, and placebo administered once daily for the treatment of seasonal allergic rhinitis. Clin Exp Allergy 30, 891–9. van Cauwenberge, P., De Belder, T. & Sys, L. (2004) A review of the second-generation antihistamine ebastine for the treatment of allergic disorders. Exp Opin Pharmacother 5, 1807–13. Vythoulka, A., Pitsios, C. & Kompoti, E. (2006) Allergic reactions due to mizolastine. Ann Allergy Asthma Immunol 97, 262–3. Wahn, U., Meltzer, E.O., Finn, A.F. et al. (2003) Fexofenadine is efficacious and safe in children (aged 6–11 years) with seasonal allergic rhinitis. J Allergy Clin Immunol 111, 763–9. Warner, J.O., on behalf of the ETAC study group (2001) A doubleblind, randomized, placebo-controlled trial of cetirizine in preventing the onset of asthma in children with atopic dermatitis: 18 months’ treatment and 18 months’ post-treatment follow-up. J Allergy Clin Immunol 108, 929–37. Weiler, J.M., Bloomfield, J.R., Woodworth, G.G. et al. (2000) Effects of fexofenadine, diphenhydramine, and alcohol on driving performance. A randomized, placebo-controlled trial in the Iowa driving simulator. Ann Intern Med 132, 354–63. Wilson, A.M. (2006) The role of antihistamines in asthma management. Treat Respir Med 5, 149–58. Woosley, R.L. (1996) Cardiac actions of antihistamines. Annu Rev Pharmacol Toxicol 36, 233–52. Wyngaarden, J.B. & Seevers, M.H. (1951) The toxic effects of antihistaminic drugs. JAMA 145, 277–82. Yap, Y.G. & Camm, A.J. (2002) Potential cardiac toxicity of H1antihistamines. In: Simons, F.E.R., ed. Histamine and H1-Antihistamines in Allergic Disease, 2nd edn. Marcel Dekker, New York, pp. 389–419. Zhou, S., Chan, E., Pan, Shen-Q., Huang, M. & Lee, E.J.D. (2004) Pharmacokinetic interactions of drugs with St John’s wort. J Psychopharmacol 18, 262–76. Zuberbier, T., Bindslev-Jensen, C., Canonica, W. et al. (2006) EAACI/ GA2LEN/EDF guideline: management of urticaria. Allergy 61, 321– 31.

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Lipid Mediators: Leukotrienes, Prostanoids, Lipoxins, and Platelet-activating Factor Sophie P. Farooque, Jonathan P. Arm and Tak H. Lee

Summary The eicosanoids are metabolites of the 20-carbon fatty acid arachidonic acid. The best-known eicosanoids are the prostanoids (prostaglandins and thromboxane) and leukotrienes, which are implicated in a large number of physiologic and pathologic processes and are synthesized during allergic and asthmatic reactions. Monocytes, alveolar macrophages, and peripheral blood neutrophils preferentially generate leukotriene (LT)B4, whereas eosinophils, mast cells, and basophils preferentially generate the cysteinyl leukotrienes (LTC4, LTD4 and LTE4). In addition to production by inflammatory leukocytes, structural cells within the lung can produce prostaglandins. Lipoxin are also eicosanoids, and are endogenous antiinflammatory mediators. Arachidonic acid is the parent compound from which leukotrienes and prostanoids are synthesized de novo upon cellular activation. It is cleaved from the cell membrane by one of the isoforms of the enzyme phospholipase A2 and if metabolized through the 5-lipoxygenase (5-LO) pathway from an unstable intermediary LTA4 yields LTB4 or the cysteinyl leukotrienes (CysLTs), whereas if metabolized through the cyclooxygenase (COX) pathway yields the prostanoids. 5-LO is the rate-limiting step in leukotriene formation but is dependent on 5-LO-activating protein, which serves as a necessary presentation molecule. Arachidonic acid release is the rate-limiting step for prostanoid formation.

orchestrate amplification of the allergic inflammatory response in the airway, and influencing neural transmission. Release of CysLTs has been found to be a final common response to mast cell activation regardless of the specific route of inflammation. Four leukotriene receptors have been cloned: BLT1 and BLT2 which both bind LTB4, and CysLT1 and CysLT2 which bind the cysteinyl leukotrienes. The expression of BLT1 is limited to leukocytes whilst BLT2 is believed to be more widely expressed, with BLT2 mRNA found in several tissue types. Biological activities of LTB4 include chemotaxis for mast cell progenitors and neutrophils, weak chemotactic activity for eosinophils, and a role in increasing leukocyte adherence to endothelium. The role of the LTB4 in the pathogenesis of airway inflammation in humans is less well elucidated compared with the CysLTs. Patients with aspirin-sensitive respiratory disease demonstrate particularly high levels of CysLTs and increased CysLT1 receptor expression compared with nonaspirin-sensitive cohorts. Most of the actions of the CysLTs are believed to be mediated through the CysLT1 receptor, which is highly expressed in human leukocytes and is upregulated in the airways of asthmatics. It has been hypothesized that CysLTs may act on more than two receptors, as several studies have recorded that the biological properties of the CysLTs cannot be satisfactorily explained on the basis of recognized pharmacologic properties of CysLT1 and CysLT2.

Prostanoids Leukotrienes Originally described as the slow-reacting substances of anaphylaxis (Brocklehurst et al., 1956), the CysLTs are strongly implicated in the pathogenesis of both allergic rhinitis and asthma. CysLTs were first recognized for their airway bronchoconstrictor activity and appear to play a central role in the airway smooth muscle hypertrophy and hyperplasia found in chronic severe asthma. Other biological properties include stimulation of airway mucus secretion, recruitment and activation of eosinophils, upregulation of the cytokines that


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The generation of prostanoids is a two-step process involving COX activity to convert arachidonic acid to PGG2 and a peroxidase reaction to produce PGH2. PGH2 serves as a substrate for the prostaglandin synthase enzymes, which are responsible for the production of the five physiologically bioactive prostaglandins generated in vivo, PGE2, PGF2α, PGD2, PGI2, and TXA2 (thromboxane). At least two isoforms of COX exist. COX-1 is constitutively expressed and is responsible for basal prostanoid production and hence is often referred to as the “housekeeping” COX isoform. COX-2 is expressed in leukocytes following cellular activation by inflammatory stimuli and is involved in inflammation. In the asthmatic human airway smooth muscle, eosinophils, bronchial epithelial cells, mast cells, and macrophages all exhibit COX-2 immunoreactivity. The expression of COX-1 and COX-2 in human lungs results in the production of two different classes of prostanoids. These are

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broadly divided into the bronchoconstrictive prostaglandins (PGD2 and PGF2α) and the bronchoprotective prostaglandins (PGE2 and PGI2). Although COX is present in most cells including the respiratory tract, airway smooth muscle and epithelial cells, the COX metabolites released from a particular cell are cell specific, reflecting the isomerase and synthase components of that cell. Prostanoid receptors are specific cell surface G-protein-coupled receptors and are classified according to the prostanoid causing selective activation.

Lipoxins Perhaps the lesser known of the eicosanoids, lipoxins are endogenous antiinflammatory mediators distinct from leukotrienes and prostaglandins in structure and function. They are double lipoxygenase products and may require separate cell sources for the two lipoxygenases and are synthesized via transcellular cooperation. Their synthesis has been strongly implicated in the resolution of inflammation and they are generated in normal and asthmatic subjects. Exogenous lipoxin A4 has been shown to block asthmatic responses in both human and animal models.

Introduction The eicosanoids are metabolites of the 20-carbon fatty acid arachidonic acid. The best-known eicosanoids are the leukotrienes and prostanoids (prostaglandins and thromboxane), which form part of a family of oxygenated fatty acids found in virtually every mammalian cell. Physiologically these products of arachidonic acid metabolism participate in many normal processes, which include body temperature regulation, the coagulation cascade, the control of parturition, blood pressure maintenance, and mediation of the immune system. Pathologically, these lipid mediators have been incriminated in a wide range of diseases, including asthma, psoriasis, rheumatoid arthritis, inflammatory bowel disease, and malignancy. Much of the early work on prostaglandins was carried out by three groups led by Sune Bergstrom, Bengt Samuelsson, and John Vane. Bergstrom determined the structure and biological role of the prostaglandins and discovered that they are synthesized in vivo from dietary polyunsaturated fatty acids; Samuelsson determined the metabolic fate and disposition of the prostaglandins; and Vane demonstrated the ability of antiinflammatory substances such as aspirin to inhibit prostaglandin synthesis. However, the original investigation of these compounds began in 1930, with the work of Raphael Kurzok and Charles Lieb, who studied the ability of human semen either to relax or to contract isolated strips of uterine tissue. Three years later, Goldblatt and von Euler independently observed a similar phenomenon using human seminal plasma and extracts from sheep seminal

vesical glands. Von Euler therefore coined the term “prostaglandin,” believing that he had isolated a product of the prostate gland. Leukotrienes were discovered during the elucidation of a mixture of compounds referred to as the slow-reacting substance of anaphylaxis or SRS-A. In 1938, Feldberg and Kellaway coined the term SRS to define the smooth muscle contracting activity found in the effluent of the perfused lungs of guinea pigs and cats following treatment with cobra venom. In 1940, Kellaway and Trethewie found that smooth muscle contractions in anaphylaxis were caused not only by histamine but also in part by a slow-reacting substance. Brocklehurst (1956) unmasked the SRS by demonstrating that the contraction it produced on the isolated guinea-pig ileum was not inhibited by an antihistamine added to the organ bath. He also showed that the pattern of contraction was different from bradykinin, substance P, and 5-hydroxytryptamine and added the suffix “A” to indicate that this was a particular slow-reacting substance associated with anaphylaxis. The 1960s and 1970s saw the purification and description of many biological and physicochemical properties of SRS-A by Orange, Austen, and Murphy (Orange & Austen 1969; Orange et al. 1973), Morris et al. (1980), Lewis et al. (1980a), and others. The covalent structure and total synthesis of SRS-A and its identification as a mixture of the sulfidopeptide leukotrienes, leukotriene (LT)C4, and its biologically active metabolites LTD4 and LTE4 was achieved by Samuelsson et al. (1980) and Corey et al. (1980). The leukotrienes and the prostanoids are not preformed, but upon cell activation are synthesized de novo. The primary substrate fatty acid which acts as a precursor for leukotrienes and prostanoids is 5,8,11,14-eicosatetraenoic acid (arachidonic acid). Leukocyte activation initiates the rapid generation of eicosanoids via the release of arachidonic acid from cell membrane phospholipids by one of the isoforms of phospholipase A2 (PLA2). Subsequent oxidative metabolism of this polyunsaturated fatty acid by the lipoxygenase pathway yields the leukotrienes, and by the cyclooxygenase (COX) pathway leads to prostanoid synthesis. Asthma exacerbations are characterized by increased leukotriene biosynthesis (Green et al. 2004). In 1984, Serhan, Samuelsson and their coworkers observed that labeled arachidonate was transformed by suspensions of mixed human leukocytes into polar compounds with physical properties distinct from prostaglandins, thromboxanes, and leukotrienes (Serhan et al. 1984). These compounds were named “lipoxins” (an abbreviation for “lipoxygenase interaction products”) as they are generated from arachidonic acid via sequential action of two or more lipoxygenases. Structurally distinct, the lipoxins carry a tetraene chromophorein and, in contrast to proinflammatory prostanoids and leukotrienes, have antiinflammatory actions and are known to have an important role in inhibiting the host response and promoting the resolution of asthma exacerbations.

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Phospholipase A2 PLA2 hydrolyzes the sn-2 ester bond of cell membrane glycerophospholipids to release free polyunsaturated fatty acids and generate lysophospholipids. The first mammalian PLA2 enzymes to be structurally characterized were those isolated from the pancreas and from the synovial fluid of rheumatoid arthritis (De Haas et al. 1968; Seilhamer et al. 1989). These were termed group IB and group IIA PLA2, respectively, based on their structural similarity to the already-characterized snake venom PLA2 enzymes. They were also termed secretory PLA2 due to the presence of a leader sequence predicting their intracellular targeting to the secretory pathway of the cell, which was validated by their presence in extracellular fluids (pancreatic secretions and synovial fluid, respectively). They are low-molecular-weight enzymes (∼ 14 kDa), are highly disulfide-linked with 14 cysteine residues, and use a catalytic histidine residue. They are distinguished from one another by the distribution of their cysteine residues, a carboxylterminal extension in the group IIA enzyme, and the “pancreatic loop” of the group IB enzyme (Schaloske & Dennis 2006) In addition, the group IB enzyme has an activation peptide that is cleaved by pancreatic trypsin. However, these enzymes are poor candidates for providing arachidonic acid as substrate for eicosanoid generation. They are secreted (not intracellular) enzymes, are active at high micromolar or millimolar concentrations of calcium, and have no specificity for arachidonic acid. Three major groups of PLA2 have been identified (Underwood et al. 1998; Pickard et al. 1999; Ohto et al. 2005): high-molecular-weight cytosolic PLA2 (cPLA2α), lowmolecular-weight secretory PLA2 (sPLA2), and calciumindependent PLA2 (iPLA2) isoforms.

cPLA2a When cytosolic PLA2 (cPLA2; group IV PLA2) was characterized it was considered a prime candidate for the enzyme initiating eicosanoid biosynthesis (Leslie et al. 1988; Clark et al. 1991). cPLA2α is an 85-kDa enzyme that uses a catalytic serine residue, is active at low micromolar concentrations of Ca2+, is present in the cytosol of cells that generate eicosanoids, and preferentially hydrolyzes phospholipids with arachidonic acid in the sn-2 position. cPLA2α translocates from the cytosol to the nuclear envelope, a prominent site of eicosanoid biosynthesis (Schievella et al. 1995). The activity of cPLA2α is regulated by phosphorylation at serine residues 505 and 727 by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 (BorschHaubold et al. 1998). The generation of mice in which the gene encoding cPLA2α was deleted by recombinant DNA technology confirmed its essential role in eicosanoid biosynthesis by leukocytes in response to a diverse array of stimuli (Bonventre et al. 1997; Uozumi et al. 1997). Mice lacking

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cPLA2α have attenuation of allergic pulmonary inflammation (Uozumi et al. 1997), acute lung injury in response to acid aspiration (Nagase et al. 2000), cerebral infarction following ischemia–reperfusion injury (Bonventre et al. 1997), and bleomycin-induced pulmonary fibrosis (Nagase et al. 2002a), as well as difficulties with fertilization and parturition (Uozumi et al. 1997). Five other isoforms of cPLA2α have been characterized whose biological roles have yet to be determined (Underwood et al. 1998; Pickard et al. 1999; Ohto et al. 2005).

sPLA2 The explosion of information on the human genome and the widespread use of in silico cloning in the 1990s led to the identification of multiple mammalian sPLA2 enzymes, each sharing a common catalytic motif (CCXXHDXC). The nomenclature built on what already existed (Schaloske & Dennis 2006), each new structure being assigned to a new group (Schaloske & Dennis 2006). Thus, the group III enzyme was so named due to the homology of its core to the group III hymenopteran PLA2, although the mammalian group III enzyme has long amino-terminal and carboxy-terminal extensions. Group V sPLA2 contains the six core disulfidelinked cysteine pairs of the group IB and IIA enzymes and no other cysteines (Chen, J. et al. 1994), whereas group X sPLA2 contains the six core cysteine pairs and both cysteine pairs characteristic of group IB and group IIA sPLA2, respectively (Cupillard et al. 1997). Group III sPLA2 and group XII sPLA2 are cysteine-rich but have a completely novel distribution of cysteine residues (Valentin et al. 2000; Ho et al. 2001). The diverse isoforms of sPLA2 enzymes do not merely serve redundant functions. They have widely differing tissue, cellular, and subcellular distributions (Bingham et al. 1999; Valentin et al. 1999). In contrast to cPLA2α most sPLA2 enzymes are released in the extracellular environment on appropriate cell activation. They also have different biochemical properties. PLA2 must be able to gain access to its substrate, which is an integral structural component of cell membranes. The group V and group X enzymes are the only isoforms of sPLA2 capable of directly binding to phosphatidylcholine, so-called interfacial binding, and can therefore release arachidonic acid from cell membrane phospholipids when applied exogenously or when secreted from the cell. Thus they have the potential for acting in a transcellular manner, being released from one cell and stimulating eicosanoid generation in a neighboring cell (Bezzine et al. 2000; Kim et al. 2002; Munoz et al. 2003). Three sPLA2 enzymes, groups IIA, IID, and V, are able to bind to cell surface proteoglycans. When transfected into transformed kidney cells, they are released from the cell on stimulation with A23187 or with interleukin (IL)-1β and serum, bind to glypican in caveolae, are internalized, and couple to COX for prostaglandin (PG)E2 biosynthesis (Murakami et al. 1998). The idea that sPLA2 enzymes can augment the essential function of cPLA2α in providing arachidonic acid for eicosanoid generation was indicated in transfection studies

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and by the actions of exogenous group V and group X sPLA2 (Murakami et al. 1998; Kim et al. 2002; Han et al. 2003; Munoz et al. 2003). Recent studies with transgenic mice lacking group V sPLA2 have demonstrated that it not only augments cPLA2αdependent eicosanoid generation in zymosan-stimulated macrophages (Satake et al. 2004) but it also regulates phagocytosis of zymosan (Balestrieri et al. 2006).

Nuclear membrane phospholipids Phospholipase A2 Arachidonic acid 5-lipoxygenase

5(S)-HPETE

Cyclooxygenase

iPLA2

Calcium-independent iPLA2 isoforms, like cPLA2α, use a catalytic serine residue but do not require calcium for activity (Wolf & Gross 1996; Balboa et al. 1997). They have been implicated in membrane remodeling (Balsinde et al. 1997), regulation of store-operated calcium channels (Smani et al. 2003) and the ion channel TRPM8 (Vanden Abeele et al. 2006), apoptosis (Zhang et al. 2006), regulation of insulin secretion (Ramanadham et al. 1999), and spermatogenesis (Bao et al. 2004), as well as in the release of arachidonic acid.

5(S)-HETE

Prostaglandins and thromboxanes

5-lipoxygenase Leukotriene A4 LTA4 hydrolase

LTC4 synthase

Leukotriene B4

Leukotriene C4

Platelet-activating factor acetylhydrolase The acetylhydrolases of platelet-activating factor (PAF) are PLA2 enzymes that cleave acetate from the sn-2 position of PAF. They also release F2-isoprostanes from phospholipids (Stafforini et al. 2006) and hydrolyze oxidized phospholipids (Marathe et al. 2003).

Gammaglutamyl transpeptidase Leukotriene D4

Slow reacting substance of anaphylaxis

Dipeptidases

Leukotrienes Biosynthesis Samuelsson conceived the term “leukotriene” to describe a family of compounds each containing three conjugated double bonds and derived from the conversion of arachidonic acid by 5-lipoxygenase (5-LO) in leukocytes. He subjected arachidonic acid to the actions of various lipoxygenases and found that rabbit polymorphonuclear leukocytes (PMNs) metabolized arachidonate to a family of dihydroxy acids that showed triple spectrophotometric absorption peaks at 259, 269 and 279 nm. These triple peaks suggested the existence of triple conjugated double bonds in these compounds. Arachidonic acid is a 20-carbon polyunsaturated fatty acid released from membrane phospholipids by the action of PLA2. The enzyme 5-LO catalyzes the first step in arachidonic acid metabolism (Fig. 26.1) by inserting oxygen at C-5 to produce the unstable intermediate 5S-hydroperoxyeicosatetraenoic acid (5-HPETE). This is either reduced to the alcohol 5Shydroxyeicosatetraenoic acid (5-HETE) or converted by 5-LO via a dehydrase step to a C-5,6-transepoxide with three conjugated (7,9-trans,11-cis) olefinic bonds, and a fourth, unconjugated double bond at C-14. This compound was the first leukotriene described and designated LTA4 (the subscript “4” refers to the number of carbon–carbon double bonds in the molecule). Subsequent metabolism of LTA4 takes place via two alternative enzymatic pathways: (i) via an epoxide

Leukotriene E4 Fig. 26.1 Leukotriene biosynthesis from nuclear membrane phospholipids and arachidonic acid via the 5-lipoxygenase pathway. 5(S)-HPETE, 5(S)-hydroperoxyeicosatetraenoic acid; 5(S)-HETE, 5(S)hydroxyeicosatetraenoic acid.

hydrolase to 5S,12R-dihydroxyeicosatetraenoic acid (LTB4) (S and R denote the chirality of the molecule at the 5 and 12 carbon atoms, respectively); or (ii) by opening the epoxide and conjugation of the tripeptide, glutathione, at C-6 by a glutathione S-transferase (S in this case refers to the sulfur moiety of glutathione) termed LTC4 synthase to LTC4. In the absence of either activated enzyme system, LTA4 degrades spontaneously to 6-trans-LTB4, which has significantly less bioactivity. LTC4 is exported from the cell by the multidrug resistance-associated protein (MRP)1 (Leier et al. 1994; Muller et al. 1994). After its export from the cell, LTC4 is subsequently cleaved to form the 6R-S-cysteinylglycine analog LTD4 by removal of glutamic acid from the peptide by γglutamyl-transpeptidase (Leier et al. 1994; Muller et al. 1994). LTD4 is further cleaved by a dipeptidase to remove glycine to form its 6R-S-cysteinyl analog (LTE4). These latter three compounds are known collectively as the cysteinyl leukotrienes, because of the presence of a cysteine linked to the eicosanoid backbone through a thiolether link at C-6. The chemical

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O COOH

Leukotriene A4 OH

OH COOH

Leukotriene B4 OH COOH S Leukotriene C4

Cys

Gly

Glu OH COOH S

Cys

Gly

Leukotriene D4 OH COOH S

Cys

Leukotriene E4 Fig. 26.2 Chemical structure of the leukotrienes.

structure of the leukotrienes is shown in Fig. 26.2. The cysteinyl leukotrienes comprise the activity previously recognized as SRS-A. Although the enzyme 5-LO is the rate-limiting step for leukotriene formation, it requires the association of an additional factor. Mammalian osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations but no leukotriene metabolites on stimulation with the calcium ionophore A23187 (Rouzer et al. 1988). In addition, a novel indole leukotriene biosynthesis inhibitor MK-886 was found to act only on intact PMNs but had no direct inhibitory effect on soluble 5-LO activity (Gillard et al. 1989). An 18-kDa membrane protein with high affinity for MK-886 was identified from rat and human leukocytes, and it was demonstrated that expression of both 5-LO and this MK-886 binding protein was necessary for leukotriene synthesis. This was confirmed by its specific labeling with a 125I-radiolabeled photoaffinity

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probe and by its retention on agarose gels to which analogs of MK-886 had been bound. This protein was termed “5lipoxygenase-activating protein” (FLAP) (Miller et al. 1990). In osteosarcoma cells transfected with 5-LO or FLAP alone and stimulated with the calcium ionophore A23187, no arachidonic acid metabolites were detected. In contrast, A23187 treatment of cell lines expressing both 5-LO and FLAP resulted in significant production of 5-LO products (Dixon et al. 1990). The human promyelocytic cell line U937 expresses FLAP but not 5-LO, and is unable to synthesize leukotrienes after A23187 stimulation but does so after transfection by a retroviral vector encoding 5-LO mRNA (Kargman et al. 1993). Human B-lymphocyte lines and normal human tonsillar B lymphocytes have 5-LO activity and can produce LTB4 on cell activation (Jakobsson et al. 1991), but five T-cell lines were found to express only FLAP and not 5-LO and therefore were unable to generate leukotrienes on cell activation (Jakobsson et al. 1992). The mechanism of cellular activation and subsequent leukotriene generation is believed to arise as a result of translocation of 5-LO from cytosolic to membrane compartments in order to bring the enzyme into close proximity with FLAP, which acts as an anchor for 5-LO and provides access to substrate and other components of leukotriene synthesis. In osteosarcoma cells expressing 5-LO but not FLAP, 5-LO is able to associate with cell membranes following A23187 stimulation and this is not inhibited by MK-886. This led the authors to conclude that 5-LO membrane association and activation can be divided into a two-stage process: (i) Ca2+dependent movement of 5-LO to membrane without product formation which can occur in the absence of FLAP; and (ii) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (Kargman et al. 1992). High levels of FLAP were expressed in SF9 insect cells transfected with recombinant baculovirus, and this system was used to demonstrate that FLAP specifically binds [125I]L739,059, a photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886 and suggests that FLAP may activate 5-LO by bringing enzyme and substrate together (Mancini et al. 1993). Ultracentrifugation (Peters-Golden & McNish 1993) and immunoelectron microscopy studies have demonstrated that on cellular activation 5-LO translocates to the nuclear envelope, which is also the location of FLAP (Woods et al. 1993). The situation in resting cells is still variable, and 5-LO may be located in the cytosol (Rouzer & Kargman 1988) or nuclear euchromatin (Woods et al. 1994).

Cellular sources Because of the requirement to express both 5-LO and FLAP, the range of cells known to synthesize leukotrienes is limited. Cells of myeloid lineage provide the major source of leukotriene production, although B lymphocytes have also been shown to be capable of generating small quantities of

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Table 26.1 Cellular source of human leukotriene generation. Predominant leukotriene product

Activating stimulus

Approximate amount generated/106 cells

Neutrophils

LTB4 LTC4

A23187 A23187

50 ng 10 ng

Eosinophils

LTB4 LTC4

A23187 A23187

10 ng 40 ng

Peripheral blood monocytes

LTB4 LTC4

A23187 A23187

70 ng 30 ng

Alveolar macrophages

LTB4

A23187

30 ng

Mast cells

LTC4

A23187/ or IgE antigen

20 ng

Basophils

LTC4

A23187/ or IgE antigen

10 ng

Cell

A23187, divalent calcium ionophore.

leukotrienes (Jakobsson et al. 1991). Cells can be divided by their ability to preferentially synthesize LTB4 or the cysteinyl leukotriene LTC4, depending on the intracellular predominance of either LTA4 epoxide hydrolase or LTC4 synthase. Monocytes, alveolar macrophages, and peripheral blood neutrophils preferentially generate LTB4, whereas eosinophils, mast cells, and basophils preferentially generate cysteinyl leukotriene products (Table 26.1). Monocytes and macrophages have the capacity to generate both LTB4 and LTC4. Human peripheral blood monocytes in adherent monolayers produce 68 ng LTB4 and 30 ng LTC4 per 106 cells on stimulation with the calcium ionophore A23187 (Williams et al. 1984). LTB4 is the major lipoxygenase product of human alveolar macrophages, which generate 17–30 ng per 106 cells in response to ionophore A23187 stimulation (Fels et al. 1982). Peripheral blood neutrophils from normal donors produce about 48 ng LTB4 per 106 cells, with only oneseventh as much LTC4 per 106 cells after A23187 stimulation. Eosinophils from the same subjects preferentially synthesize LTC4, producing 38 ng LTC4 per 106 cells and about 6 ng LTB4 per 106 cells in response to A23187 stimulation (Weller et al. 1983). Activation of leukocytes is accompanied by respiratory burst activity, with reduction of oxygen to form superoxide anion (O 2–) and hydrogen peroxide (H2O2) and the release of cytoplasmic contents, including eosinophil peroxidase (EPO) from eosinophils and myeloperoxidase (MPO) from neutrophils or monocytes. This leads to oxidative degradation of the cysteinyl leukotrienes and acts as a control on their biological effects. EPO or MPO catalyze the oxidation of halides by H2O2 to form hypohalous acids such a HOCl, HOBr, or HOI, which readily degrade cysteinyl leukotrienes to their inactive sulfoxide derivatives and to 6-trans-stereoisomers

of LTB4 (Henderson et al. 1982; Henderson & Klebanoff 1983). Human lung mast cells release 22 ng LTC4 per 106 cells, with similar amounts released in response to either IgE- or calcium ionophore A23187-mediated degranulation (Peters et al. 1984). Basophils obtained from the peripheral blood of patients with chronic myelogenous leukemia were found to release LTB4 (Rothenberg et al. 1987). Basophils also release LTC4 in response to cross-linking of FcεRI or engagement of leukocyte immunoglobulin-like receptor A2, but unlike mast cells do not produce PGD2 (MacGlashan et al. 1986; Sloane et al. 2004). Local instillation of specific allergen into the nasal mucosa of sensitive subjects leads to recovery of histamine, PGD2, and LTC4 from nasal secretions during the early-phase reaction, but only histamine and LTC4 during the late-phase reaction. These findings suggest that mast cell degranulation is important in the early-phase response to allergen and that basophils mediate the late reaction (Naclerio et al. 1983; Creticos et al. 1984). B lymphocytes produce only small amounts of LTB4 and 5-HETE on stimulation with A23187, in comparison with the amounts produced by the sonicates of these cells. However, preincubation with a glutathionedepleting agent prior to stimulation leads to similar amounts of LTB4 generated by the intact cells as are formed by the sonicated cells. T lymphocytes express the FLAP gene but not the 5-LO gene and therefore do not generate leukotrienes (Jakobsson et al. 1992). Guinea-pig lung parenchyma has been shown to convert LTA4 to LTB4, LTC4, LTD4, and LTE4 (Sirois et al. 1985) and human lung parenchyma converts LTC4 to LTD4 and LTE4 (Aharony et al. 1985; Conroy et al. 1989). Unlike 5-LO, LTC4 synthase and LTA4 hydrolase, which convert LTA4 to LTC4 and LTB4 respectively, are widely distributed. Thus, LTA4 released by a cell capable of generating

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15–LO

Endothelial cell

AA

AA LTB4

15–HPETE

5–HPETE

trans locat

ion

5–LO/FLAP complex (active)

15–LO

5–LO

AA

LTA4 hydrolase

Erythrocyte LTA4 LTA4

FLAP LTB4

Epithelial cell

LTC4 synthase

5–LO

Platelet

12–LO

Neutrophil

LXA4 LXB4

LTC4 LXA4

LXB4

Fig. 26.3 Transcellular biosynthesis of leukotrienes and lipoxins by cell–cell interaction between polymorphonuclear (PMN) leukocytes and platelets for cysteinyl leukotriene production or PMN leukocytes and either platelets, epithelial or endothelial cells for lipoxin generation. Human 5-hypoxygenase (5-LO) is located in the cytosol or nuclear euchromatin of the resting cell, and on activation is translocated to form a complex with 5-LO activating protein (FLAP) to express enzymatic activity. Endothelial and epithelial cells contain 15-LO and platelets contain both 12-LO and leukotriene C4 (LTC4) synthetase activity.

eicosatetraenoic acid (20-COOH-LTB4). 20-CHO-LTB4 can be converted via an alternative pathway by an aldehyde reductase in the microsomes back to 20-OH-LTB4, thereby allowing tightly regulated LTB4 degradation. LTC4 can be bioconverted by three major metabolic pathways. The first comprises conversion of LTC4 by peptide cleavage to LTD4 and then to LTE4 by the action of γ-glutamyl transpeptidase and one of a variety of dipeptidases, respectively. This does not require activation of the cells involved and may only result in some loss of biological activity. The second pathway involves oxidative metabolism of the cysteinyl leukorienes and depends on the production of hydrogen peroxide by the respiratory burst and secretion of cell-specific peroxidase, which occurs only in the extracellular microenvironment of activated neutrophils, eosinophils, and monocytes. A peroxidase–hypochlorous acid reaction then transforms LTC4, LTD4, and LTE4 to their respective Sdiastereoisomeric sulfoxides and to the 6-trans-diastereoisomers of LTB4. Each of the S-diastereomeric sulfoxides is then converted to its respective sulfone, which results in considerable loss of bioactivity. The 6-trans-LTB4 diastereoisomers are not immunoreactive in a sulfidopeptide leukotriene assay and are nonspasmogenic. The sulfoxides are fully immunoreactive but possess less than 5% spasmogenic activity. The third pathway involves β-oxidation and elimination leading to carboxylation, hydroxylation, and the gradual shortening of the ω-segment of the molecule. A fraction of infused LTE4 is excreted unchanged in urine and because this fraction remains constant, urinary concentrations of LTE4 have been used to reflect systemic synthesis of LTC4. It is likely that cellular activation leads not only to enhanced leukotriene release but also to increased capacity to degrade them.

Regulation of leukotriene generation 5-LO products may be utilized for further metabolism by other cells without the enzyme 5-LO. Such transcellular metabolism can lead to the generation of LTC4 by platelets (Edenius et al. 1988; Maclouf & Murphy 1988), mast cells (Dahinden et al. 1985), airway epithelium and vascular endothelial cells (Feinmark & Cannon 1986), or to generation of LTB4 by erythrocytes from LTA4 supplied by neutrophils (McGee & Fitzpatrick 1986) (Fig. 26.3).

Leukotriene metabolism and elimination Removal of leukotrienes from sites of inflammation requires rapid inactivation by specific and appropriately located enzymes. LTB4-20-hydroxylase (P450LTB) is located exclusively in neutrophil microsomes and converts LTB4 by ωoxygenation to 20-OH-LTB4, which has substantially less PMN chemotactic and activating activity than LTB4. Further metabolism takes place by oxidation to 5S,12R-dihydroxy20-aldehyde-6,14-cis-8,10-trans-eicosatetraenoic acid (20CHO-LTB4), which has no biological activity. This is followed by irreversible conversion to 20-carboxy-6,14-cis-8,10-trans-

572

The generation of lipoxygenase products is tightly controlled and is subject to regulation at several levels. Firstly, the expression of individual lipoxygenase pathway enzymes is subject to developmental and cytokine regulation. This is illustrated by the requirement for IL-3 for the full expression of 5-LO, FLAP, and LTC4 synthase in developing mouse mast cells (Murakami et al. 1995). The generation of LTC4 in human culture-derived mast cells was upregulated by IL-4, which markedly induced the expression of LTC4 synthase (Hsieh et al. 2001). Leukotriene generation was further upregulated by IL-5, which induced the redistribution of 5-LO to the nucleus (Hsieh et al. 2001). 5-LO is a pivotal point of the cross-talk between the 5-LO and COX pathways. PGE2, through the induction of adenosine 3′,5′-cyclic monophosphate (cAMP) and phosphorylation of 5-LO, regulates both the activity of the enzyme and its subcellular location. Thus, phosphorylation of 5-LO by protein kinase A (PKA) at serine 523 leads to inhibition of 5-LO activity (Luo et al. 2004). Substitution of Ser with Ala abolished the effect of PKA, whereas substitution with glutamate

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to mimic the effects of phosphorylation led to inhibition of 5-LO activity and cellular leukotriene generation. In addition to reducing the intrinsic catalytic activity of the enzyme, PKA phosphorylation at Ser 523 leads to redistribution of 5-LO from the nucleus to the cytoplasm (Luo et al. 2005), an event associated with reduced cellular capacity for leukotriene biosynthesis (Luo et al. 2003). It may be of note that Ser 523 is located within a putative nuclear import sequence (Jones et al. 2003).

In addition to the perinuclear region, lipid bodies have been implicated as a site of eicosanoid biosynthesis. The evidence for this is based on the following observations: 1 Lipid body formation is induced in leukocytes in response to cis-unsaturated fatty acids (Bozza et al. 1996) and in eosinophils in response to PAF (Bozza et al. 1997) and the chemokines eotaxin and RANTES (Bandeira-Melo et al. 2001). IL-16 via release of endogenous chemokines (Bandeira-Melo et al. 2002a) or PGD2 (Mesquita-Santos et al. 2006) is associated with increased leukotriene generation. 2 The enzymes of eicosanoid biosynthesis (cPLA2α, 5-LO, LTC4 synthase, COX) can be detected in lipid bodies by immunofluorescence labeling (Bozza et al. 1997; Yu et al. 1998). 3 Immunostaining of carbodiimide-fixed cells reveals cysteinyl leukotriene formation in lipid bodies, for example in chemokine-primed eosinophils in response to A23187 (Bandeira-Melo et al. 2001). In addition, the upstream signaling molecules phosphoinositide 3-kinase, ERK, and p38 MAPK are present in lipid bodies and are required for lipid body-associated leukotriene generation (Yu et al. 1998; Bandeira-Melo et al. 2001).

Sites of leukotriene biosynthesis As noted above, the perinuclear region is well established as a prominent site of eicosanoid biosynthesis. cPLA2α translocates from its cytosolic location to the nuclear envelope or Golgi apparatus in response to a calcium flux in a manner that depends on its C2 (calcium-binding) domain. Likewise, 5-LO translocates from either the cytosol or the nucleosol to the nuclear envelope in response to intracellular calcium mobilization. FLAP and LTC4 synthase are integral perinuclear membrane proteins that are able to form heterotrimers (Mandal et al. 2004) (Fig. 26.4).

Plasma membrane

Cytosol

Nuclear envelope

Nucleus

ER

COX-1

export PGD2

PGH2 hPGDS

SCF

AA

c-kit AA cPLA2

Ca2+

IgE Ag

AA

PO4

FceRI

FLAP 5-LO

FceRI

2+

LTA4 GSH

IgE export

Fig. 26.4 Subcellular regulation of eicosanoid biosynthesis depicting the pathways of LTC4 and PGD2 in the mast cell. The calcium flux elicited by cross-linking of FceRI or c-kit elicits translocation of cytosolic phospholipase A2a (cPLA2a) to the nuclear envelope where it releases esterified arachidonic acid (AA) from the sn-2 position of membrane glycerophospholipids; the enzymatic activity of cPLA2a is augmented by phosphorylation by MAP kinases. 5-Lipoxygenase (5-LO) likewise translocates from either a nuclear or a cytoplasmic location to the nuclear envelope in response to Ca2+ flux. AA is presented to 5-LO by the integral

Ca LTC4 synthase

LTC4

perinuclear membrane protein 5-LO activating protein (FLAP), and metabolizes AA to the unstable epoxide intermediate leukotriene LTA4. LTC4 synthase, also an integral perinuclear membrane protein, conjugates glutathione (GSH) to LTA4 to generate LTC4 that is exported from the cell by the multidrug resistance transporter, MDR1. Constitutively expressed cyclooxygenase (COX)-1 and inducible COX-2 are present in the nuclear envelope and contiguous endoplasmic reticulum (ER); they metabolize AA to the unstable intermediate PGH2 which is metabolized to PGD2 by hematopoietic PGD synthase (hPGDS).

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The ability of different stimuli to elicit LTC4 formation at different subcellular location in the eosinophils is illustrated by the observation that CCL11 (eotaxin) elicits cysteinyl leukotriene formation in lipid bodies where stimulation via stimuli that elicit calcium flux, such as A23187 or leukocyte immunoglobulin-like receptor A2, elicits cysteinyl leukotriene formation in a perinuclear distribution (Tedla et al. 2003). The relevance of lipid body formation as a site of leukotriene biosynthesis is suggested by the observations that lipid bodies are induced in eosinophils within the lungs of allergenchallenged mice, and in the leukocytes of lipopolysaccharide (LPS)-challenged mice (Vieira-de-Abreu et al. 2005) and the leukocytes of patients with sepsis (Pacheco et al. 2002). The evidence supports the concept that lipid bodies are rapidly inducible, specialized cytoplasmic domains for eicosanoid generation at sites of inflammation. Recent reports reveal that cPLA2α, 5-LO, FLAP, LTC4 synthase, and COX-2 translocate to the phagosome of mouse resident peritoneal macrophages after ingestion of zymosan (Girotti et al. 2004; Balestrieri et al. 2006) and that LTC4 formation was revealed at the phagosome by immunostaining of cardbodiimide-fixed macrophages after ingestion of zymosan.

Biological activity LTB4 LTB4 acts at the level of the microvasculature by increasing leukocyte adherence to endothelium (Hoover et al. 1984) and enhancing cutaneous microvascular permeability (Bray et al. 1981; Issekutz 1981). Other activities include potent chemotaxis for neutrophils (Ford-Hutchinson et al. 1980), mediated by a subset of high-affinity receptors, and chemokinetic and weak chemotactic activity for eosinophils (Palmer et al. 1980; Nagy et al. 1982) (Table 26.2). Consistent with the

cheomotactic activity of LTB4 for PMNs, mice transgenic for the high-affinity LTB4 receptor (BLT1) exhibited increased PMN recruitment in sites of ischemia–reperfusion (Chiang et al. 1999), whereas PMN accumulation was attenuated in mice lacking 5-LO. Infusion of aspirin-triggered lipoxins attenuated PMN accumulation into the lungs following hindlimb ischemia–reperfusion in BLT1 transgenic mice, and topical lipoxin A4 attenuated PMN accumulation at the site of dermal inflammation elicited by LTB4 (Chiang et al. 1999). Recent studies have extended our understanding of the chemotactic capacity of LTB4 beyond granulocytes and cells of the monocyte lineage. Thus LTB4 shows chemotactic activity to progenitor, but not mature, mast cells (Weller et al. 2005). Mature mast cells are not normally found in the circulation. Rather, they circulate as immature progenitors (Arinobu et al. 2005) that differentiate further on recruitment to tissues (Gurish et al. 2001) where they undergo phenotypic maturation that is dependent on the tissue microenvironment (Friend et al. 1996). These studies suggest that LTB4 may regulate the trafficking of progenitor mast cells and reactive mastocytosis at sites of inflammation. BLT1 is expressed at low levels on naive circulating T cells, and is upregulated substantially in CD8+ effector T cells (TEFF) (Goodarzi et al. 2003) and on CD4+ T cells challenged with antigen in nonskewing conditions, or in conditions that favor the development of Th1 or Th2 cells (Tager et al. 2003). LTB4 is a potent chemotactic factor both in vitro and in vivo for effector but not memory CD8+ T cells (Goodarzi et al. 2003). LTB4 augmented adhesion, but not rolling, of both TEFF and central memory CD8+ T cells in the microvasculature of tissue inflammation induced by tumor necrosis factor (TNF)-α and of Th1 and Th2 cells on mouse lung endothelial cells. LTB4 is chemokinetic for monocytes (Palmer et al. 1980), enhances expression of CR1

Table 26.2 Biological activity of LTB4. Activity

Reference

Increase leukocyte adherence to endothelium Increase microvascular permeability (secondary action) Neutrophil chemotaxis and weak eosinophil chemotaxis Monocyte chemokinesis Enhanced expression of CR1 and CR3 receptors on neutrophils and eosinophils Neutrophil aggregation Lysosomal release and superoxide generation Bronchoconstriction via secondary release of TXA2 Enhanced NK cell cytotoxicity for virus-infected and tumor cells Induction of suppressor T lymphocytes Enhances IgE production by human B lymphocytes and augments IL-2 and IFN-g production by T lymphocytes

Hoover et al. (1984) Bray et al. (1981); Issekutz (1981) Palmer et al. (1980) Palmer et al. (1980) Nagy et al. (1982); Lee et al. (1988)

See text for definition of abbreviations.

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Ford-Hutchinson et al. (1980) Goldman & Goetzl (1984) Sirois et al. (1982) Rola-Pleszczynski (1985) Atluru & Goodwin (1986) Rola-Pleszczynski et al. (1986)

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and CR3 (CD11b/CD18) receptors on neutrophils and eosinophils (Nagy et al. 1982; Lee et al. 1988), aggregates neutrophils (Ford-Hutchinson et al. 1980), and releases lysosomal enzymes and generates superoxide via a subset of low-affinity receptors (Goldman & Goetzl 1984). LTB4 is bronchoconstricting, especially when administered intravenously, and this action is mediated indirectly through the action of thromboxane (TX)A2 synthesized locally in lung tissue (Sirois et al. 1982). LTB4 also has immunoregulatory functions. At concentrations of 10 −12 to 10 −8 mol/L, LTB4 significantly enhances natural killer cytotoxicity for virus-infected cells and the tumor cell line K-562 (Rola-Pleszczynski 1985), and induces increased numbers of suppressor T lymphocytes from precursors (Atluru & Goodwin 1986). It also directly increases IgG production by highly purified human tonsillar B lymphocytes and augments IL-2 and interferon (IFN)-γ production in human T lymphocytes (Rola-Pleszczynski et al. 1986). The importance of leukotrienes in PAF-induced shock was initially revealed in 5-LO knockout mice (Chen, X.S. et al. 1994). Interestingly, survival in response to PAF was increased in female but not male mice lacking BLT1 (Haribabu et al. 2000). The importance of autocrine LTB4 in regulating leukocyte responses to PAF was revealed in human PMNs in which the second phase of Ca2+ mobilization was inhibited by LTB4 pretreatment (that would desensitize LTB4 receptors) or a BLT1 receptor antagonist (Gaudreault et al. 2005). BLT1 receptor antagonists also inhibited PAF-induced PMN degranulation and chemotaxis. In human PMNs, LTB4 acts in an autocrine manner to elicit Ca2+ flux, phospholipase D activation, and translocation of the small GTPases ARF1 and RhoA (Grenier et al. 2003).

Cysteinyl leukotrienes The cysteinyl leukotrienes have numerous biological effects (Table 26.3). LTD4, and with lesser potency LTC4 and LTE4, augment postcapillary dermal venular permeability when

administered locally to guinea pigs, as shown by the leakage of intravenously administered dye (Drazen et al. 1980; Lewis et al. 1980b). The cysteinyl leukotrienes increase microvascular permeability influenced by the contraction of adjacent endothelial cells (Drazen et al. 1980; Joris et al. 1987) and augment leukocyte adhesion to endothelial cells (McIntyre et al. 1986). Local application of the cysteinyl leukotrienes to the buccal mucosa produces submucosal edema in hamsters (Dahlén et al. 1981) and subcutaneous injection in humans produces dermal edema sustained for 2–4 hours (Soter et al. 1983). As little as 1 nmol/L LTC4 and LTD4 have been postulated to stimulate mucus secretion from studies of bronchial mucosal explants, and this may contribute to the excess mucus secretion found in bronchial asthma (Marom et al. 1982). The cysteinyl leukotrienes are potent contractile agonists for bronchial smooth muscle in isolated human lobar and segmental bronchi, with LTC4 and LTD4 reported to be approximately 1000 times more potent than histamine in contracting human bronchi in vitro (Dahlén et al. 1980) (Table 26.3). Studies using inhaled cysteinyl leukotrienes have demonstrated the potency of these mediators, in both normal and asthmatic subjects, in causing bronchoconstriction, with LTC4 being the most potent and LTE4 the least active (Adelroth et al. 1986; Drazen 1988; Arm et al. 1990). LTC4 and, to a lesser extent, LTD4 have demonstrated vasoconstrictor properties in guinea-pig skin after intradermal injection (Drazen et al. 1980; Lewis et al. 1980b) and in normal human skin by blanching of an elicited weal at the injection site (Camp et al. 1983; Soter et al. 1983). Coronary vasoconstriction has been demonstrated in sheep after direct infusion into a coronary vessel in vivo (Michelassi et al. 1982). In the rat, intravenous infusion of LTC4 led to systemic vasoconstriction (Pfeffer et al. 1983) and renal vasoconstriction (Badr et al. 1984). LTE4 has been demonstrated to recruit granulocytes and particularly eosinophils into the lamina propria of asthmatic airways (Laitinen et al. 1993) (Fig. 26.5).

Table 26.3 Biological activity of cysteinyl leukotrienes. Activity

Reference

Augment postcapillary dermal vascular permeability in guinea pigs Increased microvascular permeability Increase leukocyte adhesion to endothelial cells Increase mucus secretion Potent contractile agonists for bronchial smooth muscle Vasoconstriction Coronary vasoconstriction in sheep Systemic vasoconstriction in rats Renal vasoconstriction in rats Recruit granulocytes into the lamina propria of asthmatic airways (LTE4)

Drazen et al. (1980); Lewis et al. (1980a) Drazen et al. (1980); Joris et al. (1987) McIntyre et al. (1986) Marom et al. (1982) Dahlén et al. (1980) Camp et al. (1983); Soter et al. (1983) Michelassi et al. (1982) Pfeffer et al. (1983) Badr et al. (1984) Laitinen et al. (1993)

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Pharmacology and superoxide generation in the presence of cytochalasin B in chemotactically deactivated PMNs, which do not express high-affinity receptors, and the requirement of significantly higher concentrations of LTB4 than for chemotaxis implied that degranulation is mediated by the low-affinity receptor for LTB4 (Goldman & Goetzl 1984). Using differentiated HL-60 cells, which respond by chemotaxis to LTB4, it was found that LTB4 stimulated a dose-dependent increase in guanosine triphosphate (GTP) hydrolysis and guanosine 5′[γ-thio]triphosphate binding, indicating that LTB4 receptors on HL-60 cells are coupled to a G protein. Both pertussis and cholera toxin were able to inhibit this effect, consistent with action through Gαi (McLeish et al. 1989).

1000

Cell numbers/mm2

100

10

1

Pre

Post LTE4

Pre Post Methacholine

Fig. 26.5 Change in the number of eosinophils (closed circles) and neutrophils (open circles) in the lamina propria of airways mucosa before and after provocation with inhaled LTE4 or methacholine. (From Laitinen et al. 1993, with permission.)

Receptors The leukotriene receptors were first characterized based on their relative potencies in tissue and cellular assays and on the actions of novel pharmacologic antagonists. As in the case of the prostanoid receptors, that classification has been retained with the molecular characterization of the leukotriene receptors (Coleman et al. 1995).

LTB4 receptors The LTB4 receptor was initially identified in PMNs and later demonstrated in eosinophils, monocytes, and lymphocytes (Rola-Pleszczynski et al. 1986). Scatchard analysis of the binding of [3H]LTB4 to freshly isolated PMNs demonstrated the expression of two distinct subsets of receptors: a highaffinity (Kd 0.5– 5 nmol/L), low-density (20 × 103 sites per neutrophil) receptor and a low-affinity (Kd 15–500 nmol/L), high-density (40– 400 × 103 receptors per neutrophil) receptor (Goldman & Goetzl 1982). These two receptor classes for LTB4 also differ in their stereospecificity for stereoisomers of 5,12-diHETE. A decreased chemotactic response to subsequent LTB4, termed “deactivation,” was achieved by preincubating PMNs with 10 nmol/L LTB4 followed by washing and this was paralleled by a selective loss of high-affinity receptors, indicating that the high-affinity receptor mediates chemotactic migration and aggregation. The elicitation of degranulation

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BLT1 The high-affinity cell-surface receptor for LTB4, BLT1, was identified using a cDNA subtraction strategy (Yokomizo et al. 1997). HL-60 cells treated with retinoic acid differentiate into granulocytes that exhibit increased LTB4 binding. Therefore a subtraction cDNA library was generated using HL-60 cells cultured with and without retinoic acid. Of 66 clones that were sequenced, one encoded the 3′ untranslated region of an orphan G protein-coupled receptor (GPCR). Using this original cDNA as a probe, two identical full-length cDNA clones were isolated from a cDNA library of retinoic-acid differentiated HL-60 cells. Northern blotting revealed that mRNA for BLT1 was strongly expressed in peripheral blood leukocytes. The cDNA for BLT1 was most closely related to the type 3 and type 5 somatostatin receptors, the human IL-8 receptor, the formyl-peptide-related receptor/lipoxin A4 receptor, and the formyl peptide receptor. A variety of cell lines, including COS-7 cells and HEK-293 cells transfected with the BLT1 cDNA and retinoic acid-differentiated HL-60 cells, demonstrated high-affinity specific binding of LTB4 (Kd 0.1–0.25 nmol/L). Binding of [3H]LTB4 to membrane fractions of transfected COS-7 cells revealed competition by LTB4, 20OH-LTB4, 12-oxo-LTB4, 12-(R)-HETE, and 20-COOH-LTB4, with KI values of 0.38, 7.6, 7.6, 30, and 190 nmol/L, respectively. In CHO cells stably expressing BLT1, LTB4 inhibited forskolin-induced elevations in cAMP and this inhibition was blocked by pertussis toxin. LTB4 also elicited calcium flux and inositol 1,4,5-trisphosphate (IP3) accumulation in a manner that was partially sensitive to inhibition by pertussis toxin. In contrast, calcium fluxes induced in HL-60 cells by LTB4 are completely blocked by pertussis toxin, indicating that BLT1 can couple to different G proteins. LTB4 was both chemotactic and chemokinetic for transfected CHO cells and was inhibited by pertussis toxin and by the BLT1 receptor antagonists U-75302 and ONO-4057. The cDNA for BLT1 was identical to that for a putative purinergic receptor, P2Y7. However, C6-15 glioma cells expressing the BLT1 cDNA responded with intracellular calcium flux to LTB4 but not to ATP. The BLT1 gene lacks TATA and CAAT boxes in the 5′ flanking region. Rather, there is a GC element in the promoter

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region that binds SP1 (Kato et al. 2000). Expression of the gene is regulated by methylation. Thus there is methylation of the CpG sites in the promoter region in nonhematopoietic cells that lack expression of BLT1. Structure and function of BLT1 A low-resolution structural characterization of the structure of BLT1 expressed in Escherichia coli was compatible with seven transmembrane helices (Baneres et al. 2003), with evidence for conformational change on ligand binding. In addition to its seven transmembrane helices, BLT1 is predicted to have a C-terminal helix similar to that observed in the crystal structure of rhodopsin, but without the cysteine residue that is palmitoylated in the rhodopsin receptor to tether it to the plasma membrane. Absence of helix 8 (Okuno et al. 2003) increases the affinity of BLT1 for LTB4 and prevents ligandinduced lowering of the affinity of BLT1 for its ligand, leading to prolonged calcium flux in response to LTB4 (Okuno et al. 2003). Mutation of the di-leucine motif (Leu 304/305) in the C-terminal tail of BLT1 led to constitutive activation and failure of ligand-dependent internalization (Gaudreau et al. 2004). BLT1 is desensitized by protein kinase C (PKC) and G protein receptor-coupled kinases (GRKs). Thus internalization of BLT1 expressed in RBL-2H3 cells was blocked by dominant negative GRK2 and promoted in HEK-293 cells cotransfected with GRK2 (Chen et al. 2004). Deletion mutants revealed the importance of the C-terminal tail for association with GRK2 and internalization (Chen et al. 2004). Substitution of Thr 308 within helix 8 prevented GRK-6 mediated inhibition of LTB4-induced phospholipase C activation (Gaudreau et al. 2002). The importance of dimerization of BLT1 for its function was demonstrated using a mutant carrying C97S, which has 100-fold reduced affinity for LTB4 compared with the wild-type receptor, and a fluorescent 5-hydroxytryptophan probe at residue 234. The mutant receptor was expressed as a dimer with the wild-type receptor. Ligation of the wild-type receptor with low concentrations of LTB4 elicted a conformational change in the mutant, unliganded receptor, revealing cooperativity of the dimers. Reconstitution of the BLT1/Gprotein complex using partially purified cell preparations of each (Igarashi et al. 1999) or recombinant proteins expressed in E. coli or in insect cells (Baneres & Parello 2003; Masuda et al. 2003) demonstrated that the affinity of BLT1 is regulated by its association with heterotrimeric G proteins (Igarashi et al. 1999; Baneres & Parello 2003), specifically Gi and Go but not Gs (Masuda et al. 2003). Cross-desensitization of BLT1 by CXC chemokines was suggested in a study of BLT1 expression in patients with trauma. BLT1-dependent chemotaxis of human PMNs was reduced in patients after severe trauma, concomitant with increased circulating levels of CXCL8 (IL-8). Pretreatment of human PMNs with CXCL8 or CXCL1 (Gro-α) led to reduced Ca2+ flux in response to subsequent stimulation with subnanomolar concentrations of LTB4, suggesting cross-desensitization

of BLT1 by CXCR1 and CXCR2 (Tarlowe et al. 2003). LTB4 did not downregulate Ca2+ flux to either CXCL8 or CXCL1. The expression of BLT1 is limited to leukocytes. Expression of BLT1 is increased on human PMNs in response to dexamethasone and also, surprisingly, in response to LTB4 (Stankova et al. 2002). Both dexamethasone and LTB4 prolong the survival of PMNs in culture, and their actions are inhibited by a BLT1 antagonist. Expression on monocytes is reduced on CD14+/CD16+ cells (Pettersson et al. 2005), and is diminished on exposure to the proinflammatory cytokines IFN-γ, TNF-α, and LPS, and increased in response to IL-10 or dexamethasone (Pettersson et al. 2005). In contrast, expression of BLT1 in human umbilical vein endothelial cells (HUVECs) was increased more than 10-fold by LPS or IL-1β (Qiu et al. 2006) and LTB4 elicited the generation of NO and CCL2 (MCP-1) from HUVECs (Qiu et al. 2006).

BLT2 The gene encoding BLT2 was identified during characterization of the gene encoding BLT1 (Kamohara et al. 2000; Tryselius et al. 2000; Wang et al. 2000; Yokomizo et al. 2000). The gene encoding BLT2 was identified within the promoter region of the BLT1 gene. In contrast to the restricted distribution of BLT1 in leukocytes, Northern analyses indicate that BLT2 mRNA is more widely expressed, with prominent expression in spleen, ovary, and liver as well as leukocytes, with lower expression in most tissues examined. [3H]LTB4 bound specifically to membranes of HEK-293 cells transfected with BLT2 cDNA (Kd 23 nmol/L). Binding was inhibted by the BLT1 receptor antagonist ONO 4057, but not by U-75302. In CHO cells stably expressing BLT2, LTB4 elicited calcium flux. The maximal response of cells expressing BLT2 was approximately one-third that of cells expressing BLT1 and required about 100-fold higher concentration of LTB4. BLT2, in addition to its lower affinity for LTB4 compared with BLT1, also recognizes a wider range of ligands (Yokomizo et al. 2001). Thus, LTB4 binding was competed at BLT2 by 12-(S)-HETE, 12-(R)-HETE, 15-(S)-HETE, as well as 20-OH-LTB4 and 20-epi-LTB4. 20-OH-LTB4 and 12-HETE and 15-HETE at concentrations up to 1 μmol/L did not elicit calcium flux from CHO cells transfected with BLT2. LTB4induced chemotaxis of CHO cells transfected with BLT2 was completely inhibited by pertussis toxin, whereas calcium fluxes were only partially inhibited. As with BLT1, LTB4 did not elicit an increase in cAMP in BLT2-transfected CHO cells, but did inhibit the rise in cAMP elicted by forskolin that was pertussis toxin insensitive (in contrast to BLT1). Thus, BLT2 signals by coupling to Gi and Gq and Gz (Kamohara et al. 2000).

Cysteinyl leukotriene receptors The evidence for separate cysteinyl leukotriene receptors was initially supported by differences in biological activity of the individual leukotrienes, the effects of leukotriene receptor

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antagonists, and by radioligand binding studies. Much of this evidence came from work carried out in the guinea-pig lung, which supported the existence of distinct receptors for LTC4, LTD4, and LTE4. The evidence for human tissue was more limited but was supported by the demonstration in human lung of a ligand-specific and stereospecific LTD4 receptor regulated by GTP. In guinea-pig lung the existence of separate receptors for LTC4, LTD4, and LTE4 was demonstrated by physiologic and radioligand binding studies. The rank order of potency of the sulfidopeptide leukotrienes for contraction of tracheal spirals (LTE4 > LTD4 = LTC4) is different from that for contraction of parenchymal strips (LTD4 > LTE4 > LTC4), thereby suggesting three separate receptors (Drazen et al. 1983; Lee, T.H. et al. 1984). Furthermore, whereas LTC4 and LTE4 elicited monophasic contraction in peripheral airway strips, LTD4 evoked a biphasic response (Drazen et al. 1980). Evidence for a separate LTE4 receptor in guinea-pig lung is provided by the capacity of LTE4 to enhance histamine responsiveness in a time- and dose-dependent manner, an effect that could not be reproduced by LTC4 and LTD4 despite eliciting the same magnitude of contraction of tracheal smooth muscle as LTE4 (Lee, T.H. et al. 1984). Studies in guinea-pig lung tissue in the presence of L-serine borate, which blocks the conversion of LTC4 to LTD4, showed that the LTD4/LTE4 antagonist FPL 55712 was unable to antagonize the contractile activity of LTC4 (Snyder & Krell 1984). Other selective LTD4 antagonists were shown to antagonize LTD4-induced contraction of guinea-pig tracheal strips but had little or no effect on the contractile effects mediated by LTC4 (Fleisch et al. 1985; Snyder et al. 1987). In guinea-pig uterus, specific binding of [3H]LTD4 could not be detected but specific and saturable binding of [3H]LTC4 was observed and reversed with unlabeled LTC4 (Levinson 1984). The binding of [3H]LTD4 and [3H]LTE4, but not [3H]LTC4, in guinea-pig lung was enhanced by divalent ions and the rate of dissociation was accelerated by either NaCl or GTP, providing further evidence for a multireceptor theory (Pong & DeHaven 1983). Several studies provided evidence for the heterogeneity of LTD4 receptors, both in the guinea-pig lung and across other tissues and species (Fleisch et al. 1982; Krell et al. 1983; Hua et al. 1985). Radioligand receptor binding assays with guinea-pig trachea (Krell et al. 1983) and lung membranes (Aharony et al. 1989), using specific LTD4 receptor antagonists, supported the existence of at least two distinct receptors for LTD4, with LTE4 preferentially interacting with only a subset of LTD4 receptors. This contrasted with the evidence in studies of human airways. Responses elicited by LTC4 and LTD4 are both inhibited to a similar degree by agents that are selective LTD4 receptor antagonists, with the inference that all the biological effects attributed to LTC4 can be explained by the bioconversion of LTC4 to LTD4 (Drazen & Austen 1987). Using radiolabeled LTD4 binding displacement assays, it was demonstrated that LTE4 binds to the identical receptor as

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LTD4 (Cheng & Townley 1984). However, these studies may be flawed because the prevention of metabolic conversion of LTC4 to LTD4 was not demonstrated experimentally, and this could explain the discrepancy in the literature.

CysLT1 Identification of the receptor A number of expression cloning and biochemical strategies were employed unsuccessfully in the 1990s to attempt to clone a cDNA for the cysteinyl leukotriene receptor CysLT1. Rapid progress in cDNA cloning, including the generation of databases of expressed sequence tags, facilitated the molecular characterization of the cysteinyl leukotriene receptors. In 1999 a previously cloned orphan GPCR, HG55 (identical to the cDNA HMTMF81) was “de-orphanized” and shown to have the pharmacologic properties of CysLT1 (Lynch et al. 1999; Sarau et al. 1999). CysLT1 has 32% amino acid identity to a purinergic receptor, P2Y1, and 28% identity to BLT1. Xenopus laevis oocytes injected with the complementary RNA for CysLT1 responded to LTD4 with a brisk calcium-dependent chloride flux (EC50 3 nmol/L, threshold 0.01 nmol/L) that was blocked by the selective CysLT1 receptor antagonist MK-571. Xenopus laevis melanophores, which respond to GPCR signaling with either aggregation or dispersion of pigment, were used to further characterize agonist responses of CysLT1. In this system the order of potency of the cysteinyl leukotrienes was LTD4 > LTC4 > LTE4, with EC50 values of 0.4, 21, and 212 nmol/L, respectively. Similar values were obtained in transfected HEK-293 cells: 2.3, 24, and 240 nmol/L, respectively. Signaling through CysLT1 was insensitive to pertussis toxin, indicating that it is not linked to Gi. In saturation binding assays the rank order of affinities was LTD4 >> LTC4 = LTE4 >> LTB4. The CysLT1 receptor antagonists montelukast, pranlukast, and zafirlukast all demonstrated highaffinity binding to membranes transfected with CysLT1 and dose-dependently displaced [3H]LTD4 from HEK-293 cells transfected with the cDNA encoding CysLT1. Molecular characterization of CysLT1 facilitated studies of its regulated expression and function in cells of both hematopoietic and nonhematopoietic origin, allowed the characterization of its gene, and permitted the generation of transgenic mice lacking expression of its gene, Cysltr1, extending our understanding of the function of the cysteinyl leukotrienes and CysLT1 beyond contraction of airway and vascular smooth muscle. CysLT1 gene The gene encoding CysLT1 was mapped to the human X chromosome at Xq13–q21 (Lynch et al. 1999). It contains five exons, the fifth exon containing the open-reading frame and 5′ untranslated sequence (Woszczek et al. 2005). There are multiple transcription starts and variable splicing of the first four exons, providing transcripts with variable 3′ untranslated sequence. Various polymorphisms of the human CysLT1

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gene have been described in association with allergy. There is increased frequency of the −634T/− 475C/−336G haplotype in a Korean population with a history of aspirin-sensitive respiratory disease (Kim, S.H. et al. 2006) . The T927C polymorphism was reported to be increased in Spanish males with atopic asthma and atopic dermatitis (Arriba-Mendez et al. 2006) and to be associated with more severe atopy in females in the UK (Hao et al. 2006). Expression and function Northern blot analysis showed high expression of CysLT1 in spleen and leukocytes, with lower expression in several other tissues including lung and small intestine (Lynch et al. 1999; Sarau et al. 1999). In situ hybridization revealed CysLT1 not only in smooth muscle of lung tissue but also in lung macrophages (Lynch et al. 1999). The expression of CysLT1 has been demonstrated in human nasal polyps (Chao et al. 2006), with markedly increased expression in individuals with aspirin-exacerbated respiratory disease that was decreased following desensitization to aspirin (Sousa et al. 2002). CysLT1 expression is increased in the airways in asthma (Zhu et al. 2005) and is further increased in severe asthma, with immunohistochemical identification of its expression in eosinophils, PMNs, mast cells, macrophages, B cells and plasma cells, but not T cells (Figueroa et al. 2001; Zhu et al. 2005). The promoter region of the human CysLT1 gene contains an active STAT6 binding site and expression is regulated by IL-4 and IL-13 (Woszczek et al. 2005). Thus THP-1 cells primed with IL-4 demonstrated increased generation of CCL2 (MCP-1) in response to LTD4 and LTC4 (Woszczek et al. 2005). IL-4 primed human cord blood-derived mast cells (hCBMCs) for increased Ca2+ flux (Mellor et al. 2001) and release of IL-5, TNF-α, and CCL4 (MIP-1β) in response to LTC4, LTD4, and UDP without concomitant release of histamine or PGD2 (Mellor et al. 2002). Pretreatment of hCBMCs with the CysLT1 antagonist MK-571 inhibited the generation of IL-5 and TNF-α in response to cross-linking of FcεRI, implying an autocrine role for CysLTs in amplifying IgE-mediated cytokine generation by mast cells (Mellor et al. 2002). Expression of CysLT1 by human monocytes is upregulated by IL-13 and IL-4, with a concomitant increase in Ca2+ flux and chemotaxis to LTD4 (Thivierge et al. 2001). Treatment of human fetal lung fibroblasts with IL-13 increased the expression of CysLT1 (Chibana et al. 2003). The regulation of CysLT1 is not confined to IL-4 and IL-13. The expression and function of CysLT1 in human airway smooth muscle cells is augmented by IFN-γ (Amrani et al. 2001). Treatment of eosinophilic HL-60 cells with IL-5 increased the expression of CysLT1 (Thivierge et al. 2000). CysLT1 is expressed on human T cells and its expression is increased in response to T-cell receptor (TCR) engagement (Spinozzi et al. 2004). Montelukast inhibited T-cell proliferative responses to TCR engagement with concomitant increase in IFN-γ release and increased apoptosis (Spinozzi et al. 2004). T cells from mice carrying a mutant form of linker for activa-

tion of T cells have increased numbers of CysLT1 and exhibit increased Ca2+ flux and chemotaxis to LTD4 (Prinz et al. 2005). In addition to its ability to contract bronchial smooth muscle, LTD4 augmented the mitogenic response to human airway smooth muscle cells to epidermal growth factor (Panettieri et al. 1998). With respect to the function of CysLT1 in cells of hematopoietic origin, in addition to eliciting Ca2+ flux and release of cytokines, LTD4 upregulated the expression of CD11b/CD18 on human eosinophils leading to increased adhesion to intercellular adhesion molecule (ICAM)-1 (Nagata et al. 2002). The regulation of dendritic cell migration by CysLTs was revealed in mice carrying targeted disruption of multidrug resistance-associated protein 1 (MRP1), which is essential for export of LTC4 from cells (Leier et al. 1994; Muller et al. 1994). Thus there was impaired migration of dendritic cells lacking MRP1 from the skin to the lymphatics which was restored by exogenous cysteinyl leukotrienes (Robbiani et al. 2000). Maturation of human monocytes to dendritic cells in the presence of LPS led to decreased expression of CysLT1 and increased expression of CysLT2 in a COXdependent manner that was reproduced by TNF-α and PGE2 (Thivierge et al. 2006). In contrast, dendritic cells differentiated in the presence of the TLR3 agonist poly I:C exhibited chemotaxis to LTD4, which also enhanced migration to CCL19 (MIP-3) (Thivierge et al. 2006). CysLT1 undergoes agonist-induced internalization that depends on phosphorylation by PKC of critical serene residues in the carboxyl tail of the receptor (Naik et al. 2005). Deletion of residues 310–321, mutation of serines 313–316, or inhibition of PKC leads to reduced agonist-induced internalization, with reduced generation of inositol phosphates, and reduced Ca2+ flux to CysLT stimulation (Naik et al. 2005).

CysLT2 Identification of the receptor The screening of public databases for novel orphan GPCRs led to the identification of CysLT2 (Heise et al. 2000; Nothacker et al. 2000; Takasaki et al. 2000). When the cRNA encoding the full-length open reading frame of CysLT2 was injected into X. laevis oocytes the cells responded to LTC4 and LTD4 with Ca2+-dependent chloride flux that was ablated by pretreatment with either LTC4 or LTD4 and was resistant to pertussis toxin. The response was resistant to inhibition by MK-571, but was inhibited by the CysLT1/CysLT2 antagonist Bay u9773. The rank order of potency of the cysteinyl leukotrienes in eliciting Ca2+ flux in HEK-293T cells transfected with CysLT2 and in radioligand binding studies of membranes of COS-7 cells transfected with CysLT2 was LTC4 = LTD4 >> LTE4. EC50 values for LTC4, LTD4, and LTE4 in eliciting Ca2+ flux from transfected HEK-293 cells were 8.9, 4.4, and 293 nmol/L, respectively. IC50 values for displacing [3H]LTC4 from transfected COS-1 cells by LTC4, LTD4, and LTE4 were 1.5, 59, and 1890 nmol/L, respectively (Takasaki et al. 2000). Northern blotting revealed widespread expression of CysLT2 in human

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tissues, including leukocytes, heart, spleen, lymph nodes, and neural tissues. In situ hybridization revealed expression of mRNA for CysLT2 in lung macrophages and peripheral blood leukocytes including eosinophils, in pheochromocytes and ganglion cells of the adrenal gland, and in Purkinje cells of the heart. The gene encoding CysLT2 was localized to human chromosome 13q14. Transgenic mice At present there are no specific inhibitors of CysLT2, so transgenic mice lacking expression of CysLT2 may be informative as to the role of the receptor. As in mice lacking CysLT1, the vascular leak in response to passive cutaneous anaphylaxis (PCA) was attenuated in Cysltr2-null mice. Unlike mice lacking CysLT1, the vascular leak in response to intraperitoneal zymosan was intact in mice lacking CysLT2 (Beller et al. 2004a), suggesting tissue-specific roles for the two cysteinyl leukotriene receptors in regulating vascular permeability. Interestingly, the pulmonary fibrotic response to bleomycin was augmented in Cysltr2-null mice (Beller et al. 2004a), suggesting a protective role for cysteinyl leukotrienes acting at CysLT2, in contrast to the attenuated fibrotic response to bleomycin in Cysltr1-null mice (Beller et al. 2004b). The effects of CysLT2 on vascular permeability were confirmed in mice with transgenic overexpression of the receptor in endothelial cells under the control of the Tie 2 promoter (Hui et al. 2004). These mice showed increased vascular leak in response to PCA and LTC4, but a decreased pressor response to intravenous LTC4 associated with increased NO generation. Expression and function Compared with CysLT1, there is limited information on the regulation of the expression and function of CysLT2. CysLT2 is prominently expressed by endothelial cells. Gene profiling of HUVECs treated with LTD4 yielded 37 genes, most of which were also upregulated by thrombin (Uzonyi et al. 2006). Genes induced by LTD4 included EGR1, E-selectin, CXCL2, CXCL8 (IL-8), and COX-2. Transcript induction was not blocked by CysLT1 receptor antagonism. The combined results suggest that CysLT2 may act in synergy with protease-activated receptor 1 to elicit vascular injury. Polymorphisms of the gene encoding CysLT2 The –1220 A/C polymorphism of the human gene encoding CysLT2, CYSLTR2, has been associated with asthma in a Japanese population (Fukai et al. 2004). Four SNPs in the 5′ and 3′ flanking region of the CysLT2 gene and five haplotypes were identified in a Korean population (Park et al. 2005). The frequency of certain rare alleles (− 819 T/G, 2078 C/T, and 2534 A/G) was higher in individuals with a history of aspirin-exacerbated respiratory disease. The M202V SNP encodes a receptor that has attenuated Ca2+ flux to LTD4 and is associated with atopy in the population of Tristan da Cunha (Thompson et al. 2003).

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Is there another CysLT receptor? Several studies have reported biological properties of the cysteinyl leukotrienes that cannot be explained on the basis of the recognized pharmacologic properties of CysLT1 and CysLT2, whether encoded on separate genes or generated by heterodimerization of cysteinyl leukotriene and/or related receptors. The cysteinyl leukotrienes were first recognized for their ability to potently constrict smooth muscle. While bronchoconstriction elicited by LTD4 was inhibited by the CysLT1 antagonists pranlukast, pobilukast, and zafirlukast, the co-mitogenic activity of LTD4 with epidermal growth factor was inhibited by pranlukast and pobilukast but was resistant to zafirlukast, suggesting that it might be mediated by a receptor separate from CysLT1 (Panettieri et al. 1998). Studies suggesting that there is a receptor with selective sensitivity to LTE4 have been referred to above. Cysteinyl leukotrienes and the pyrimidinergic agonist UDP elicited concentration-dependent calcium fluxes in hCBMCs that were blocked by the CysLT1 receptor antagonist MK571 (Mellor et al. 2001). Priming of hCBMCs with IL-4 for 5 days enhanced their sensitivity to each agonist, but preferentially lowered the threshold for activation by LTC4 and UDP by approximately 3 logs without altering CysLT1 receptor mRNA or surface protein expression, implying the induction of a second cysteinyl leukotriene receptor. IL-16, eotaxin, and RANTES stimulate vesicular transportmediated release of preformed, granule-derived IL-4 and RANTES from eosinophils and the synthesis of LTC4 at intracellular lipid bodies (Bandeira-Melo et al. 2002b). 5-LO inhibitors blocked IL-16-, eotaxin-, and RANTES-induced IL-4 release. After membrane permeabilization, LTC4 and LTD4 stimulated the release of IL-4 but not RANTES. This was blocked by pertussis toxin, implying the involvement of a GPCR. Inhibitors of CysLT1 and CysLT2 did not block LTC4elicited IL-4 release. LTC4 was 10-fold more potent than LTD4 and at low concentrations (0.3–3 nmol/L) elicited, and at higher concentrations (> 3 nmol/L) inhibited, IL-4 release from permeabilized eosinophils. These results demonstrate that LTC4, well recognized as a paracrine mediator, may also dynamically govern inflammatory and immune responses as an intracrine mediator of eosinophil cytokine secretion mediated by an intracellular GPCR distinct from CysLT1 and CysLT2. Ciana et al. (2006) recently characterized GPR17, an orphan GPCR intermediate in phylogeny between the cysteinyl leukotriene receptors and the P2Y pyrimidinergic receptors, as a dual UDP/CysLT receptor. Using a [35S]GTP-γ-S binding assay, the authors demonstrated responses to LTC4, LTD4, UDP, UDP-glucose, and UDP-galactose in the 1321N1 astrocytoma cell line transfected with GPR17. There was no response to ATP, ADP, 2-methyl-thio-ADP, UTP, α,β-methylene ATP, and guanosine, indicating that GPR17 responds to a different array of agonists than the already cloned cysteinyl leukotriene and P2Y receptors. Similar responses were seen to

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cysteinyl leukotrienes in COS-7 cells and to nucleotides in CHO cells and HEK cells transfected with GPR17. Agonist responses were inhibited by both P2Y antagonists and CysLT1 antagonists. Gi linkage was demonstrated by inhibition of agonist responses by pertussis toxin and by the ability of UDP and LTD4 to inhibit forskolin-induced elevations of cAMP. Immunoreactive GPR17 was seen within and at the borders of rat ischemic cerebral cortex, and ischemic damage in rats was inhibited by the P2Y inhibitor cangrelor, by the CysLT1 inhibitor montelukast, and by antisense knockdown of GPR17 expression in the brain. Thus, GPR17 is a dual UDP/CysLT receptor.

Actions in bronchial asthma It was the lasting contractile property of SRS-A at very low concentrations that originally generated interest in these lipid compounds as putative mediators in bronchial asthma. Isolated tracheal, bronchial, or parenchymal tissues from guinea pigs (Piper & Samhoun 1981), dogs (Johnson et al. 1983) and rats (Szarek & Evans 1988) have been found to contract in response to LTC4 at nanomolar concentrations. In guinea-pig tissue, LTC4 and LTD4 are approximately equipotent as contractile agonists, with EC50 values of 0.1–1 nmol/L. LTE4 is less potent in the same model, with EC50 values of 30–100 nmol/L (Dahlén 1983; Drazen & Austen 1987). Similar effects of the cysteinyl leukotrienes have been demonstrated in studies in vitro of human bronchus (Dahlén et al. 1980) and tracheal smooth muscle (Jones et al. 1982). The contractile action of LTC4 is at least 1000 times more potent than histamine in causing muscle contraction, and LTD4 has a similar potency to LTC4. Hanna et al. (1981) found a lower contractile activity of LTC4 and LTD4 than that of histamine. This contrasts with the findings of Chagnon et al. (1985), who showed that LTA4, LTC4, and LTD4 are at least 200 times more potent than histamine in human lung parenchyma in vitro. The release of LTC4, LTD4, and LTE4 was demonstrated in vitro in isolated bronchi from two birch pollen-sensitive asthmatic subjects after antigen challenge, and the amount of leukotriene released was found to correlate with the contraction evoked by allergen (Dahlén et al. 1983). In the guinea pig, pretreatment with a COX inhibitor led to a diminished LTC4- and LTD4-induced contractile response in isolated perfused lung, suggesting that bronchoconstrictor prostanoids are released by leukotriene action and contribute significantly to their contractile activity (Piper & Samhoun 1981). In contrast, in isolated human lung tissue indomethacin pretreatment had no effect on the contractile response to antigen induced by leukotriene release, which could be abolished by a leukotriene biosynthesis inhibitor (Dahlén et al. 1983). In humans, in vivo studies of inhaled leukotrienes have demonstrated potent stimulation of contractile activity in the airways in both normal and asthmatic subjects. Inhalation of nebulized solutions of leukotrienes leads to airway obstruc-

tion measured by falls in specific airway conductance or flow as measured from full or partial expiratory flow–volume curves. In five normal subjects the potency of LTC4 was 600– 9500 times greater than that of histamine, with 20 μg/mL LTC4 and 2–10 mg/mL histamine concentrations required to produce a 30% fall in expiratory flow rate at 30% of baseline vital capacity above residual volume (Vmax30) (Weiss et al. 1982a). In the same study, LTD4 was 6000-fold more potent than histamine (Weiss et al. 1983). If data on normal subjects from different sources are combined (Holroyde et al. 1981; Weiss et al. 1982a, 1983; Barnes et al. 1984a; Smith et al. 1985; Adelroth et al. 1986; Kern et al. 1986; Bel et al. 1987), inhaled LTC4 and LTD4 in normal subjects are 2000 times more potent than histamine or methacholine in producing airway obstruction. LTE4, on the other hand, is only 40–60-fold more potent than histamine but produces longer-lasting bronchoconstriction (Davidson et al. 1987; O’Hickey et al. 1988). Despite the similar potencies for LTC4 and LTD4, the timecourse of their action is different. LTC4 has a slower onset of action (10–15 min) than LTD4 and LTE4 (4–6 min), but the response for LTC4 is more prolonged (20–40 min) (Weiss et al. 1983; Drazen 1988). The reason for this difference in timecourse is unclear, but may be related either to the action of the cysteinyl leukotrienes at different receptors or because LTC4 requires metabolism to LTD4 prior to action at a specific receptor site. The discrepancy in the literature on the potency of the cysteinyl leukotrienes using a variety of measures of bronchoconstriction is likely to be due to differences in these parameters as measures of constriction at different sites within the lung. Changes in Vmax30 are mediated by bronchoconstricting agonists considered to act at a peripheral site of action, whereas forced expiratory volume in 1 s (FEV1) and specific airway conductance are measures of a central site of action. Inhalation of LTC4 and LTD4 in normal subjects produced a fall in Vmax30 with little effect on FEV1 (Holroyde et al. 1981). Similarly, Weiss et al. (1982b) demonstrated that a 50-fold greater concentration of LTC4 was required to achieve a 20% fall in FEV1 compared with the concentration required to cause a 30% fall in Vmax30. These studies on normal subjects suggested a predominantly peripheral site of action for LTC4 and LTD4. In asthmatic subjects, inhalation of LTC4 and LTD4 showed similar effects on specific airway conductance and Vmax30 (Barnes et al. 1984b; Smith et al. 1985; Kern et al. 1986), indicating that the site of LTC4 and LTD4 bronchoconstrictor activity in asthma is likely to be in both central and peripheral airways (Barnes et al. 1984a; Pichurko et al. 1989; Molfino et al. 1992). Asthmatic airways also respond by bronchoconstriction to inhaled leukotrienes, but in contrast to normal airways they exhibit smaller responses when comparison to a reference agonist is made. LTC4 and LTD4 have been reported to be of the order of 40-fold (Adelroth et al. 1986) to 140-fold (Griffin et al. 1983) more potent than histamine or methacholine in their bronchoconstrictor effects on asthmatic airways, which

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compares with 600– 9500-fold for LTC4 (Weiss et al. 1982a) and 6000-fold for LTD4 (Weiss et al. 1983) in normal airways compared with histamine. A correlation between airway responsiveness to methacholine (Adelroth et al. 1986) and histamine (Barnes et al. 1984c) and airway responsiveness to LTC4 and LTD4 has been established, but these studies also confirmed the relative lack of airway responsiveness to LTC4 and LTD4 in asthmatic subjects compared with normal subjects (Barnes et al. 1984c). The data for LTE4 is more limited. Using Vmax30 as a measure of airway obstruction, LTE4 was 39-fold more potent than histamine in normal subjects and 14-fold more potent than histamine in asthmatic subjects (Davidson et al. 1987). However, using a 35% fall in specific airway conductance as a measure of bronchoconstriction, the relative potency of LTE4 was found to be two to three times greater in asthmatic than in normal individuals (O’Hickey et al. 1988). In view of the discordance in the literature, a more extensive study was performed to compare the relative potencies of LTC4, LTD4, and LTE4 and the reference agonists histamine and methacholine in the same normal and asthmatic individuals (Arm et al. 1990). The airways of the subjects with asthma were approximately 14-fold, 15fold, sixfold, ninefold, and 219-fold more responsive to histamine, methacholine, LTC4, LTD4, and LTE4, respectively, than for normal subjects. Furthermore, as airway hyperresponsiveness (AHR) to histamine and methacholine increased, so too did the potency of LTE4, in contrast to LTD4 and LTC4 which decreased. Therefore, the results of these studies taken together suggest that, compared with normal airways, asthmatic airways are relatively less responsive to LTC4 and LTD4 but have a disproportionate hyperresponsiveness to the bronchoconstricting effects of LTE4. This indicates an important role for LTE4 in bronchial asthma, which may be due to its relative stability among the cysteinyl leukotrienes and because it persists for the longest time at the site of release (Lam et al. 1988). A subset of asthmatic individuals develop bronchospasm on ingestion of aspirin and other COX inhibitors. These subjects are unusually sensitive to the bronchoconstrictor effects of inhaled LTE4, which is 1870 times more potent than histamine in aspirin-sensitive subjects and only 145 times more potent than histamine in asthmatics who are aspirin tolerant (Arm et al. 1989). Following aspirin desensitization, there was a mean 20-fold decrease in airway responsiveness to LTE4 but no change in histamine responsiveness. In aspirin-sensitive subjects, this selective hyperresponsiveness is exclusive to LTE4 and not found with LTC4 (Christie et al. 1993), suggesting an important role in this form of asthma.

Airway responsiveness Bronchial hyperresponsiveness to contractile agonists such as histamine and methacholine and to nonspecific irritants is a key pathophysiologic feature of asthma. Studies in vitro on guinea-pig ileum have demonstrated that SRS-A enhanced

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the contractile response to histamine (Brocklehurst 1962). Pretreatment of guinea-pig spirals with a contracting dose of LTE4 accentuated the subsequent contractile response to histamine in a time- and dose-dependent fashion (Lee, T.H. et al. 1984). LTC4 and LTD4 did not enhance histamine responsiveness in parenchymal strips. Indomethacin pretreatment abolished the hyperresponsiveness to histamine, despite a lack of effect on the contractile activity of LTE4. A further study in guinea-pig tracheal spirals demonstrated enhanced contractile activity to histamine following LTC4 only in the presence of 0.1 mmol/L Ca2+ ions and this effect was blocked by the leukotriene antagonist FPL 55712 (Creese & Bach 1983). This AHR found in guinea-pig tracheal spirals was specific for histamine and was not found with carbachol or substance P. Furthermore, the effect was blocked not only by indomethacin but also by a TXA2/PGH2 (TP) receptor antagonist (GR32191). Pretreatment with LTE4 induces a similar effect on isolated human bronchus, with a fourfold leftward displacement of the histamine dose–response curve. This effect is blocked by GR32191, suggesting that the observed LTC4/ LTE4-induced hyperresponsiveness to histamine found in human and guinea-pig trachea is mediated by the secondary generation of COX products (Jacques et al. 1991). In normal subjects, inhalation of either a subthreshold dose of LTD4 (Barnes et al. 1984c) or bronchoconstricting doses of LTC4, LTD4, or LTE4 (Arm et al. 1988; O’Hickey et al. 1991) did not significantly enhance airway responsiveness to subsequent histamine inhalation. Inhalation of a bronchoconstricting dose of LTD4 produced an approximately twofold increase in airway responsiveness to methacholine (Kern et al. 1986). In an earlier study, inhalation of a bronchoconstricting dose of LTD4 led to significantly increased airway methacholine responsiveness, which was maximal at 7 days in six of eight subjects and persisted for 2–3 weeks in five subjects (Kaye & Smith 1990). Interestingly, in the same study the degree and duration of changes in methacholine airway responses were similar to those found after PAF inhalation. In asthmatic subjects, pre-inhalation of LTE4 that caused a 41% fall in specific airway conductance produced a doseand time-dependent increase in histamine responsiveness, which reached a peak of 3.5-fold at 7 hours after LTE4 inhalation (Arm et al. 1988). A subsequent study by the same authors found that each of the cysteinyl leukotrienes, LTC4, LTD4 and LTE4, produced an approximately threefold to fourfold increase in histamine responsiveness at 4 hours after inhalation in seven asthmatic individuals (O’Hickey et al. 1991) (Fig. 26.6). The magnitude of this enhanced histamine responsiveness is similar to that observed after inhaled allergen challenge (Cockcroft et al. 1977; Cartier et al. 1982). Prior inhalation of LTC4 was found to have no effect on airways response to inhalation of distilled water in nine asthmatic subjects (Bianco et al. 1985). Predosing with indomethacin significantly inhibited the LTE4-induced hyperresponsiveness to histamine in

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0.5

increase the extent of maximal airway narrowing, two cardinal features of airway responses in bronchial asthma.

0.4

Airway secretions

PD35 histamine (mmol)

0.3

0.2

0.1

0.05 1

4 Time (hours)

7

Fig. 26.6 Time-course of changes in airway histamine responsiveness after inhalation of phosphate-buffered saline (closed triangles), methacholine (closed circles), LTC4 (open triangles), LTD4 (closed squares), and LTE4 (open circles). Each point represents the geometric mean for seven subjects with asthma. (From O’Hickey et al. 1991, with permission.)

eight asthmatic subjects (Christie et al. 1992a). Indomethacin was also found to inhibit the increase in airway responsiveness following inhaled allergen provocation in seven atopic subjects, without an effect on either the early or late asthmatic reaction (Kirby et al. 1989). This suggests that the cysteinyl leukotrienes make a significant contribution to the enhanced airways responsiveness that is so characteristic of asthma and they may exert part of this effect through the secondary generation of COX pathway-derived products. Asthmatic airways are more sensitive to bronchoconstricting agonists such as histamine and methacholine than normal airways, with a leftward shift in the dose–response curve and a greater absolute response. Normal airways are found to reach a plateau of maximum bronchoconstriction at mild degrees of airway narrowing. In a study of eight normal subjects, LTD4 was found to produce a maximal response plateau at a higher level than methacholine. The addition of methacholine at the top of the LTD4 plateau caused a further fall of 6.6% and 4.8% in FEV1 and expiratory flow at 40% vital capacity (V40P), respectively (Bel et al. 1987). This effect could be demonstrated for at least 3 days following LTD4 inhalation. A later study by the same group reported that prior administration of budesonide for 6 days could diminish the maximal response to LTD4 in eight nonasthmatic subjects by 7.9% and 8.4% for FEV1 and V40P, respectively (Bel et al. 1989). Thus, the cysteinyl leukotrienes increase nonspecific AHR and also

In bronchial asthma, abnormalities of airway mucociliary function are suggested by the clinical observation of excessive tracheobronchial secretions, which are difficult to clear and may contribute to bronchial obstruction. The cysteinyl leukotrienes can influence both mucus secretion and composition as well as mucociliary transport within the airways. Cultured human airway explants when exposed to LTC4 and LTD4 increased the rate of secretion of radiolabeled glucosamine as part of a high-molecular-weight glycoprotein by 15 and 26%, respectively (Marom et al. 1982; Coles et al. 1983). Repeated stimulation of the explants led to diminished radiolabeled product secretion with the ratio of bound radiolabel to protein remaining unchanged, suggesting that LTC4 and LTD4 stimulate secretion rather than de novo mucus synthesis (Marom et al. 1982). LTC4 was found to have 1000fold more potent secretagogue effects on mucus secretion than LTD4 when injected into the artery supplying the cervical canine trachea (Johnson & McNee 1983). In vitro studies on cultured airway goblet cells demonstrated enhanced mucin release by the action of physiologic concentrations of LTC4 and LTD4 (Kim et al. 1989). LTC4 and LTD4 also mediate an increase in chloride flux of isolated canine tracheal epithelium, which induces an increase in the short-circuit ionic current (Leikauf et al. 1986) and is accompanied by enhanced fluid secretion (Johnson et al. 1983). In sheep allergic to Ascaris suum, concentrations of LTD4 as low as 25 μg/mL produced significant decreases in tracheal mucus velocity, with the maximum effect observed at 3 hours after leukotriene challenge (Russi et al. 1985). The overall effect of the leukotrienes therefore leads to a thickening of the mucous gel layer and a decrease in tracheal mucus velocity. Cysteinyl leukotrienes were detected in the sputum of 16 of 25 patients with cystic fibrosis and one of five patients with chronic bronchitis (Cromwell et al. 1982). In a study of 30 children with cystic fibrosis, urinary LTE4 levels were found to correlate with sputum LTE4 values, and one-third of the children with cystic fibrosis had urinary LTE4 greater than 200 pmol per mmol creatinine compared with only 3% of normal children (Sampson et al. 1990). The same group reported that in a study of 13 children with cystic fibrosis, the logarithm of sputum LTE4 levels and total cysteinyl leukotriene levels correlated with the overall severity of pulmonary disease, as assessed by Chrispin–Norman chest radiograph scores (Spencer et al. 1992). These findings suggest that cysteinyl leukotrienes may have a role in the sputum abnormalities characteristic of cystic fibrosis and chronic bronchitis.

Leukotriene release in disease Leukotrienes have been detected in a variety of biological fluids by employing sensitive assay systems to detect picogram

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Table 26.4 Release of leukotrienes into biological fluids. Disease

Biological fluid

Leukotriene release

Asthma Clinical disease Symptomatic Acute

BAL BAL Urine

LTE4 detected in mild/severe disease LTB4/LTC4 LTE4: highest levels in patients responding

BAL Urine Urine

LTC4 increases ninefold LTE4 increases 2–4 hours after allergen challenge LTE4 sixfold higher at baseline and increases a further fourfold after aspirin challenge LTC4 levels increase after lysine–aspirin challenge LTB4 rises 12-fold and LTC4 rises fivefold after isocapnic hyperventilation LTE4 found to increase 1.7-fold in only one study of children with severe asthma

Allergen challenge Early-allergen response Aspirin sensitive Exercise induced

Nasal lavage BAL Urine

Cryptogenic fibrosing alveolitis

BAL

LTB4 detected

Persistent pulmonary hypertension

BAL

LTC4 and LTD4 detected in newborn infants

Adult respiratory distress syndrome

Pulmonary edema fluid

LTD4 increased fourfold

Rheumatoid arthritis

Synovial fluid

LTB4 detected in active disease

Gouty arthritis

Joint fluid

LTB4 levels significantly increased

Psoriasis

Skin chamber fluid

LTB4, LTC4 and LTD4 levels detected in increased quantities

Inflammatory bowel disease

Intestinal mucosal fluid

LTB4 detected in both ulcerative colitis and Crohn disease

Acute myocardial infarction

Urine

LTE4 levels raised and return to normal day 3

Hepatorenal syndrome

Urine

LTE4 levels elevated threefold

BAL, bronchoalveolar lavage.

quantities, such as high-performance liquid chromatography (HPLC), radioimmunoassay, and mass spectrometry (Table 26.4). Bronchoalveolar lavage (BAL) has been used as a tool to obtain fluid in pulmonary disease states. Because the ratio of the volume of lavage fluid instilled to that recovered is not always constant, BAL suffers from the drawback that it is not easy to interpret the absolute values given and the results are usually expressed in terms of amounts per volume recovered. Similar concerns have been expressed regarding measurements of mediators in exhaled breath condensates. Urine is a more reliable biological fluid and amounts of mediator are easily standardized by expressing quantities per unit of creatinine, which is excreted in a relatively constant fashion throughout a 24-hour period (Smith et al. 1992). Maltby et al. (1990) found that by infusing three subjects with three doses of radiolabeled LTC4, it was possible to recover a constant 4.1–6.3%, regardless of the amount infused, in the form of LTE4, the most stable of the cysteinyl leukotrienes. Radiolabeled LTC4 instilled into the airways of asthmatics, nonasthmatics, and asthmatics challenged with allergen was found to be excreted in a constant fashion in all three groups,

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with LTE4 as the major metabolite (Westcott et al. 1993). Christie et al. (1994) found a significant correlation between the dose of inhaled LTC4 and the amounts excreted as urinary LTE4. These studies provide strong evidence for the utility of urinary LTE4 as a marker of pulmonary cysteinyl leukotriene release.

Clinical asthma On BAL, 15 of 17 asthmatic subjects with mild to severe disease were found to have detectable LTE4 in BAL fluid, but there was no correlation between LTE4 levels and pulmonary function. None of the group of nine control subjects had detectable LTE4 (Lam et al. 1988). In a study by Wardlaw et al. (1989), eight symptomatic asthmatic subjects had significantly higher levels of LTB4 and LTC4 in BAL fluid than a control group of 14 without asthma. A comparison of 11 healthy with 11 atopic subjects with mild asthma found no difference in cysteinyl leukotriene, histamine, or PAF levels between the two groups, but there was a higher level of PGD2 and a lower level of LTB4 in the asthmatic group (Crea et al. 1992). These findings are in contrast to the two studies above and

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may be due to selection of very mild asthmatic subjects. In a study using urinary LTE4 as a marker for pulmonary cysteinyl leukotriene release, Smith, C.M. et al. (1992 found no difference in baseline urinary levels of LTE4 between 17 normal and 31 asthmatic subjects. In addition, there was no correlation between urinary LTE4 levels as measured in picograms per milligram of creatinine and baseline airway responsiveness to histamine or FEV1 as a percentage of predicted values (Crea et al. 1992). Drazen et al. (1992) examined 72 subjects presenting to accident and emergency departments and classified 22 patients with a doubling of peak expiratory flow rate (PEFR) following nebulized salbutamol as responders, and 19 patients with < 25% increase in PEFR as nonresponders. Urinary LTE4 levels were assayed by precolumn extraction, HPLC, and radioimmunoassay in these two groups and compared with 13 normal controls. Urinary LTE4 levels were significantly higher in responders compared with nonresponders or normal subjects. The authors concluded that the highest levels of LTE4 found in those with acute reversible airflow obstruction were consistent with a bronchospastic role for cysteinyl leukotrienes in spontaneous acute asthma.

Allergen-induced asthma Leukotriene release has been measured in studies employing various bronchial challenge procedures. Wenzel et al. (1990) reported that using HPLC the predominant leukotriene in BAL fluid in atopic asthmatic subjects after endobronchial allergen challenge was LTC4, with an approximate ninefold rise in levels compared with baseline. Measurable levels of LTC4 at baseline were found in 9 of 11 atopic asthmatic subjects but in only one of seven atopic nonasthmatic subjects and one of six nonatopic subjects. There was only a slight rise in BAL LTC4 levels after allergen in the atopic nonasthmatic group and no change in the nonatopic samples. In a further study by Wenzel et al. (1991), the levels of PGD2, TXB2, LTC4, and histamine were measured in BAL fluid before and 5 min after endobronchial allergen challenge in three groups of atopic subjects: seven nonasthmatic subjects, six asthmatic subjects without a late asthmatic response, and six asthmatic subjects with a late asthmatic response. LTC4 was detected in 9 of 12 asthmatic subjects but in only one of seven subjects without asthma. Significant increases in all mediator levels were observed in both groups with asthma post allergen challenge compared with the nonasthmatic group. Interestingly, the asthmatic group without a late asthmatic response recorded significantly higher levels of all four mediators post challenge than the groups with a late response and the nonasthmatic controls. In a study of 17 allergic asthmatic subjects undergoing allergen provocation, there was a fall in FEV1 within the first 2 hours of 25– 59%, and this was accompanied by a rise in urinary LTE4 levels from 46 to 92 ng over a 12-hour collection period. Methacholine challenge alone, which led to similar falls in FEV1, did not significantly change urinary LTE4 excretion. There was a significant correlation

between the decrease in FEV1 during the early asthmatic response, the excretion of urinary LTE4, and airways reactivity. No correlation was found between urinary LTE4 excretion and the severity of late response to allergen, but there was significant prolonged elevated urinary LTE4 excretion in those patients with the most severe late asthmatic responses (Westcott et al. 1991). Manning et al. (1990a) studied 18 asthmatic subjects who were divided into three groups: those with an isolated early asthmatic response (EAR), those with an isolated late asthmatic response (LAR), and those with a dual asthmatic response (DAR). Urinary LTE4 rose significantly from baseline values only in the two groups with an EAR, with a rise from 150 to 1816 pg/mg creatinine in the group with an isolated EAR and a rise from 66 to 174 pg/mg creatinine in the group with an EAR preceding an LAR. No increase in urinary LTE4 was found in the group with an isolated LAR. Furthermore, the degree of maximum bronchoconstriction during the EAR correlated with urinary LTE4 release, suggesting that the cysteinyl leukotrienes are only released during the EAR and that they contribute significantly to the bronchoconstriction found during this phase of the asthmatic response. Several subsequent studies have also documented urinary LTE4 release 2–4 hours following specific bronchial allergen challenge (Taylor et al. 1989; Hui & Barnes 1991; Kumlin et al. 1992a; Nasser et al. 1994b).

Exercise-induced asthma Pliss et al. (1990) found increases in BAL fluid levels of LTB4 (from 10 to 121 pg/mL) and immunoreactive cysteinyl leukotrienes (from 46 to 251 pg/mL) following isocapnic hyperventilation as a model for exercise-induced asthma. There were also increases in eosinophil and epithelial cell numbers, but no changes in prostaglandin or histamine levels were detected. A further study was unable to find evidence for mast cell-derived mediator release, with no increase in BAL fluid histamine, tryptase, PGD2 or LTC4 following treadmill exercise in seven atopic asthmatic subjects. However, the sensitivity of the LTC4 assay in this study may not have been sufficient to detect any LTC4 released (Broide et al. 1990). Urinary levels of LTE4 were not found to increase in a study of six asthmatic subjects after treadmill exercise, leading to a mean 22% fall in FEV1 (Smith, C.M. et al. 1991). However, small increases in urinary LTE4 (14.3 ng/mg creatinine before and 24.3 ng/mg creatinine after exercise) were found in 8 of 10 children with severe asthma following exercise, which produced a 60% fall in FEV1 but no increases in urinary LTE4 in seven children with moderate asthma who experienced only a 24% fall in FEV1 (Kikawa et al. 1991). Despite the conflicting evidence, pharmacologic studies have indicated an important role for cysteinyl leukotrienes in exercise-induced bronchospasm (Makker et al. 1993). It is likely that our inability to detect these mediators consistently in biological fluids may be because whole-body cysteinyl leukotriene release does not change significantly after exercise, which may be

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further modified by changes in bronchial or pulmonary blood flow in response to airway cooling and local changes in pH and osmolarity. Furthermore, the modest local increases in cysteinyl leukotriene release in exercise-induced asthma are likely to be difficult to detect with the assays employed.

Aspirin-sensitive asthma The cysteinyl leukotrienes have considerable importance in the pathogenesis of aspirin-sensitive asthma (ASA). Mean resting urinary LTE4 levels in ASA subjects are significantly higher than in normals or non-ASA subjects. Christie et al. (1991a) found sixfold higher urinary LTE4 levels in ASA subjects compared with non-ASA controls. Furthermore, oral aspirin challenge which led to a mean 21% fall in FEV1 in six ASA subjects resulted in a fourfold increase in urinary LTE4 values over baseline values. There was no such increase in urinary LTE4 levels in the control subjects and no fall in FEV1 on aspirin ingestion. Smith et al. (1992) reported that baseline urinary LTE4 levels in 10 ASA subjects were 101 pg/mg creatinine compared with 43 pg/mg creatinine in 31 nonASA subjects and 34 pg/mg creatinine in 17 normals. There was substantial overlap between the groups and no correlation was found between urinary LTE4 and histamine PD20 or baseline FEV1, and so measurement of LTE4 in a single sample of urine does not predict the degree of resting airflow obstruction, the degree of bronchial hyperresponsiveness, or diagnose aspirin sensitivity. Subsequent studies have affirmed these findings (Knapp et al. 1992; Kumlin et al. 1992a; Sladek & Szczeklik 1993). Studies have also confirmed increased urinary LTE4 levels in ASA subjects following lysine-aspirin bronchial challenge (Christie et al. 1992b; Sladek et al. 1994). Nasal lavage mediator levels have been studied in aspirinsensitive rhinosinusitis. Following lysine-aspirin challenge increased levels of both histamine and LTC4 were detected in nasal lavage samples in three of four ASA subjects with both naso-ocular symptoms and a bronchospastic reaction. No increases in these mediators were found in normals or nonaspirin-sensitive subjects or aspirin-sensitive subjects in whom lysine-aspirin did not provoke naso-ocular symptoms (Ferreri et al. 1988; Picado et al. 1992).

Biological roles of leukotrienes and their receptors in asthma revealed through gene disruption in mice Potent inhibitors of CysLT1 have been used extensively in humans with minimal toxicity and have unequivocally demonstrated a role for the cysteinyl leukotrienes acting at CysLT1 in bronchial asthma, rhinitis, and chronic idiopathic urticaria (see Chapter 90). However, there are no existing BLT or CysLT2 antagonists approved for use in humans, and information on their biological activities is imputed largely from models of disease in mice engineered to lack one or more receptor. These studies also allow for dissection of putative mechanisms that is not possible in humans.

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Various models have been developed to study pulmonary allergic inflammation (Boyce & Austen 2005), each revealing different aspects of the disease. The actions of LTB4 in recruiting effector CD8+ and CD4+ T cells has been noted above (Goodarzi et al. 2003; Tager et al. 2003). In a mouse model of allergic pulmonary inflammation, there was substantially reduced recruitment of CD3+, CD4+, and CD8+ T cells to the BAL fluid of ovalbumin-sensitized mice after the first two of three sequential challenges with inhaled ovalbumin (Tager et al. 2003). No differences were seen in recruitment of T cells to the lung of ovalbumin-sensitized and -challenged mice, nor to the BAL fluid after a third ovalbumin challenge (Tager et al. 2003). A role for CD8+ T cells has been demonstrated in allergen-induced AHR and pulmonary inflammation (Hamelmann et al. 1996). Both AHR and pulmonary inflammation were restored by the adoptive transfer of antigen-primed CD8+ T cells from BLT1-sufficient but not from BLT1-null mice (Miyahara et al. 2005a), suggesting an important function for LTB4 acting at BLT1 in the recruitment of CD8+ effector T cells in allergic pulmonary inflammation. Similar findings were reported in a passive sensitization and ovalbumin challenge model of allergic pulmonary inflammation that is mast cell dependent (Taube et al. 2006), suggesting that the mast cell may be the source of LTB4. Two different strains of BLT1-null mice, sensitized and challenged with ovalbumin, developed less AHR and less goblet cell hyperplasia than wild-type BLT1-sufficient control mice, with reduced IL-5 and IL-13 in the BAL fluid and reduced staining for these cytokines in both CD4+ and CD8+ T cells (Miyahara et al. 2005b; Terawaki et al. 2005). Antigen restimulation of peribronchial lymph node cells from sensitized mice elicited less Th2 cytokine generation in vitro (Terawaki et al. 2005). Transfer of antigen-primed but not naive BLT1-sufficient T cells restored antigen-induced AHR consistent with the increased expression of BLT1 on effector compared with naive T cells. In a separate model of ovalbumin sensitization and challenge, a role for LTB4 acting at BLT1 was revealed for the early recruitment of neutrophils and eosinophils into the airways of ovalbumin-challenged mice (Medoff et al. 2006). That the findings regarding BLT1 on effector T cells in mice may be relevant to human disease is indicated by the finding that in humans BLT1-positive T cells, which are normally few in number, are found in increased numbers in individuals following infection with Epstein–Barr virus and in the lungs of individuals with asthma compared with normal controls (Islam et al. 2006). In addition to BLT1, these cells express IFN-γ and IL-4 and the chemokine receptors CCR1, CCR2, CCR6, and CXCR1. A role for the cysteinyl leukotrienes acting at CysLT1 is conclusively proven in human asthma through the use of CysLT1 antagonists (see Chapter 30). Studies in mice lacking LTC4 synthase suggest a distinct role for the cysteinyl leukotrienes in the epithelial reactive mastocytosis that is seen in asthma (Kim, D.C. et al. 2006). These mice have markedly

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attenuated AHR, eosinophilic pulmonary inflammation, goblet cell hyperplasia, and mucus hypersecretion compared with wild-type control mice following ovalbumin sensitization and airway challenge. The expansion of activated mast cells in the epithelium, but not in the submucosa, of allergic mice was markedly attenuated in mice lacking LTC4 synthase. Antigen restimulation of peribronchial lymph node cells from sensitized LTC4 synthase-null mice elicited less Th2 cytokine generation in vitro compared with wild-type control mice. It is also of note that the induction of allergen-specific IgE was attenuated in mice lacking LTC4 synthase (Kim, D.C. et al. 2006), as has also been seen in mice lacking 5-LO (Hashimoto et al. 2005) and BLT1 (Terawaki et al. 2005). A role for the cysteinyl leukotrienes in airway remodeling is suggested in a mouse model of chronic allergen challenge. Administration of the CysLT1 receptor antagonist montelukast to mice sensitized with ovalbumin and then subjected to repetitive ovalbumin challenge over several weeks attenuated the induction of airway goblet cell metaplasia, smooth muscle hypertrophy, and subepithelial fibrosis (Henderson et al. 2002). The increase in mRNA for IL-4 and IL-13 in the lung induced by allergen challenge was also attenuated by montelukast (Henderson et al. 2002). Of note, montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition (Henderson et al. 2006). Thus studies in mice engineered to lack enzymes of leukotriene biosynthesis and specific leukotriene receptors suggest that the cysteinyl leukotrienes have potent inflammatory properties and are not merely constrictors of smooth muscle and mucus secretogogues. Mice models of allergic inflammatory disease address specific aspects of the asthma phenotype and can be manipulated to address selective aspects of the inflammatory process. Furthermore, mice lacking a specific protein from conception cannot address the role of leukotrienes in established disease. To our knowledge there have been no studies of leukotriene-modifying drugs in the prevention of asthma or allergic disease. Whether findings of allergic pulmonary inflammation in mice will be effectively extended to human asthma requires the development of specific inhibitors of downstream enzymes and receptors beyond the current generation of CysLT1 receptor antagonists.

Other diseases Leukotrienes have also been recovered in a number of other nonallergic disease states. Increased quantities of LTB4 were detected in lavage fluid of patients with cryptogenic fibrosing alveolitis and correlated with the recovery of PMNs in BAL fluid, suggesting that leukotriene release may be responsible for granulocyte recruitment in this disease (Wardlaw et al. 1989). LTC4 and LTD4 have been found in increased concentrations in the lung lavage fluids of newborn infants with persistent pulmonary hypertension requiring assisted ventilation (Stenmark et al. 1983). In the adult respiratory distress

syndrome (ARDS), LTD4 was detected in fourfold higher concentrations in pulmonary edema fluid compared with patients with cardiogenic pulmonary edema. Furthermore, the LTD4 levels correlated with the ratio of edema fluid to plasma concentrations of albumin, suggesting that cysteinyl leukotrienes contribute to the permeability defect that allows accumulation of protein-rich fluid in ARDS (Matthay et al. 1984). Small quantities of LTB4 have been detected in the synovial fluid of joints from patients with active rheumatoid arthritis (Davidson et al. 1983). In gouty joint effusions, significantly higher quantities of LTB4 are detected compared with those found in either normal or rheumatoid joints. This may be partly due to monosodium urate crystals, which have been shown to inhibit metabolism and biological deactivation of LTB4 by polymorphonuclear granulocytes (Rae et al. 1982). Skin chamber fluid from abraded psoriatic skin lesions contains significantly higher quantities of LTB4 (Brain et al. 1984) and immunoreactive LTC4 and LTD4 (Brain et al. 1985) than that from normal skin, and this may partly explain the characteristic intraepidermal neutrophil infiltration and local vasodilatation found in psoriasis. By measuring the incorporation of radiolabeled thymidine, picomolar to nanomolar concentrations of LTB4 were found to increase DNA synthesis in cultured epidermal keratinocytes in vitro. The peptide leukotrienes LTC4 and LTD4 were less potent than LTB4, but also stimulated keratinocyte DNA synthesis. This finding may relate to the excessive epidermal hyperplasia seen in psoriasis (Kragballe et al. 1985). Both LTB4 and the cysteinyl leukotrienes are generated and released in vitro in greater quantities from intestinal mucosa obtained from patients with ulcerative colitis and Crohn disease (Sharon & Stenson 1984; Peskar et al. 1986; Lauritsen et al. 1987). Intestinal mucosa from patients with inflammatory bowel disease was found to contain 50-fold higher quantities of LTB4 per gram of tissue than that from normal mucosa (Sharon & Stenson 1984). The use of rectal pouches has allowed the measurement of mediator levels in vivo in inflammatory bowel disease and has demonstrated significantly higher LTB4 levels in active ulcerative colitis (Lauritsen et al. 1987). Treatment with either prednisolone or aminosalicylic acid decreased the generation of LTB4 in vivo, which was associated with clinical improvement. Pretreatment levels of both PGE2 and LTB4 were significantly higher in patients not responding to 5-aminosalicylic acid or prednisolone treatment, and the authors of the study suggested that intraluminal levels of these mediators are more useful predictors of relapsing ulcerative colitis than clinical indices of disease severity (Lauritsen et al. 1986). Levels of urinary LTE4 were found to be raised in a study of 16 patients presenting with acute myocardial infarction, with a fall to normal levels by day 3. Similar elevated levels of urinary LTE4 were found in unstable angina, with a reduction to normal levels once the chest pain had resolved. Treatment with thrombolytic agents leading to early coronary

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COOH

Arachidonic acid

COOH O

COOH

O

O COOH O

O

Prostaglandin H synthase (cyclooxygenase)

TXA2

OH

OOH HO

PGI2

OH

HO O HO

COOH

COOH

O

COOH

O

HO OH

HO

O

PGH2

TXB2

OH

HO

OH 6-keto-PGF1a

HO COOH COOH

O

HO

HO

O OH

COOH

HO

HO OH

PGE2 COOH

HO

HO

COOH

COOH

O HO

OH

O

O

15-keto-PGF2a

COOH

HO

2,3-dinor TXB2

O

OH

PGD2

O OH 11-dehydro TXB2

15-keto-PGE2 HO COOH

HO

OH

9a, 11b-PDF2a

Fig. 26.7 Prostanoid biosynthetic pathway. Cyclooxygenase catalyzes the first two steps and converts arachidonic acid to the endoperoxides PGG2 and PGH2, which are then enzymatically transformed to the prostaglandins, prostacyclin and thromboxanes by the action of the respective synthesis.

reperfusion resulted in a faster decline in LTE4 levels compared with patients who did not receive such treatment, suggesting that cardiac ischemia and necrosis were responsible for cysteinyl leukotriene release (Carry et al. 1992). In hepatorenal syndrome and decompensated liver disease, the rate of urinary LTE4 excretion per hour is threefold higher than that found in compensated liver disease. The additional finding of reduced renal clearance of a radiolabeled LTC4 infusion in one such patient suggested that the increased excretion of urinary LTE4 is due to excessive production or reduced metabolism of LTE4 and not due to reduced renal clearance of LTE4 (Moore et al. 1990). The fact that LTC4 and LTD4 are potent renal vasoconstrictors suggests that these mediators may modulate glomerular function and be involved in the pathogenesis of this disease.

Prostaglandins and thromboxanes Biosynthesis and cellular source TXA2 and prostaglandins belong to the series 2 prostanoids

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since two double bonds are present in the side chains attached to the cyclopentane ring; they are formed by the metabolic pathway outlined in Fig. 26.7. Arachidonic acid, a 20-carbon polyunsaturated fatty acid, is hydrolyzed from phospholipid cell membranes by PLA2, in response to various stimuli. This is followed by the subsequent enzymatic conversion of arachidonic acid to PGH2 by the COX enzymes (also termed prostaglandin H2 synthases). This is a two-step process involving COX activity to oxidise arachidonic acid to PGG2 at the cyclooxygenase site of the COX and a peroxidase reaction to reduce PGH2 at the peroxidase site of COX (Garavito & deWitt, 1999) . These two reactions occur at distinct but structurally and functionally interconnected sites. The peroxidase reaction occurs at a heme-containing active site located near the protein surface. The cyclooxygenase reaction occurs in a hydrophobic channel in the core of the enzyme (Smith et al., 1999). PGH2 serves as a substrate for the prostaglandin synthase enzymes, which are responsible for the production of the five principal bioactive prostaglandins generated in vivo: PGE2, PGF2α, PGD2, PGI2 (prostacyclin), and TXA2 (thromboxane).

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Hence it is the release of arachidonic acid that is the ratelimiting step in prostanoid synthesis. It is now well recognized that there are at least two COX enzymes, COX-1 and COX-2, both are homodimeric, heme-containing, glycosylated, integral membrane associated enzymes found in the endoplasmic reticulum (ER) and the nuclear envelope (Smith et al., 1996). Immunostaining suggests that COX-2 is expressed both in the cytoplasm and at the nuclear membrane whilst COX-1 localises to the cytoplasm alone (Morita et al., 1995). Both forms are expressed in normal human respiratory epithelium. COX-1 is constitutively expressed in most tissues and in blood platelets and the metabolites derived from COX-1 are involved in cellular housekeeping functions. COX-2 is the predominant isoform expressed in injured tissue and a main source of prostanoids during inflammation and is expressed in leukocytes only following cellular activation by inflammatory stimuli (Sousa et al. 1997). In the asthmatic human airway: airway smooth muscle (Belvisi et al. 1997), eosinophils (Sousa et al. 1997), bronchial epithelial cells (Mitchell et al. 1994), mast cells (Sousa et al. 1997), and macrophages (Hempel et al. 1994) all exhibit COX-2 immunoreactivity. The expression of COX-1 and COX-2 in human lungs results in the production of two different classes of prostanoids. These are broadly divided into the bronchoconstrictive prostaglandins (PGD2 and PGF2α) and the bronchoprotective prostaglandins (PGE2 and PGI2) (Samuelsson et al. 1978; Uotila & Vapaatalo 1984). Although COX is present in most cells including the respiratory tract, airway smooth muscle and epithelial cells, the COX metabolites released from a particular cell are cell specific reflecting the isomerase and synthase components of that cell and the quantity and variety of prostanoids produced determined by the nature and the activation state of the cells present at the site of inflammation (Gilroy et al., 1999).

Role of cyclooxygenase COX-1 was originally characterized from bovine seminal vesicles (Miyamoto et al. 1976) and subsequently the inducible form called COX-2 was isolated in a number of species and tissues (Simmons et al. 1991). These isoforms are the products of distinct genes. The gene for COX-1 is situated on chromosome 9q32– 33.3 (Funk et al. 1991) whereas the gene for COX-2 is localized to chromosome 1q25.2–25.3 (Tay et al. 1994). The differences between these two isoforms of COX appear to involve their regulation and cell expression. COX-1 is known to be constitutively expressed at varying levels in virtually all mammalian tissues and is responsible for the basal production of prostanoids. In contrast, COX-2 is undetectable in most tissues but its expression increases substantially in situations associated with cell replication and differentiation and in response to inflammation and mitogenic stimuli, with typical increases of 10–80-fold (Kujubu et al. 1991,

1993). The potential role of COX-2 in the pathogenesis of inflammation and cancer is suggested by its upregulation by bacterial LPS, cytokines, growth factors, and tumor promoters (Kujubu et al. 1991; Lee et al. 1992; O’Banion et al. 1992; O’Sullivan et al. 1992). The differences in expression of both isoforms of COX is reflected in their gene structure. The human COX-2 gene is 8.3 kb whereas the COX-1 gene is much larger at 22 kb (Kraemer et al. 1992; Tazawa et al. 1994). COX-1 has no TATA or CAAT box in its promoter region and is GC-rich, features consistent with its role as a housekeeping gene providing a continuously transcribed stable message (Tanabe & Tohnai 2002). In contrast, the gene structure of COX-2 suggests that it is an immediate early gene product that can be switched on rapidly during inflammation. The COX-2 gene promoter contains a canonical TATA box 31 bases upstream of the transcriptional start site and several functionally important enhancer elements, including a cAMP-response element (CRE), E box and AP-1 regulatory element complex situated very close to TATA, a C/EBP site and two nuclear factor (NF)-κB sites (Appleby et al. 1994). The 72-hour kinetics of the expression of COX-isoform mRNA has been examined in ovalbumin-sensitized and -challenged guinea-pig lungs (Oguma et al. 2002). The sensitized animals demonstrated a robust transient induction of COX-2 mRNA expression within 1 hour after ovalbumin challenge, while their COX-1 mRNA levels remained unchanged. Upregulation of the level and activity of COX-2 protein followed the induction of COX-2 mRNA. Polymorphisms in COX-1 appear not to play a substantial role in genetic predisposition for asthma or asthma severity (Shi et al. 2005) and to date no COX-2 polymorphism has been shown to be associated with asthma or atopy. Despite the differences in gene structure between the two COX isoforms, the catalytic activities and tertiary structures of COX-1 and COX-2 are remarkably similar: there is 61% homology between their amino acid sequences (Hla & Neilson 1992; Filizola et al. 1997), but COX-2 has a broader specificity for substrates because the hydrophobic channel leading to the active site of the enzyme is more accommodating. More recently, more COX enzymes have been discovered: COX-3 and two smaller forms of COX-1. All these are derived from the COX-1 gene by alternative splicing of COX-1 mRNA. COX-3 is also constitutively expressed (Chandrasekharan et al. 2002); it remains unclear whether the splice variants of COX-1 are constitutively expressed or are inducible.

Cyclooxygenase enzymes and asthma In humans, COX-1 and COX-2 immunoreactivities have been reported in bronchial mucosa of normal and asthmatic lungs (Demoly et al. 1997; Sousa et al. 1997). The relative expression of COX-1 and COX-2 in the airways of patients with asthma remains unresolved. Demoly et al. found that neither COX-1 nor COX-2 is upregulated in stable asthma.

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Sousa et al. found enhanced expression of COX-2 but not COX-1 in asthmatic airways, whereas Taha et al. (2000) found increased immunoreactivity of both COX-1 and COX-2 in induced sputum cells from asthmatics. In normal and asthmatic human lung COX-2 is expressed in activated eosinophils, mast cells, and macrophages but not T cells (Sousa et al. 1997). It is thought that increased COX-2 expression in alveolar macrophages from asthmatic subjects may contribute to enhanced eosinophil survival through an increase in PGE2 production (Profita et al. 2003). In human bronchial smooth muscle cells, COX-2 expression is easily induced by proinflammatory IL-1β, leading to increased PGE2 formation (Bonazzi et al. 2000. In human airway epithelial cells, COX-2 expression is also upregulated by inflammatory stimuli via a mechanism that appears to involve ERK and possibly p38 MAPK (Lin et al. 2002) and JNK as well (Newton et al. 2000; Mizumura et al. 2003). In human neutrophils LTB4 is the sole eicosanoidable to up-regulate COX-2 expression (St-Onge et al. 2007). A further regulatory property of COX-2 is that its induced expression can be completely inhibited by antiinflammatory glucocorticoids such as dexamethasone (Masferrer et al. 1992). Inhibitors of transcription and translation such as cycloheximide and actinomycin D also inhibit COX-2 induction in response to either cytokines (Pang & Knox 1997a) or bradykinin (Pang & Knox 1997b) The most likely mechanism for the effect of dexamethasone is inhibition of the transcription factor NF-κB (Cembrzynska-Nowak et al. 1993). It has therefore been hypothesized that the constitutively expressed COX-1 is a housekeeping gene involved in the production of prostanoids that regulate normal cellular function, and that inducible COX-2 is involved in the production of prostanoids that mediate inflammatory and mitogenic responses. Data from mice expressing neither COX-1 nor COX-2 have suggested that COX-1 attenuates airway responsiveness under basal conditions and is important in lung protection, probably due to more efficient coupling with constitutive cytosolic isomerase producing PGE2 (Gavett et al. 1999; Zeldin et al. 2001). Under basal conditions, mice deficient in either COX-1 or COX-2 showed no difference compared with wild-type mice with respect to basal lung function and lung histology, although mice not expressing COX-1 had significantly lower PGE2 levels in BAL fluid (Zeldin et al. 2001). Following allergen sensitization and exposure, lung inflammatory indices (BAL cells, proteins, IgE, lung inflammation score) and airway hyperreactivity were significantly greater in COX-1-deficient compared with COX-2-deficient mice and both were far greater than in wild-type mice (Zeldin et al. 2001), suggesting that in allergic mouse lung COX-1 products have mainly bronchprotective effects. In contrast, much increased airway inflammation in mice not expressing COX-2 was not associated with bronchial hyperreactivity to methacholine, thus dissociating airway inflammation and methacholine responsiveness (Zeldin et al. 2001).

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Recently, further understanding of the relative contribution of the COX enzymes to allergic lung inflammation has been facilitated by the development of transgenic mice that overexpress human COX-1 on airway epithelium. No differences in basal respiratory or lung mechanical parameters were observed between COX-1 transgenic and wild-type mice. However, consistent with Zeldin’s data, COX-1 transgenic mice had almost three times increased PGE2 content in BAL fluid compared with wild-type littermates and exhibited decreased airway responsiveness to inhaled methacholine (Card et al. 2006). In response to ovalbumin challenge, comparable upregulation of COX-2 protein was observed in the lungs of allergic wild-type and COX-1 transgenic mice. In order to eliminate the presumed confounding effects of COX-2 upregulation, COX-1 transgenic mice were bred into a COX-2 null background. In these mice, the presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly reduced allergen-induced AHR, coincident with reduced airway leukotriene levels (Card et al. 2006). The role of COX-1 and COX-2 in the pulmonary response to bacterial LPS has also been examined. Bacterial LPS is a known risk factor for exacerbation of asthma and a cause of airway inflammation. Following exposure to LPS, all mice exhibited increased bronchoconstriction and methacholine responsiveness; however, these changes were much more pronounced in mice that did not express COX-1 or COX-2 relative to wild-type mice (Zeldin et al. 2001). The degree of airway inflammation did not differ between the genotypes, indicating that airway inflammation and bronchial hyperreactivity remain dissociated in COX-1- and COX-2-deficient mice exposed to LPS. Lung fibrotic responses following vanadium pentoxide exposure have also been investigated in COX-1- or COX-2-deficient mice (Bonner et al. 2002). Mice deficient in COX-2 but not COX-1 exhibited severe inflammatory responses by 3 days following exposure and developed pulmonary fibrosis 2 weeks after vanadium exposure. Exposure to vanadium pentoxide led to an increase in PGE2 levels in BAL fluid from wild-type and COX-1-deficient mice within 24 hours, whereas PGE2 was not upregulated in COX2-deficient mice. COX-2 appears therefore to be protective against pulmonary fibrogenesis. This finding is significant, as chronic asthma is characterized by airway remodeling, in part secondary to subepithelial fibrosis.

Cyclooxygenase and aspirin sensitivity Aspirin-sensitive respiratory disease (ASRD) refers to a particular phenotype of adult asthma that is characterized by chronically severe asthma, rhinosinusitis, and nasal polyposis (Widal et al. 1922; Samter & Beers 1968). This phenotype affects around 10% of asthmatics (Sturtevant 1999) and like other forms of adult asthma affects more women than men, with an average age of onset at around 34 years (BergesGimeno et al. 2002). It is characterized by a potentially lifethreatening exacerbation of asthma following the ingestion

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of nonsteroidal antiinflammatory drugs (NSAIDs). Often ASRD is aggressive and refractory to treatment and patients are overrepresented in the severe asthma group, with 50% of patients with aspirin hypersensitivity being steroid dependent (Stevenson & Simon 1998). The ability of NSAIDs to exacerbate symptoms in ASRD is directly related to their COX-1 inhibitory activity. COX-1 inhibitors induce acutely elevated local and systemic cysteinyl leukotriene release in aspirin-sensitive patients (Christie et al. 1992b; Nasser et al. 1995) and in the event of COX-1 inhibition, it has been hypothesized that there is insufficient upregulation of COX-2 to produce sufficient PGE2 to compensate for loss of production. With the advent of selective COX-2 inhibitors, it has been demonstrated that inhibition of COX-1 but not COX-2 precipitates asthma attacks in ASRD (Stevenson & Simon 2001; Szczeklik et al. 2001; Gyllfors et al. 2003; Senna et al. 2004). COX-2 expression is known to be diminished and its activity reduced in nasal polyps from patients with ASRD (Pujols et al. 2004). It has therefore been hypothesised that selective COX-2 inhibitors are well tolerated by patients with ASRD because COX-2 expression is diminished in the lung and does not contribute much to eicosanoid balance (Szczeklik & Sanak 2006).

Receptors Following intracellular synthesis, prostanoids leave the cell probably by facilitated diffusion (Smith 1986). Outside the cell prostanoids act either on the parent or neighboring cells in an autocrine or paracrine fashion through specific G proteinlinked prostanoid receptors to stimulate or inhibit changes in the levels of second messengers (Smith 1989). Pharmacologically, prostanoid receptors are classified according to the prostanoid that causes selective activation of GPCRs. In the early 1980s, five different receptors were described, designated DP, EP, FP, IP, and TP on the basis of preferential sensitivity to PGD2, PGE2, PGF2α, PGI2, and TXA2 (Coleman et al. 1984). Within each receptor type there may be distinct subtypes, many of which have been identified using radioligand binding studies and cloning. Individual prostanoid receptors share 20–30% sequence identity with each other and encode specific motifs common only to members of the subfamily (Hata & Breyer 2004). Recently, a ninth prostaglandin receptor has been identified, CRHT2 (chemoattractant receptor homologous molecule expressed on Th2 cells), which binds PGD2 (Hirai et al. 2001). Activation of a given prostaglandin receptor by its cognate ligand may elicit varying responses depending on the cell type and tissue in which it is expressed. Evidence is also emerging that prostanoid receptors may be expressed not only on the cell surface but also on the nuclear membrane (Gobeil et al. 2002). Increased understanding of prostaglandin receptor function has started to reveal a complicated picture in which prostaglandins serve to both promote and inhibit inflammation.

Pharmacologic properties of prostanoids in asthma PGD2 PGD2 has long been associated with inflammation and allergy. It is mainly produced by activated mast cells or T cells (Roberts et al. 1980). PGD2 is the most abundant eicosanoid generated during IgE-mediated allergic responses (Lewis et al. 1982) and by ionophore-stimulated (Holgate et al. 1984) human pulmonary mast cells. In a murine model of asthma, PGD2 was found to increase the recruitment of eosinophils and CD4+ Th2 cells into the lung to induce expression of macrophage-derived chemokine, a chemoattractant for Th2 cells in (Honda et al. 2003). In patients with exercise-induced asthma, dietary supplementation with fish oil has been demonstrated to improve pulmonary function and decrease symptoms and β2-agonist use (Mickleborough et al. 2006. These findings were associated with a reduction in induced sputum differential cell count percentage. Concentrations of cysteinyl leukotrienes, PGD2, IL-1β, and TNF-α were also significantly reduced before and following exercise on the fish-oil diet compared with normal and placebo diets. PGD2 is synthesized by PGD synthase, which catalyzes simple nonoxidative rearrangements (isomerization) of PGH2 (Urade et al. 1995). The neural form of PGD synthase is glutathione (GSH) independent and is a member of the lipocalin superfamily (Nagata 1991). In contrast, the hematopoietic form of PGD synthase is GSH dependent and is a member of the sigma class of glutathione-S-transferases (Kanaoka et al. 1997). In the united airways theory, both allergic rhinitis and asthma are viewed as manifestations of a single allergic airway. It is therefore of note that in both normal controls and patients with allergic rhinitis the hematopoietic form of PGD synthase, but not lipocalin-type PGD synthase, is the predominant form expressed in inflammatory cells (mast cells, eosinophils, macrophages, and lymphocytes) and structural cells (epithelial cells and fibroblasts). Higher levels of the hematopoietic form of PGD synthase are also found in patients with allergic rhinitis compared with normal controls (Okano et al. 2006). In murine asthma models, mice that overexpress lipocalin-type PGD synthase have elevated PGD2 levels and an increased allergic response in the ovalbumininduced model of airway hyperreactivity (Fujitani et al. 2002). PGD2 is able to bind with similar high affinity and to activate two distinct GPCRs: DP (Boie et al. 1995) and CRHT2 (Hirai et al. 2001; Nagata et al. 1999a,. The proinflammatory effects of PGD2 therefore appear to be mediated by its actions on both DP and CRHT2 receptors. PGD2 may also contribute to the nonimmune pathogenesis of asthma. Hyperplasia of airway epithelial goblet cells leading to mucus hypersecretion is a feature of airway remodeling. Mucus secretion by colonic goblet cells has been linked to the DP receptor (Wright et al. 2000). The DP receptor is also expressed on bronchiole epithelial cells in antigen-challenged mice (Matsuoka et al. 2000) and is found on nasal epithelial goblet cells in normal human volunteers (Nantel et al. 2004).

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Taken together, these observations suggest that the DP receptor may evoke mucus secretion in response to PGD2 in asthma and allergic rhinitis. PGD2 is a potent contractile agent and elicits a rapidly developing bronchoconstriction (Hardy et al. 1984; Beasley et al. 1987a,b). In asthmatic subjects it has a potency of approximately 3.5 and 10 times greater than PGF2α and histamine, respectively, with the maximal effect evident 3 min following inhalation. However, these effects are unlikely to be mediated by the DP receptor since an increase in intracellular cAMP is typically associated with smooth muscle relaxation (Hall 2000). Despite the fact that PGD2 has relatively low affinity for the TP receptor (Abramovitz et al. 2000), it has been suggested that TP receptors mediate the bronchoconstrictive response to PGD2 (Beasley et al. 1989; Francis et al. 1991; Johnston et al. 1992). The TP antagonist ramatroban is a CRHT2 antagonist (Sugimoto et al. 2003) and the TXA2 metabolite 11-dehydro-TXB2 has been demonstrated to be a CRHT2 agonist (Bohm et al. 2004). Exposure to respiratory RNA viruses often aggravates the airway inflammation and bronchial responsiveness, characteristic of asthma. In a rat model of asthma, dsRNA (a nucleotide synthesized during viral replication) was instilled into the trachea and an increase in airway eosinophilia and enhanced bronchial hyperresponsiveness to methacholine in ovalbumin challenge was observed. These changes were associated with induction of COX-2 expression and COX-2-dependent PGD2 synthesis in the lungs, particularly in alveolar macrophages – suggesting that COX-2-dependent production of PGD2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenetic factors responsible for the dsRNAinduced enhancement of airway inflammation and responsiveness (Shirashi et al. 2008). These observations raise the possibility that activation of the CRHT2 receptor may contribute to bronchoconstriction in asthma. The DP receptor is expressed on bronchial epithelium. Its activation leads to increased intracellular concentrations of cAMP and mediates physiologic and pathologic events, including the allergic response, by producing chemokines and cytokines that recruit inflammatory lymphocytes and eosinophils (Matsuoka et al. 2000) to the airway. BAL fluid analyses have demonstrated that PGD2 is released in the airways following antigen challenge during acute allergic response (Murray et al. 1986). In 2000, a series of landmark experiments by Matsuoka and colleagues with DP receptordeficient mice clearly demonstrated that PGD2 is a proinflammatory prostanoid in allergic asthma. Sensitization and aerosol challenge with ovalbumin induced increases in the serum concentration of IgE that was similar in both DP receptor-deficient and wild-type mice. However, the concentrations of Th2 cytokines and the extent of lymphocyte accumulation in the lung of ovalbumin-challenged DP receptor-deficient mice was greatly reduced. Furthermore, DP receptor-deficient mice showed only marginal infiltration of eosinophils and did not develop airway hyperreactivity.

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These findings were strongly suggestive that the DP receptor plays a key role in mediating the effects of PGD2 released by mast cells during an asthmatic response (Matsuoka et al. 2000). DP antagonists have also been shown to inhibit ovalbumin-induced airway hyperreactivity in mice (Arimura et al. 2001). Furthermore, PGD2 may inhibit eosinophil apoptosis via the DP receptor (Gervais et al. 2001). In humans, the CRHT2 receptor is expressed on Th2 lymphocytes, eosinophils, and basophils (Nagata et al. 1999a,b; Hirai et al. 2001), key cells in the allergic response. The CRHT2 receptor (also known as the DP2 receptor) is a GPCR but shares little sequence homology with prostanoid DP receptor and structurally is closely related to chemoattractant receptors. As with many chemoattractant receptors, CRHT2 couples to a Gi-type protein leading to inhibition of intracellular cAMP and to an increased intracellular concentration of calcium (Hirai et al. 2001; Sawyer et al. 2002), with the specific response being influenced by cell type (Nagata et al. 2003). The CRHT2 receptor binds an overlapping but distinct set of ligands compared with the DP receptor. For example, the CRHT2 receptor binds the PGD2 metabolite 15-deoxy-Δ12,14-PGJ2 with equally high affinity as PGD2 itself (Sawyer et al. 2002), raising the possibility that metabolites of PGD2 may differentially exert effects through CRHT2 but not the DP receptor. The TP antagonist ramatroban (BAY u4305) is also a CRHT2 antagonist (Sugimoto et al. 2003) and this may explain the reduction in eosinophilic infiltration observed following antigen challenge in ramatroban-treated animals (Nagai et al. 1995; Narita et al. 1996), since TP receptor activation has no effect on eosinophil migration (Monneret et al. 2001). In allergic rhinitis, expression of the CRHT2 but not the DP receptor has been found to strongly correlate with eosinophilic inflammation in the nasal mucosa (Okano et al. 2006). Activation of CRHT2 by PGD2 has been shown to induce a modulation of eosinophil morphology, and an increase in both eosinophil and Th2 cell motility (Nagata et al. 2003). Shiraishi et al. (2005) have also underlined the significant role of PGD2 in eosinophil trafficking from the bloodstream to the airways during bronchial inflammation. Therefore, PGD2 acting through both DP and CRHT2 receptors is likely to contribute to the eosinophilic infiltration that is a hallmark of allergic asthma and rhinitis. Recent studies using two in vivo models of allergic inflammation (atopic dermatitis and allergic asthma) showed that CRHT2 plays a critical role in allergic diseases and underlined the interest of CRHT2 antagonists in human therapy (Spik et al. 2005). Characterization of mice deficient in the CRHT2 receptor will begin to clarify the exact role that each receptor plays in mediating the effects of PGD2 in allergic inflammation.

PGF2αα Human lung parenchyma, and to a lesser extent bronchial tissue fragments, spontaneously release PGF2α under resting conditions, leading to the suggestion that this prostanoid,

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together with PGE2, may be important in maintaining bronchial tone (Schulman et al. 1982; Cuthbert & Gardiner 1983). PGF2α is synthesized by PGE 9-ketoreductase via three pathways from PGE2, PGD2, or PGH2, PGD 11-ketoreductase, or PGH 9,11-endoperoxide reductase (Watanabe 2002). It binds the FP receptor, which was originally cloned from human kidney, uterus, and placental cDNA libraries (Abramovitz et al. 1994) and has two isoforms, FP(A) and FP(B), which differ from each other in C-terminal tail length (Pierce et al. 1997). Recently, a six-transmembrane splice-variant FP receptor mRNA from human heart and placenta cDNA, named hFP(S), was described (Vielhauer et al. 2004). The FP receptor is the least selective of the prostanoid receptors in binding the principal endogenous prostaglandins and binds both PGD2 and PGE2 at nanomolar concentrations (Abramovitz et al. 2000). When activated the FP receptor signals via Gq-mediated IP3 generation and increases in intracellular calcium (Abramovitz et al. 1994; Sugimoto et al. 1994). FP receptor expression has not been observed on immune cell populations (Tilley et al. 2003. Therefore, unlike the other prostaglandins, there is very little evidence to support a role for PGF2α–FP receptor signaling in inflammatory and immunologic processes. Nonetheless the inhalation of PGF2α has contractile activity on isolated human airway tissue (Sweatman & Collier 1968; Orehek et al. 1973) and causes bronchoconstriction in vivo, with asthmatic subjects demonstrating greater sensitivity to the effects of inhaled PGF2α than normal control subjects. In a study of 10 asthmatic and 10 healthy controls, it was reported that asthmatic subjects were 10 times more sensitive to histamine but 8000 times more sensitive to the bronchoconstrictor action of inhaled PGF2α (Mathe et al. 1973). Furthermore, whereas the healthy controls had reproducible decreases in specific airway conductance in response to repeated PGF2α challenge, the asthmatic subjects demonstrated tachyphylaxis (Mathe & Hedqvist 1975). Other studies have also reported similar responses to PGF2α inhalation in asthmatic subjects but with wide interstudy variation in the degree of hyperreactivity, ranging from 150-fold (Smith et al. 1975) to 8000-fold (Mathe et al. 1973) greater sensitivity compared with nonasthmatic subjects. In studies of asthmatic individuals that examined the effect of PGF2α on airway responsiveness, inhalation of a nonbronchoconstricting dose of PGF2α led to a fourfold enhancement of bronchial responsiveness to histamine (Walters et al. 1981; Heaton et al. 1984), but had no effect on subsequent methacholine provocation (Heaton et al. 1984).

TXA2 The majority of TXA2 produced in vivo is generated from platelets (Hamberg et al. 1975) but it is also produced after activation of a number of inflammatory cells, including alveolar macrophages (Nusing et al. 1990) and eosinophils (Kroegel et al. 1993. Due to its prothrombotic and vasoconstrictor effects, TXA2 has been described as the physiologic

antagonist of prostacyclin (Rolin et al. 2006). In addition to its roles as a mediator of platelet function and its association with heart failure (Castellani et al. 1997) and ischemic heart disease (Oates et al. 1988), TXA2 is a potent bronchoconstrictor and is increased in the airways of asthmatic patients on antigen challenge (Wenzel et al. 1991). Its other pharmacologic actions include increased microvascular leakage, impairment of mucociliary clearance, (Kurashima et al. 1995), and induction of AHR (Fujimura et al. 1995). TXA2 can also stimulate proliferation of airway smooth muscle cells, suggesting a role in the pathogenesis of asthma (Devillier & Bessard 1997). Increased concentrations of TXA2 and its metabolites have been demonstrated in BAL fluid, urine, plasma, and exhaled breath condensate from asthmatic patients (Wenzel et al. 1989; Kumlin et al. 1992a; Huszar et al. 2005). In a murine asthma model, blockade of either thromboxane synthesis or TP receptor activation reduced ovalbumin-induced cytokine production, which in turn inhibited eosinophil infiltration into the murine airway, with splenic mononuclear cells from treated mice exhibiting impaired antigeninduced cytokine production (Shi et al. 1998). TXA2 also appears to facilitate allergen-induced cough in a guinea-pig asthma model (Xiang et al. 2002). These data suggest that TP receptor signaling may promote cytokine production and allergic inflammation, although the exact mechanism and target cell population(s) responsible are not clear. In humans, the administration of the TXA2 receptor antagonist seratrodast is associated with a decrease in the concentration of eosinophilic cationic protein in the sputum of asthmatic patients (Fukuoka et al. 2003). The enzymes responsible for catalyzing the conversion of PGH2 to TXA2 are called the TXA synthases and have now been cloned from human platelets (Yokoyama et al. 1991) and lung (Ohashi et al. 1992). A member of the cytochrome P450 family, TXA synthase localizes to the epithelial goblet cells and subtracheal gland in a guinea-pig model (Xiang et al. 2002). In vivo, the half-life of TXA2 is only about 30 s and therefore much of the information about its biological activity comes from studies employing synthetic compounds that are stable analogs of the TXA2 structure, such as the long-acting thromboxane mimetic U-46619. TXA2 is readily degraded to its more stable and relatively inactive metabolite TXB2 which can be measured in biological fluids such as BAL and urine. Prostanoid TP receptors are distributed in both plasma membrane and cytosolic compartments and are found in tissues rich in vasculature such as lung, heart, and kidney (Rolin et al. 2006). The human TXA2 receptor, termed TP, was the first eicosanoid receptor cloned (Hirata et al. 1991). Two G proteincoupled isoforms of this receptor have been subsequently described, TPα (placental/platelet) and TPβ (endothelial), and these two splice variants differ in the length and sequence of the carboxyl-terminal tail distal to Arg328 (Raychowdhury

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et al. 1994, 1995). Both TPα and TPβ were initially characterized as functionally coupling to a Gq protein (Shenker et al. 1991), leading to phospholipase C activation, calcium release, and activation of PKC. Subsequently, differential G-protein coupling (Gi, Gs, Gh) of the two isoforms has been noted in cultured cells, where TPα and TPβ show similar ligand binding characteristics and phospholipase C activation but oppositely regulate adenylyl cyclase activity. TPα activates adenylyl cyclase, whereas TPβ inhibits it (Hirata et al. 1996). However, the physiologic role for differences in coupling between the TP receptor splice variants remains to be determined. Intrareceptor differences in C-terminal tail sequence also allow for significant differences in their ability to internalize in response to agonist exposure. For example, in HEK-293 cells, TPβ but not TPα undergoes U46619-induced GRK phosphorylation and internalization (Parent et al. 1999), whereas the C-terminus of TPα is not capable of being phosphorylated by GRKs (Zhou et al. 2001). TXA2 is a potent stimulator of airway smooth muscle cell proliferation (Devillier & Bessard 1997), smooth muscle constriction, and bronchial hyperresponsiveness (Arimura et al. 1994). In human airway smooth muscle cells the TP receptor, predominantly coupled to Gi/Go proteins, activates the Ras/ERK pathway to induce mitogenesis, probably with the involvement of nonreceptor tyrosine kinases and PKC (Citro et al. 2005). In bronchiole smooth muscle cells stimulation by U46619 leads to an increase in mitogenesis that can be blocked with the TP antagonist SQ29548 (Capra et al. 2003). It is therefore not inconceivable that TP receptor signaling partly mediates the airway smooth muscle hyperplasia seen in the airway remodeling of asthma (Vignola et al. 2003). The mechanism by which TXA2 induces bronchoconstriction and an increase in lung resistance remains unclear. Activation of TP receptors expressed on bronchiole smooth muscle cells leads to intracellular calcium mobilization (Capra et al. 2003), the predominant pathway mediating constriction of airway smooth muscle cells (Hall 2000). However, although TXA2 appears to cause airway constriction by TP receptor activation, it has been demonstrated that this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor antagonists (Allen et al. 2006). TXA2 may also exacerbate AHR by potentiating airway smooth muscle contraction elicited by cholinergic parasympathetic neurotransmission (Chung et al. 1985; Munoz et al. 1986). Pretreatment with ipratropium bromide, a muscarinic receptor antagonist, attenuates U46619-induced bronchoconstriction in asthmatic subjects, suggesting that TXA2-mediated bronchoconstriction may itself be mediated by cholinergic neurons (Saroea et al. 1995). TXA2 receptor antagonists and thromboxane synthase inhibitors have been developed that inhibit the physiologic actions of TXA2. Double-blind, placebo-controlled clinical trials have shown limited clinical benefit of the thromboxane receptor antagonist seratrodast and the selective thromboxane

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synthase inhibitor ozagrel in the treatment of patients with asthma. In 1992, ozagrel was released onto the Japanese market (Nakazawa et al. 1994). In humans, oral and inhaled administration of ozagrel hydrochloride reduced bronchial hyperresponsiveness to acetylcholine and leukotriene D4 in asthmatic subjects (Fujimura et al. 1986, 1990a,b). Seratrodast (AA-2414) was the first thromboxane receptor antagonist to be developed as an antiasthmatic drug and has received marketing approval in Japan (1997). Since 1999, four relevant clinical studies have been published with seratrodast that confirm its effect in asthma. Seratrodast treatment has been shown to decrease daytime asthma symptoms, bronchial hyperresponsiveness to methacholine, and supplemental use of inhaled β2 agonists but did not change baseline pulmonary function (Fukuoka et al. 2003). These results support the concept that TXA2 may play a part in the pathogenesis of bronchial hyperresponsiveness in asthma. Furthermore, antagonsim of the TXA2 receptor, while having minimal effects on pulmonary function, has also been shown to decrease the amount of sputum produced by asthmatics along with alterations in its physicochemical properties (Tamaoki et al. 2000). The TP antagonist, BAY u3405 or ramatroban, is also active on a wide range of airway smooth muscles cells across species, including human bronchial smooth muscle (McKenniff et al. 1991). It is highly effective in reducing antigen-, LTC4- and LTD4-induced bronchoconstriction in guinea pigs (Francis et al. 1991; Iwamoto et al. 1995) and PGD2-, exercise-, bradykinin- and histamine-induced bronchoconstriction in humans (Magnussen et al. 1992; Rajakulasingam et al. 1996). Moreover, Aizawa et al. (1996) demonstrated that oral ramatroban (75 mg/day) attenuated bronchial hyperresponsiveness to methacholine in asthmatics. Ramatroban is currently under clinical evaluation as an antiasthmatic agent (Rolin et al. 2006) and has been shown to partially attenuate PGD2induced bronchial hyperresponsiveness in humans, probably through its actions as an antagonist at the CRHT2 receptor and as an inhibitor of PGD2-induced chemotaxis of eosinophils (Sugimoto et al. 2003). TP receptors can be activated by high levels of other eicosanoids, including PGD2 and 8-iso-PGF2α, which act as TP receptor agonists (Elmhurst et al. 1997; Audoly et al. 2000) and may play an important role in activating prostaglandin receptors in settings of oxidative stress. The isoprostane 8-iso-PGF2α, a chemically stable product of oxidative stress, is also known to activate TP receptors, albeit as a partial agonist (Kinsella 2001). This perhaps limits the clinical use of thromboxane synthase inhibitors.

PGE2 PGE2 and allergic inflammation Prostaglandin E synthase (PGES), which generates PGE2 from COX-derived PGH2, occurs in multiple forms, two of which are membrane bound and one of which is cytosolic. Microsomal PGES (mPGES)-1 is a member of the MAPEG (microsome-

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associated proteins of eicosanoid generation) family of proteins, as are FLAP and LTC4 synthase, and like these proteins it is inhibited by MK-886, raising caution about use of this drug, originally described as a FLAP inhibitor, in discerning the role of leukotrienes in any particular process. mPGES-1 is induced by proinflammatory stimuli and downregulated by antiinflammatory glucocorticoids (Murakami et al. 2000). mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to formation of a mature cytosolic enzyme, although its cellular function remains uncertain (Tanikawa et al. 2002; Murakami et al. 2003). Whereas mPGES-1 is an inducible enzyme, cytosolic PGES is constitutively expressed, and in vitro these enzymes have been shown to be functionally coupled to COX-2 (Murakami et al. 2000; Mancini et al. 2001; St Onge et al. 2007) and COX-1 (Tanioka et al. 2000), respectively. PGE2 itself is rapidly degraded by the enzyme 15prostaglandin dehydrogenase (15-PGDH) (Bergholte & Okita 1986). Innate factors such as IL-1β suppress 15-PGDH, suggestive of overall upregulation of PGE2 and its relevant enzymes in response to innate stimuli (Mitchell, J.M. et al. 2000). It is now appreciated that IL-13 significantly decreases the inducible mPGES-1, while markedly upregulating 15PGDH, the enzyme preferentially metabolizing PGE2 (Trudeau et al. 2006). Asthma is a chronic Th2-dominated inflammatory disease. Despite both proinflammatory and antiinflammatory actions in vitro, in vivo inhaled PGE2 has been shown to attenuate exercise-induced (Melillo et al. 1994), allergen-induced (Pavord et al. 1993), and aspirin-induced bronchoconstriction (Sestini et al. 1996), as well as protecting against bronchoconstrictor agents such as methacholine and histamine (Walters et al. 1982; Manning et al. 1989). Hence, PGE2 is widely regarded as the prostanoid with the most prominent immunomodulatory, antifibrotic, and bronchodilating properties. It is spontaneously released from both human epithelial cells (Churchill et al. 1989) and lung fibroblasts (Korn 1983). Further sources include macrophages (Macdermott et al. 1984), follicular dendritic cells (Heinen et al. 1986), and endothelial cells. There are many events leading to the development of allergic inflammation which PGE2 may modulate. The adherence of inflammatory cells to endothelium is one of the initial events necessary for migration of these cells through the vascular wall. It is likely that PGE2 can modulate cell recruitment indirectly through inhibition of TNF-α. PGE2 inhibits both TNFα-induced expression of ICAM-1 and vascular cell adhesion molecule (VCAM)-1 on airway smooth muscle and the adhesion of activated T cells to TNF-α-stimulated airway smooth muscle (Panettieri et al. 1995). It also blocks the synthesis of endothelial–leukocyte adhesion molecule (ELAM)-1 and VCAM-1 in endothelial cells, which contributes to the reduced adhesion of activated T cells (Pober et al. 1993). The mechanisms by which PGE2 may regulate AHR and

airway inflammation after allergen challenge are speculative. Both eosinophils and mast cells, which are present in greater numbers in the asthmatic lung (Pin et al. 1992), are rich sources of proinflammatory cysteinyl leukotrienes and are immunomodulated by PGE2. Acting via the EP2 receptor (Kay et al. 2006), PGE2 inhibits antigen-induced human mast cell degranulation (Peters et al. 1982). Conversely, in vitro it appears to potentiate IgE-mediated histamine release from human peripheral blood-derived cultured mast cells via its actions on EP3 and/or EP1 receptors. Endogenous PGE2 inhibits PAFinduced synthesis of the potent bronchoconstrictor LTC4 by eosinophils (Tenor et al. 1996). It has been hypothesized that the ability of inhaled PGE2 to inhibit the late response is secondary to its ability to inhibit allergen-induced synthesis of LTC4. PGE2 also inhibits human eosinophil degranulation in vitro (Kita et al. 1991) chemotaxis, and cytokine-stimulated survival (Alam et al. 1993) and reduces arachidonic acid and TXA2 release from bronchial biopsies. Raised intracellular levels of cAMP generated by PGE2 have been shown to suppress leukotriene and prostaglandin production by neutrophils and eosinophils (Keuhl et al. 1987). However, increased levels of PGE2 in sputum from asthmatic patients may prolong eosinophil survival (Profita et al. 2003). Profita et al. which obtained induced sputum from 14 control and 30 asthmatic subjects, and demonstrated a striking correlation between PGE2 concentration and eosinophils numbers (P < 0.0001) and eosinophil cationic protein concentration (P < 0.0001). Immunostaining for COX-2 showed enhanced expression in macrophages of asthmatic subjects compared with control subjects, and incubation of sputum with the highly-specific COX-2 inhibitor SC58236 led to marked suppression of PGE2 levels, providing evidence that PGE2 synthesis was the result of COX-2 enzymatic activity in asthma-induced sputum cells. Incubation of peripheral blood eosinophils with induced sputum supernatant of subjects with a high eosinophil count caused decreased apoptosis of peripheral blood eosinophils compared with control subjects, but immunoprecipitation of PGE2 significantly reverted this phenomenon. PGE2 enhancement of eosinophil survival may therefore also contribute to the development of the “classic” eosinophilic inflammation of the airways of asthmatic subjects. PGE2 also inhibits leukotriene biosynthesis both directly and indirectly in certain specific cell types. In 2002, using human polymorphonucleocytes stimulated with PAF and thapsigargin, it was demonstrated that a number of cAMP-elevating agents including PGE2 inhibited leukotriene biosynthesis and 5-LO translocation to the nucleus (Flamand et al. 2002). PGE2 inhibits the production of LTB4 from human bone marrow-derived dendritic cells. This is achieved indirectly by an IL-10-dependent mechanism that inhibited expression of FLAP (Harizi et al. 2003). The presence of PGE2 during T-cell activation and maturation following priming by antigen-presenting cells (APCs) also appears to modulate the nature of the cellular immune

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response. Th1 and Th2 cells are differentiated from naive T cells after antigenic stimulation by the influence of cytokines (IL-12, IFN-γ) in the case of Th1 and IL-4/IL-13 in the case of Th2 or costimulatory molecules (CD80/CD86) provided by APCs. In vivo, PGE2 is thought to polarize the balance in favor of a Th2 response partly by EP4 receptor-mediated inhibition of IL-12 production by monocytes and macrophages (Van der Pouw Kraan et al. 1995; Nataraj et al. 2001) and partly by acting directly on T cells to suppress production of IL-2 and IFN-γ (Hilkens et al. 1995; Kabashima et al. 2003b). However, some reports suggest that PGE2 can also inhibit Th2 cytokine secretion by EP2/EP4 receptor-driven cytokine-induced class switching and IgE production by B cells (Fedyk & Phipps 1996). Despite evidence suggesting that PGE2 influences CD4+ T-cell maturation in favor of a Th2-dominant immune response, in vivo studies suggest that PGE2 suppresses Th2mediated allergic inflammation. As well as inhibiting transendothelial migration of human T cells, PGE2 also upregulates expression of Th2 cytokines following allergen challenge. In a rat model of allergic asthma, pretreating rats with PGE2 prior to challenging them with ovalbumin reduced the increase in the number of cells expressing IL-4 and IL-5 mRNA and eosinophils in lavage fluid, to levels comparable to those seen in sham-challenged animals. This suggested that in part the antiinflammatory effects of PGE2 are mediated by reducing allergen-induced Th2 cell activation (Martin et al. 2002). Dendritic cells (DCs) are thought to play an important role in the pathogenesis of allergic disorders via their interactions with T cells to initiate and amplify Th2 immune responses. DCs are the most potent APCs and are highly responsive to inflammatory stimuli such as bacterial LPS and TNF-α (Steinman 1991). DCs express all four EP receptor subtypes (Harizi et al. 2003) and PGE2 has been shown to modulate DC function. PGE2 upregulates IL-10, which downregulates IL-12 production and the antigen-presenting capabilities of bone marrow cells (Harizi et al. 2002). When activated in the presence of PGE2, DCs lose their ability to secrete cytokines such as IL-12, CCL3, and CCL4, but upregulate expression of chemokine receptors necessary for migration to lymph nodes (Luft et al. 2002; Scandella et al. 2004 Jing et al. 2003). In human monocyte-derived dendritic cells (MoDCs), stimulation of EP2 and EP4 by PGE2 promotes MoDC maturation and inhibits LPS-induced cytokine production (Kubo et al. 2004). Coculture of naive T cells with matured MoDCs showed that EP2/EP4-stimulated MoDCs preferentially induced Th2 polarization, indicating the importance of EP2 and EP4 receptors in the determination of Th1/Th2 development of naive T cells (Kubo et al. 2004). In vitro, LPS-stimulated MoDCs from individuals with asthma exhibited increased PGE2 and IL-10 production compared with DCs from normal subjects. Increased PGE2 synthesis by DCs from subjects with asthma was associated with an increase in COX-2 mRNA expression (Long et al. 2004). Alveolar macrophages represent a major source of PGE2, in

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particular after expression of COX-2 (Hempel et al. 1994 ). The resolution of inflammation in asthma and tissue homeostasis is dependent on clearance of apoptotic cells by alveolar macrophages. In animal and in vitro studies, apoptotic cell clearance induces secretion of antiinflammatory mediators such as transforming growth factor (TGF) and PGE2, with associated suppression of proinflammatory cytokines, chemokines, and eicosanoids (Fadok et al. 1998). Clearance of apoptotic cells is crucial to the resolution of inflammation and development of fibrosis, but the process is not well understood in normal or diseased human lungs. Human BAL macrophages from normal control subjects and subjects with mild/moderate or severe asthma have been examined in vitro for phagocytosis of apoptotic human T-cell line Jurkats and secretion of inflammatory mediators (Huynh et al. 2005). Alveolar macrophages from normal subjects and patients with mild/ moderate asthma were able to phagocytose apoptotic cells in response to LPS, resulting in PGE2 and 15-HETE induction; in contrast, cells from patients with severe asthma had defective LPS-stimulated uptake of apoptotic cells, with associated failure to induce PGE2 and 15-HETE. A decrease in the responsiveness of alveolar macrophages to LPS in severe asthma is manifested by defective apoptotic cell uptake and reduced secretion of inflammatory mediators and this may contribute to the chronicity of inflammation and remodeling in lungs of patients with asthma (Huynh et al. 2005). Finally, PGE2 has a fundamental role in tissue repair and fibrosis. Airway remodeling in chronic asthma is characterized by specific structural changes in the airway wall including subepithelial fibrosis, myofibroblast accumulation, airway smooth muscle hyperplasia and hypertrophy, mucous gland and goblet cell hyperplasia, and epithelial disruption. These changes are typically refractory to corticosteroids and make a major contribution to disease chronicity. Unstressed bronchial epithelium releases PGE2 and 15-HETE, which supress mesenchymal cell activation. In asthma, however, the epithelium has increased susceptibility to oxidant injury and imparied epithelial repair. PGE2 acts as a potent inhibitor of mitogenesis, collagen synthesis, and mesenchymal cell chemotaxis and can therefore suppress inflammation and fibroblast activation. In human lung explants PGE2 also reduces mucus glycoprotein secretion (Marom et al. 1981).

PGE2 receptors PGE2 activates four GPCRs (EP1–EP4) that are present in differing ratios depending on the cell system. Stimulation of EP1 receptors results in activation of phosphatidylinositol hydrolysis and elevation of the intracellular Ca2+ concentration. EP2 and EP4 ligation increases intracellular cAMP through activation of adenyl cyclase (Narumiya et al. 1999. However, direct comparison of the relative abilities of EP2 and EP4 to increase cAMP reveals much weaker Gs coupling by EP4 compared with the EP2 receptor (Fujino et al. 2002). The striking feature of the EP3 receptor is that it exists in

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multiple isoforms generated by alternative splicing of the C-terminal tail. In humans, at least eight EP3 receptor splice variants have been identified, and multiple splice variants exist for other species including mouse, rabbit, and cow (Hata & Breyer 2004). EP3 generally couples to Gi and its stimulation inhibits adenylate cyclase, leading to a reduction in intracellular cAMP (Narumiya et al. 1999) but individual splice variants can also couple to stimulation of cAMP and IP3 generation (Irie et al. 1993; Namba et al. 1993). Hence, the diverse effects of PGE2 may be accounted for in part by the existence of EP1, EP2, EP3, and EP4 receptors, and heterogeneity in the coupling of these receptors to intracellular signal transduction pathways. It is known that EP receptors are localized to the plasma membrane. However, radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver has revealed perinuclear localization of EP3 and EP4 (Bhattacharya et al. 1999) and, in humans, perinuclear EP3 receptors have been found in brain endothelial cells (Gobeil et al. 2002) and adult epidermis (Konger et al. 2005). The EP receptors in brain endothelial cells have been proven to be functional as they modulate transcription (Gobeil et al. 2002), including the gene encoding inducible nitric oxide synthase (iNOS). COX-1 and COX-2 have been shown to be present on the inner and outer nuclear membranes and in similar proportions (Spencer et al. 1998). As cyclooxygenases localize to the nuclear envelope and play a dominant role in prostaglandin production, it could be speculated that PGE2-induced regulation of genes in asthma might involve EP receptors localized intracellularly and specifically at the cell nucleus. Of the four EP receptors, the EP3 and EP4 receptors bind PGE2 with highest affinity (Kd < 1 nmol/L), whereas the EP1 and EP2 receptors bind with lower affinity (Kd > 10 nmol/L) (Abramovitz et al. 2000). To elucidate the differential effects of PGE2 on individual EP receptors, a number of EP receptor agonists and antagonists have been used. First-generation EP receptor agonists include butaprost (EP2), the mixed EP1 and EP3 agonist sulprostone, and PGE1-OH (EP4). More recently, second-generation EP receptor ligands with higher receptor selectivity have been developed and include the agonists ONO-DI-004 (EP1), ONO-AE1-259-01 (EP2), ONO-AE-248 (EP3), and ONO-AE1-329 (EP4) and antagonists ONO-8713 (EP1), ONO-AE3-240 (EP3), and ONO-AE208 (EP4) respectively (Suzawa et al. 2000; Watanabe et al. 2000; Kabashima et al. 2002; Amano et al. 2003). Analyses of expression patterns of the four EP receptors have revealed their presence on most major subsets of cells involved in the adaptive immune response including macrophages, neutrophils, CD3+ T lymphocytes, and eosinophils in asthmatic patients and normal controls (Ying et al. 2004), suggesting that PGE2 may play a role at multiple levels within the immune system. EP3-deficient mice, but not EP1-, EP2-, or EP4-deficient mice, exhibited enhanced allergic inflamma-

tion in an EP receptor knockout model of murine ovalbumininduced allergic inflammation (Kunikata et al. 2005) and the selective EP3 agonist AE-248 suppressed the increased inflammation. In humans, induced sputum from atopic asthmatic patients contained greater numbers of macrophages expressing all four EP receptors and elevated percentages of these cells expressing EP2 and EP4 (Ying et al. 2004). The elevated expression of the PGE2 receptors EP2 and EP4 on bronchial macrophages in asthma, demonstrated in this study, is particularly interesting because studies in knockout mice have shown that EP2 and EP4, but not EP1 and EP3, agonists reduce the production of proinflammatory cytokines such as TNF-α but increase that of antiinflammatory cytokines such as IL-10 by macrophages (Shinomiya et al. 2001). PGE2 also reduces chemokine production in human macrophages through action on the EP4 receptor (Takayama et al. 2002). Furthermore, EP2 and EP4 receptor agonists, including PGE2 itself, upregulates COX-2 expression in activated macrophages, which might form a mechanism for further stimulation (Hinz et al. 2004). Ying et al. (2006) have also examined the expression of EP1–EP4 in nasal biopsies from patients with ASRD. ASRD is characterized by asthma often poorly responsive to treatment, rhinosinusitis, and recurrent nasal polyps. Impaired braking of inflammatory cell cysteinyl leukotriene production by PGE2 has been implicated in the pathogenesis of aspirin-exacerbated airways disease, but the mechanism is obscure. To address the hypothesis that expression of one or more EP receptors on nasal mucosal inflammatory cells was deficient in patients with aspirin-sensitive compared with nonaspirin-sensitive polypoid rhinosinusitis, double-staining was used to phenotype inflammatory leukocytes expressing EP1–EP4. Global mucosal expression of EP1 and EP2, but not EP3 or EP4, immunoreactivity was significantly elevated in aspirin-sensitive and nonaspirin-sensitive rhinosinusitis compared with controls. In contrast, the percentages of neutrophils, mast cells, eosinophils, and T cells expressing EP2 but not EP1, EP3 or EP4 was significantly reduced (P ≤ 0.04) in aspirin-sensitive compared with nonaspirin-sensitive patients. These findings are suggestive of a possible role for PGE2 in mediating epithelial repair in rhinitis and asthma. Because PGE2 exerts a range of inhibitory actions on inflammatory leukocytes via the EP2 receptor, its reduced expression in aspirin-sensitive rhinosinusitis may be partly responsible for the increased inflammatory infiltrate and production of cysteinyl leukotrienes that characterize aspirin-sensitive disease. Further confirmation of the likely relevance of these findings in ASRD comes from a Japanese genetics study, which has shown that polymorphisms in the PGE2 receptor subtype 2 gene confer susceptibility to aspirin-intolerant asthma (Jinnai et al. 2004). To identify the receptors mediating the actions of PGE2 on bronchomotor tone, Tilley et al. (2003) developed a mouse model and examined the effects of PGE2 on the airways of

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wild-type and EP receptor-deficient mice. In conscious mice, they found that administration of PGE2 increased airway responsiveness primarily through the EP1 receptor. Pretreatment with either atropine or bupivacaine eliminated these effects and AHR was also undetectable in anesthetized mice or denervated tracheal rings and only EP2-mediated relaxation of airway smooth muscle was observed. PGE2/EP1/ EP3-induced airway constriction appeared to occur indirectly through activation of neural pathways, whereas PGE2induced bronchodilation resulted from direct activation of EP2 receptors on airway smooth muscle. These finding raised the possibility that selective EP receptor analogs may prove useful in the treatment of asthma. EP2 and EP3 alone are expressed on bronchial smooth muscle (Burgess et al. 2004). In humans the action of PGE2 at the EP2 receptor appears to mediate against proliferation of airway smooth muscle cells (Burgess et al. 2004). One of the cardinal features of chronic asthma is airway remodeling and in particular increased airway smooth muscle bulk. PGE2 has previously been reported to inhibit the proliferation of nonasthmatic airway smooth muscle cells. Burgess et al. (2004) hypothesized that the proliferative control exerted by PGE2 was altered in the airways smooth muscle of asthmatic patients. Human airway smooth muscle cells from 17 nonasthmatic and 18 asthmatic patients were cultured in the presence and absence of PGE2 and tritiated thymidine uptake used to measure cell proliferation. The asthmatic airway smooth muscle cells were significantly more sensitive to inhibition of proliferation by PGE2 than the nonasthmatic cells. EP2 and EP3 receptors were also significantly increased in the asthmatic cohort compared with control subjects. The asthmatic cells also had increased sensitivity to inhibition of proliferation by EP2-specific agonists (butaprost and CAY10399) but not by EP3-specific agonists. Characterization of the structure of the human gene encoding EP2 has identified a CCAAT enhancer binding protein (C/EBP) consensus binding site 66 bp upstream of the transcription initiation site and several other C/EBP consensus binding sites in the 5′ flanking region (Smock et al. 1999). Three consensus sequences for the C/EBPs have also been described in the 5′ flanking sequence of the mouse gene encoding the EP2 receptor (Katsuyama et al. 1998). It is possible that the increased numbers of EP2 receptors observed on asthmatic airway smooth muscle cells is because these cells lack C/EBPα (Borger et al. 2003; Roth et al. 2003), the negative regulatory C/EBP. The authors hypothesized that the absence of this transcription factor might lead to disruption of the regulation of transcription, translation, or both of the EP2 receptor mRNA and therefore could contribute to the increased numbers of EP2 receptors observed. Hence the increased growth observed in asthmatic airway smooth muscle cells is not the result of impaired responsiveness to PGE2, as unexpectedly these cells have increased sensitivity to the antiproliferative effects of PGE2.

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The role of the EP1 in modulating airway function is just beginning to be elucidated and it has been suggested that its activation may be associated with the β-agonist resistance found in asthma (McGraw et al. 2006). Using a mouse model, activation of EP1 did not elicit airway smooth muscle contraction in mouse trachea. However, EP1 activation markedly reduced the bronchodilatory function of β2-adrenoceptor agonists. Activation of EP1 reduced β2-adrenoceptor-stimulated cAMP in airway smooth muscle but did not promote or augment β2-adrenoceptor phosphorylation or alter β2adrenoceptor trafficking. Bioluminescence resonant energy transfer, indicative of receptor cross-talk, showed that EP1 and β2-adrenoceptors formed heterodimers and this ultimately affected β2-adrenoceptor-mediated bronchial relaxation (McGraw et al. 2006). It therefore appears that lone stimulation of EP1 does not directly modify airway tone but that EP1 acts as a modulator of the β2-adrenoceptor.

PGE2 in asthma Shortly after its discovery in the 1960s, PGE2 was observed to have potent relaxant effects on airway smooth muscle, making this endogenous lipid an attractive candidate drug for asthma (Main 1964; Mathe & Hedqvist 1975). Inhalation of 55 μg PGE1 and PGE2 in normal subjects increased specific airway conductance by 10 and 18%, respectively, and in asthmatic subjects by 41 and 39%, respectively (Smith et al. 1975). However, the effect of PGE2 on human smooth muscle is much more complex. Inhalation in normal subjects produces an initial bronchoconstriction within 5 min, followed by bronchodilatation peaking at 15 min and returning to baseline after 30 min (Walters & Davies 1982). In asthmatic subjects the response to inhaled PGE2 is more often biphasic, with the degree of initial bronchoconstriction inversely related to resting airway tone and enhanced following bronchodilatation with the anticholinergic agent ipratropium bromide (Walters & Davies 1982). Additionally, PGE2 has paradoxical effects against the bronchoconstrictor activity of histamine and methacholine, protecting against bronchoconstriction during the bronchodilator phase and enhancing bronchial responsiveness to these reference agents if administered after the end of this phase (Walters & Davies 1982). It is likely that some of these seemingly paradoxical effects are likely to be explained by the actions of PGE2 on four receptor subtypes. Ex vivo studies from humans and animals have also shown that PGE2 can both relax and constrict airway smooth muscle, a paradox explained when it was recognized that PGE2 could act through more than one receptor. A number of studies have examined the usefulness of PGE2 in various models of asthma. Inhaled PGE2 has also been shown to protect against the early- and late-phase bronchoconstriction and AHR induced by allergen (Pavord et al. 1993; Gauvreau et al. 1999), inhalation of ultrasonically nebulized distilled water (Pasargiklian et al. 1976), and sodium metabisulfite (Pavord et al. 1991). Exogenously administered

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PGE2 reduces exhaled nitric oxide in both asthmatic and normal subjects, an effect seen independently of PGE2-induced airway bronchoconstriction and thought to be secondary to iNOS inhibition (Kharitonov et al. 1998). Inhaled PGE2 also reduced Th2 cytokine and cysteinyl leukotriene production in a rat model of ovalbumin-induced allergic inflammation (Martin et al. 2002). Inhaled PGE2 has been shown to decrease the maximal fall in exercise-induced FEV1 by 63% and reduce the duration of exercise-induced asthma (Melillo et al. 1994). This effect is comparable to that found with leukotriene receptor antagonists (Manning et al. 1990b), sodium cromoglycate (Robuschi et al. 1992), and β2 agonists (Anderson et al. 1979). In a randomized, double-blind, crossover study, induced sputum was used to measure mast cell mediators and eicosanoids at baseline and 30 min after exercise challenge in 25 individuals with asthma with exercise-induced symptoms (Hallstrand et al. 2005). The percentage of columnar epithelial cells in induced sputum at baseline was associated with the severity of exerciseinduced asthma. After exercise challenge, histamine, tryptase, and cysteinyl leukotrienes significantly increased and PGE2 and TXB2 significantly decreased in the airways, and there was an increase in airway columnar epithelial cells. The concentration of columnar epithelial cells was associated with the levels of histamine and cysteinyl leukotrienes in the airways. PGE2 is the predominant eicosanoid synthesized by the airway epithelium and injury to the airway epithelium is known to decrease the production of PGE2 (Holgate et al. 2003). The decreased levels of PGE2 after exercise and the increased numbers of columnar epithelial cells strongly suggests that the relative underproduction of epithelial PGE2 leads to an overproduction of cysteinyl leukotrienes by leukocytes such as bronchial macrophages. Alteration in this ratio leads to a predominance of the effects of cysteinyl leukotrienes, which are counteracted in part by PGE2 under normal circumstances. In a placebo-controlled, randomized, double-blind, crossover study, Hartert et al. (2000) noted that PGE2 reduced allergen-stimulated release of PGD2 in subjects with asthma. PGE2 has been shown to affect airway physiology, inhibiting both the early and late asthmatic response, without resulting in significant bronchodilation (Pavord et al. 1993). PGE2 may attenuate the early asthmatic response by decreasing allergen-induced release of PGD2 in the airways of asthmatic patients. This process, involving the actions of histamine, PGD2, and cysteinyl leukotrienes, is thought to be largely mast cell dependent (Holgate et al. 1991) and PGD2 is thought to be principally released from activated mast cells. It also seems likely that the mechanisms of action of PGE2 in attenuating the late asthmatic response is antiinflammatory rather than bronchodilatory. There is good evidence for PGE2 ameliorating aspirin sensitive respiratory disease (ASRD) and exogenous PGE2 has been shown to prevent aspirin-induced bronchoconstriction and urinary LTE4 excretion (Sestini et al. 1996) and the release of

cysteinyl leukotrienes from cultured peripheral blood leukocytes (Celik et al. 2001). Therefore a special role for PGE2 in the pathogenesis of ASRD has been suggested (Szczeklik et al. 1995). PGE2 has the potential to reverse at least three of the cardinal features of aspirin-sensitive asthma: enhanced cysteinyl leukotriene production, smooth muscle hyperplasia (smooth muscle cells from asthmatic patients over-express PGE2 receptors) and airways epithelial damage. It has been suggested that COX-1 inhibitor induced exacerbations of ASRD are secondary to a depletion of bronchoprotective PGE2, which is more vulnerable because of a functional deficiency in COX-2 (Pujols et al. 2004). Alternatively it has been hypothesized that decreased expression of the EP2 receptor in inflammatory cells isolated from nasal biopsies in aspirin sensitive patients, may impair PGE2 in its anti-inflammatory effects (Ying et al. 2006). If deficient PGE2 production does lie at the heart of ASRD conclusively proving this hypothesis has been difficult as in vivo and in vitro studies have yielded inconsistent results. In 2005, a paper measuring PGE2 production at baseline in nasal polyps from aspirin sensitive and aspirin tolerant patients was published (Perez-Novo et al. 2005). The authors homogenised nasal polyps, in 15% methanol and 0.1M sodium phosphate buffer and measured PGE2 in the supernatant. Polyps in patients with ASRD had significantly lower PGE2 production (30 000 pg/ml) compared to aspirin tolerant patients (50 000 pg/ml). Homogenisation can disrupt the cell membrane and it is possible that the authors were measuring intracellular as well as extracellular PGE2 levels, as the PGE2 levels measured are significantly higher than in other in vitro studies. The authors also noted that local down-regulation of PGE2 was specifically observed in both nasal polyp groups compared to samples from patients with chronic rhinosinusitis (but without nasal polyps) and normal controls. This led them to question whether diminished local production of PGE2 might not be a unique characteristic of aspirin sensitivity. Other, in vitro studies, addressing PGE2 production in aspirin-sensitive patients have involved stimulation of peripheral blood cells which are remote from the site of the disease or prolonged culture of tissues in vitro. A study published in 2000, measured PGE2 production in cell culture supernatants, from nasal polyp epithelial cell explants (Kowalski et al. 2000). The authors found diminished PGE2 production at baseline in aspirin sensitive compared to aspirin tolerant patients but on stimulation with aspirin, a similar relative inhibition of PGE2 production was seen in both groups and on stimulation with calcium ionophore a similar relative increase in PGE2 production. In contrast, a study a few years later, stimulated bronchial fibroblasts with cytomix (lipopolysaccharide, il-1α, and tumour necrosis factor-α) for 18 hours. They found that after stimulation, in aspirin sensitive patients the bronchial fibroblasts did produce less PGE2 but spontaneous production was equivalent in both groups (Pierzchalska et al. 2003).

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In vivo studies, have directly quantified PGE2 production by measuring PGE2 release in nasal lavage fluid, exhaled breath condensate and bronchealveolar lavage fluid in aspirinsensitive and aspirin-tolerant patients. The findings from these studies are inconsistent. The first study measuring eicosanoid levels in nasal lavage fluid from aspirin tolerant, aspirin sensitive, aspirin desensitised and normal patients; was published in 1988. The authors found no significant decreases in PGE2 levels after aspirin challenge in patients with ASRD but levels did decrease in all other groups (Ferreri et al. 1988). Four year later, Picado et al. published a double blind placebo controlled study where they measured CysLT and PG production in nasal lavage fluid in aspirin sensitive and tolerant patients after nasal lysine aspirin challenge. They found that there was inhibition of PGE2 production in both groups of patients (Picauo et al. 1992). In 1996 Szczeklik et al. published their findings in bronchealveolar lavage fluid after segmentally challenging, aspirin sensitive and aspirin tolerant patients with lysine aspirin. They found no variation at baseline in PG, CysLT, tryptase, IL-5 or ECP levels. But, 15 minutes after aspirin instillation, there was a statistically significant rise in CysLT, IL-5, and eosinophil number in the aspirin sensitive patients only. The administration of aspirin significantly depressed PGE2 levels in aspirin sensitive and tolerant patients. Exhaled breath condensates have also been measured in patients pre- and post-aspirin challenge and no change in measurable PGE2 was noted after aspirin challenge in either group (Sanak et al. 2004).

PGI2 (prostacyclin) Prostacyclin is generated by macrophages and constitutes a major product of endothelial cells with potent cAMPdependent smooth muscle relaxant activity for the maintenance of vascular patency. The hydrolysis product of PGI2, 6-keto-PGF1α, is released into biological fluids such as BAL and provides a reliable index of PGI2 generation and release. This metabolite has been detected as the major COX product released from both resting and allergen-stimulated fragments of human bronchi, although the cells responsible remain unidentified (Schulman et al. 1982). In studies of airway smooth muscle, PGI2 was found to cause relaxation of guinea-pig tracheal strips and of isolated precontracted human bronchus, although smooth muscle contraction was also sometimes seen (Gardiner & Collier 1980). In a study of 8 asthmatic and 10 normal controls, the effects of inhaled PGI2 and 6-oxo-PGF1α on airway caliber were examined. Inhalation of both COX products caused cough and retrosternal discomfort and no consistent effect was found in either group, except for reproducible bronchodilatation in two asthmatic subjects (Hardy et al. 1985). The same group went on to examine the effect of doubling doses of both prostanoids using different parameters of airway caliber in mild asthmatic subjects. With doses of PGI2 of up to 1 mg/mL there

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was no effect on specific airway conductance, but a concentration-dependent fall in FEV1 and Vmax30 was observed in all subjects. In two of the four subjects, there was an increase in residual volume and reduction in vital capacity without a change in total lung capacity. Furthermore, PGI2 but not 6-oxo-PGF1α protected against PGD2- and methacholineinduced bronchoconstriction, as measured using any parameter of airway caliber (Hardy et al. 1988). An explanation of these seemingly paradoxical results may be that the principal action of PGI2 in the airways is to cause vasodilatory mucosal engorgement and hence a reduction in small-airway caliber as measured by FEV1 and Vmax30, but to protect against the spasmogenic effects of other mediators by increasing their clearance from receptor sites by increasing blood flow. PGI2 is less potent than PGE2 in relaxing human airways in vitro (Tamaoki et al. 1993) and, in contrast to PGE2, does not protect against histamine-induced contraction (Knight et al. 1995). Prostacyclin binds the prostanoid IP receptor, which predominantly couples to a Gs-type G protein, leading to an increase in cAMP (Boie et al. 1994; Katsuyama et al. 1994; Nakagawa et al. 1994). C-terminal modification of the IP receptor allows the receptor to couple to other signal transduction pathways such as Gq-dependent phosphoinositide turnover (Katsuyama et al. 1994 ) and Gi-dependent inhibition of cAMP (Hebert et al. 1998). The human prostacyclin IP receptor was first cloned from lung and megakaryocyte cDNA libraries (Boie et al. 1994; Katsuyama et al. 1994; Nakagawa et al. 1994) and has been shown to be expressed in many tissues including the lung. Due to its unstable nature, PGI2 has limited experimental use. Therefore, synthetic agonists such as iloprost, cicaprost, and carbacyclin are commonly used to study IP receptor function. Studies examining signaling through the IP receptor support the hypothesis that IP receptor signaling suppresses Th2-mediated allergic inflammatory responses. IP receptor mRNA is upregulated in CD4+ Th2 cells and inhibition of PGI2 formation by the COX-2 inhibitor NS-398 during antigeninduced airway inflammation results in greater lung Th2mediated lung inflammation (Jaffar et al. 2002). Prostacyclin has been suggested to exert this effect in part by enhancing Th2 production of the antiinflammatory cytokine IL-10. An immunosuppressive role for the IP receptor in Th2-mediated inflammation is supported by the observation that in the ovalbumin-induced asthma model, IP receptor-deficient mice have increased antigen-induced leukocyte accumulation in BAL fluid and peribronchiolar and perivascular inflammatory infiltration (Takahashi et al. 2002). In a murine model of asthma, IP receptor-deficient mice were challenged with ovalbumin. They developed goblet cell hyperplasia, and subepithelial fibrosis compared to wild-type animals. These changes were suggestive of allergen induced airway remodeling and were probably due to the upregulation of Th2 cytokine production, IgE production, or airway eosinophilic inflammation

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(Nagao et al. 2003). Thus, PGI2 may shift the balance within the immune system away from a Th2 dominant response and inhibits allergic inflammation. One potential mechanism by which PGI2 inhibits allergic inflammation is by increasing T-cell production of IL-10, which suppresses Th2 immunity. PGI2 or its stable analog carbaprostacyclin augmented IL-10 production by Th2 cells.

Lipoxins Nicknamed the “good” lipids for asthma (Peters-Golden 2002), lipoxins were discovered just over 20 years ago following experiments where labeled arachidonate was subjected to stimulation with a mixed leukocyte suspension (Serhan et al. 1984). Two new polar compounds with distinct physical properties from prostaglandins, leukotrienes and thromboxanes were identified using mass spectrometry. The name “lipoxin” was conceived to describe them, an abbreviation for lipoxygenase interaction product. Since then, Serhan and others have identified lipoxins in biological fluids and made structurally stable lipoxin analogs permitting further elucidation of their functions. Four years after Serhan’s initial description, lipoxins were isolated from rat alveolar macrophages (Kim 1988) and guinea-pig lung parenchyma (Cristol & Sirois 1988). Lipoxins have since been measured in a wide range of biological fluids and human tissues using enzyme-linked immunosorbent assay (ELISA), reverse-phase HPLC, and radioimmunoassay. In 1990, Lee and colleagues were the first to demonstrate the presence of lipoxins in BAL fluid from patients with a variety of pulmonary pathologies including asthma (Lee et al. 1990). Lipoxins have now been identified in patients with a range of respiratory conditions including from bronchial tissue in patients undergoing lung resection (Edenius et al. 1990), nasal lavage fluid from aspirinsensitive asthmatics (Levy et al. 1993), induced sputum from asthmatic patients (Vachier et al. 2005), and nasal polyp homogenates from both aspirin- and nonaspirin-sensitive patients (Perez-Novo et al. 2005).

Lipoxin biosynthesis, metabolism, and elimination Lipoxins are now known to be trihydroxytetraene eicosanoids that modulate leukocyte trafficking and vascular tone and are generated via the sequential actions of two or more lipoxygenases. Interaction of leukocytes with epithelium, endothelium, or platelets results in the formation of lipoxin (LX)A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) and its positional isomer LXB4 (5S,14R,15S-trihydroxy6,10,12-trans-8-cis-eicosatetraenoic acid). In contrast to cysteinyl leukotrienes, lipoxins have potent antiinflammatory effects. They are formed at sites of inflammation by transcellular cooperation. There are three principal biosynthetic pathways for their formation. The first involves cooperation between 15-LO and 5-LO, the second involves acetylation of

COX-2 by aspirin to trigger 15-epi-LXA4 biosynthesis, and the third involves interactions between 12-LO and 5-LO. 15-LO is an enzyme that is abundant in human lung and which is upregulated in the asthmatic airway (Bradding et al. 1995). It is present in airway epithelial cells and eosinophils (Nadel et al. 1991; Bradding et al. 1995), macrophages (Profita et al. 2000), and other leukocytes. In the lung 5-LO from circulating eosinophils (Steinhilber & Roth 1989), macrophages (Chavis et al. 1992), neutrophils (Chavis et al. 1996), or monocytes (Chavis et al. 1998) uses 15(S)-hydroxyeicosatetraenoic acid (15S-HETE), which is released from airway epithelial cells by airway epithelial 15-LO, as a substrate to generate lipoxins. Aspirin also interacts with the lipoxin system. Aspirin-acetylated COX-2 no longer produces prostaglandins but remains catalytically active in cells and generates 15RHETE from arachidonic acid; this becomes a substrate for leukocyte 5-LO-mediated conversion to 15-epi-LXA4 and 15-epi-LXB4 (Clària & Serhan 1995). 15-epi-LXA4 and 15epi-LXB4 are also known as aspirin-triggered lipoxins (ATL). 15R-HETE is also generated via the cytochrome P450 metabolism of arachidonic acid in epithelial cells (Clària et al. 1996). The third major route of lipoxin biosynthesis in humans involves an LTA4-dependent step, with lipoxin generation within the vascular lumen during platelet–leukocyte interactions and at mucosal surfaces via leukocyte–epithelial cell interactions. Platelets adhere to neutrophils, LTA4 is exported from leukocytes and converted rapidly to bioactive LXA4 and LXB4 in a 1 : 1 ratio by platelets rich in 12-LO and 15-LO (Serhan & Sheppard 1990). The process is similar in leukocyte– epithelial cell interactions but 15-LO from macrophages and bronchial tissue acts as a lipoxin synthase instead of 12-LO. Lipoxins are rapidly generated, act locally, and then rapidly inactivated by metabolic enzymes. The major route of lipoxin inactivation is rapid dehydrogenation and inactivation by monocyte 15-hydroxyprostaglandin dehydrogenase (PGDH). This process occurs in several stages. Firstly, 15PGDH catalyzes a dehydrogenation step that converts LXA4 to biologically inactive 15-oxo-LXA4 (Serhan et al. 1995) or LXB4 to 15-oxo-LXB4 (Maddox et al. 1998). This is followed by specific reduction of the double bond adjacent to the ketone (Serhan et al. 1995) by eicosanoid oxidoreductase to give 13,12-dihydro-15-oxo-LXA4. Further metabolism of the 15oxo group by 15-PGDH yields 13,14-dihydro-LXA4 (Clish et al. 2000). In comparison with lipoxins, the metabolic inactivation of ATLs is less efficient, with slower conversion in vitro to their 15-oxo-metabolite. Hence when ATLs are generated or cytochrome 450 15-epi-lipoxins, their biological half-life is around twofold greater than LXA4, leading to an enhanced bioavailability and duration of action (Serhan et al. 1995). However, the rapid degradation of lipoxins was a barrier to further investigation of lipoxin function, and this led to the design and synthesis of first-generation PGDH-resistant LXA4, LXB4 and ATL stable analogs by Serhan, Maddox and colleagues between 1995 and 1998 (Serhan et al. 1995; Maddox

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et al. 1998). These are relatively stable analogs that resist inactivation and retain biological activity and, together with myeloid-specific ALX-R-expressing transgenic mice, have provided powerful tools for exploring lipoxin functions in vivo.

Receptors When administered to human cells in vitro or murine systems in vivo, lipoxins act on at least two classes of receptors. Lipoxins competitively antagonize cysteinyl leukotrienes for binding at their cognate receptor CysLT1 (Gronert et al. 2001) and LXA4 binds to a seven-transmembrane-spanning GPCR designated ALX (Takano et al. 1997). Signal transduction pathways activated on stimulation of the ALX receptor (ALXR) are still being identified. In 1993 using [3H]-LXA4, Fiore et al. (1993) described the presence of a high-affinity lipoxin receptor (kd ∼ 0.5 nmol/L) on differentiated HL-60 cells. They also noted a second lowaffinity receptor (kd ∼ 10 nmol/L) on endothelial cells. A year later the same group identified the cDNA of the highaffinity receptor as a homolog of the low-affinity formylmethyl-peptide receptor (Fiore et al. 1994). The binding of other eicosanoids including LXB4, LTD4, LTB4, and PGE2 against this receptor was also tested but only LTD4 showed weak competition, with a Ki of 80 nmol/L (Fiore et al. 1994). Hence the ALXR is also known as formyl peptide receptor like-1 and formyl peptide receptor-2. The mRNA for ALXR has been isolated from a number of mammalian tissuesand cell types including human neutrophils (Fiore et al. 1994) and monocytes (Yang et al. 2001) (Table 26.5).

Table 26.5 Mammalian tissues expressing ALX mRNA. Tissue/cell type

Reference

Human cells Neutrophil Enterocytes Monocytes Synovial fibroblasts Activated T cells Bronchial epithelial cells

Fiore et al. (1994) Gronert et al. (1998) Yang et al. (2001) Sodin-Semrl et al. (2000) Ariel et al. (2003) Bonnans et al. (2006)

Human tissues Lung, placenta Spleen, liver (lesser amounts), heart (lesser amounts)

Fiore et al. (1994) Takano et al. (1997)

Murine cells Neutrophil

Takano et al. (1997)

Murine tissues Spleen, lung

Takano et al. (1997)

Rat Leukocytes

Chiang et al. (2003)

602

Mouse and rat ALXR have been cloned from a spleen cDNA library (Takano et al. 1997) and from peripheral blood leukocytes, respectively (Chiang et al. 2003). Across species, the human, mouse, and rat ALXR share a minimum of 74% homology (Serhan 2002). A second mouse lipoxin GPCR has also been identified using a macrophage cDNA library and, like its previously described counterpart, activation of this receptor is coupled to antiinflammatory responses (Vaughn et al. 2002). Using a murine model of zymosan-induced peritonitis in transgenic mice with myeloid-specific expression of the human ALXR, it has been shown that increased ALX signaling considerably abrogates the inflammatory response in vivo (Devchand et al. 2003). Adding further to the complexity of ALXR signaling, this study also showed that a 30–130% increase in human ALXR compared with endogenous murine ALXR led to a 20-fold enhancement in levels of endogenous LXA4, with a concomitant decrease in LTB4. This has led to suggestions that ALXR is not only activated by LXA4 but also amplifies lipoxin-mediated signal transduction (Parkinson 2006). ALXR is increasingly known as a “promiscuous receptor” as a number of peptide ligands can activate it in addition to LXA4 (Migeotte et al. 2006). These include serum amyloid A, synthetic peptides derived from HIV glycoprotein 120, and annexin 1, all of which bind the receptor but at much lower affinity than LXA4 (Su et al. 1999; Shen et al. 2000; Perretti et al. 2002). Differential responses depending on the ligand bound to ALXR have been noted (Sodin-Semrl et al. 2004) and may reflect “agonist-dependent trafficking.” Of interest in asthma, from murine data it is known that glucocorticoids such as dexamethasone induce expression of annexin 1 which is enzymatically cleaved to produce peptides and that these peptides interact with ALX to induce antiinflammatory cascades (Perretti et al. 2002). It has recently been hypothesized that glucocorticoids exert their antiinflammatory actions in part via upregulation of the ALXR; furthermore, in human neutrophils isolated from blood, the administration of dexamethasone was shown to upregulate the expression of ALX in a dose- and time-dependent manner (Hashimoto et al. 2007). As well as binding ALXR, lipoxins competitively antagonize the binding of cysteinyl leukotrienes to the CysLT1 receptor. In a study using human vascular endothelial cells it was demonstrated that both aspirin-triggered 15-epi-LXA4 and LTD4 bind and compete with equal affinity at the CysLT1 receptor. The clinically used CysLT1 receptor antagonist montelukast showed a lower rank order for competition with a [3H]-ATL analog (Gronert et al. 2001). In contrast, LTD4 was an ineffective competitive ligand at the recombinant ALXR compared with the [3H]-ATL analog, suggesting that lipoxin can act at the CysLT1 receptor but that the reverse does not hold (Gronert et al. 2001). It is unknown whether lipoxins also bind to and antagonize CysLT2.

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LXA4 also binds the nuclear aryl hydrocarbon receptor (Ah), a ligand-activated transcription factor (Schaldach et al. 1999) In human neutrophils, subcellular fractionation showed that tritiated LXA4 binding sites were found in the plasma membrane and endoplasmic reticulum (42.1%) and granules (34.5%) as well as nuclear-enriched fractions (23.3%) (Fiore et al. 1992). The finding that LXA4 acts as a ligand for the Ah receptor might relate to these reports of specific binding of LXA4 to intracellular recognition sites after transport and uptake (Levy 2005). In murine DCs, it has been demonstrated that lipoxins activate both Ah and ALXR and that this activation triggers expression of suppressor of cytokine signaling (SOCS)-2 (Machado et al. 2006). It is not known whether LXB4 binds to a specific high-affinity receptor, although functional studies have indicated such a putative receptor may exist (Maddox et al. 1998). However, presently there is no known receptor for LXB4 and research in this area has been slow because of lack of a suitable LXB4 radiolabel with high specificity (Brink et al. 2003).

Physiologic actions of lipoxins The lipoxins and their bioactive structural analogs have numerous biological effects, with cell-type specific actions. It is believed that the immunomodulatory role of lipoxins

may be important in regulating inflammatory processes in a number of diseases including asthma (Nasser & Lee 2002). Hence while inhibiting neutrophil chemotaxis and eosinophil activation at nanomolar concentrations (Lee et al. 1989; Soyombo et al. 1994), lipoxins stimulate monocyte trafficking (Maddox et al. 1997) and macrophage phagocytosis of apoptotic PMNs (Godson et al. 2000). Table 26.6 summarizes some of the key immunomodulatory actions of lipoxins. Increasingly the role of structural cells in the orchestration of ongoing inflammatory processes has been highlighted and a number of nonmyeloid cell functional responses have also been shown to be potently regulated by lipoxins (Serhan 2002). Chronic asthma is associated with airway remodeling, where structural changes including subepithelial fibrosis, myofibroblast accumulation, airway smooth muscle hyperplasia and hypertrophy, mucous gland and goblet cell hyperplasia, and epithelial disruption may be seen. Of particular interest in asthma, LXA4 has been shown to inhibit proliferation of human lung fibroblasts induced by connective tissue growth factor (Wu et al. 2006). Lipoxins have also been shown to stimulate cytosolic Ca2+ release in bronchial epithelium, with resultant Cl– secretion (Bonnans et al. 2002), indicating that in airway epithelial cells LXA4 is involved in ionic transport regulation. It has also been hypothesized that the

Table 26.6 Immunomodulatory activities of lipoxins/lipoxin analogs in leukocytes. Activity

Reference

Neutrophils Inhibit neutrophil transmigration across: Epithelial cells Endothelial cell monolayers Inhibition of LTB4-induced neutrophil migration Inhibition of neutrophil adhesion molecule expression Inhibit neutrophil degranulation Inhibit neutrophil superoxide anion generation Inhibit TNF-a-induced IL-1b release from neutrophils Inhibit LTB4 release from neutrophils

Colgan et al. (1990) Papayianni et al. (1996) Lee et al. (1991) Papayianni et al. (1996) Hachicha et al. (1999); Vachier et al. (2002) Hachicha et al. (1999) Hachicha et al. (1999) Vachier et al. (2002)

T lymphocytes Inhibit NK cell cytotoxicity Inhibit TNF-a secretion

Ramstedt et al. (1987) Ariel et al. (2003)

Dendritic cells Inhibit IL-12 production from murine dendritic cells

Aliberti et al. (2001)

Eosinophils Inhibit eosinophil chemotaxis and degranulation Inhibit allergen-induced trafficking

Soyombo et al. (1994) Bandeira-Melo et al. (2000)

Monocytes Stimulate monocyte adhesion Stimulate chemotaxis Stimulate phagocytosis of apoptotic neutrophils by macrophages

Maddox & Serhan (1996) Maddox et al. (1997) Godson et al. (2000)

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LXA4-induced [Ca2+]i increase might play a central role in autocrine production of LXA4 and constitute an amplification mechanism for the biosynthesis of LXA4 itself (Bonnans et al. 2002). More recently it has been demonstrated that injured bronchial epithelial cells upregulate ALX to help promote resolution of bronchial inflammation by LXA4 (Bonnans et al. 2006). In vitro, lipoxins have been shown to mediate relaxation of precontracted pulmonary arteries and bronchi (Dahlén et al. 1988). It is also worth noting that the role of LXA4 may not be solely confined to antiinflammatory actions and recently several studies have suggested that it may also have an antiproliferative role in various tissue types including human mesangial cells and HUVECs (Mitchell, D. et al. 2000; Fierro et al. 2002). Remodeling in chronic asthma is also characterized by angiogenesis, and using a murine chronic granulomatous air pouch model in vivo, ATL-1 has been shown to inhibit angiogenesis (Fierro et al. 2002), a reminder perhaps that the vascular biology of lipoxins should not be forgotten.

Lipoxin and asthma Since Lee and colleagues first measured lipoxins in BAL fluid from patients with different pulmonary pathologies including asthma, a number of groups have confirmed that lipoxins and ATLs are generated in vivo and in vitro by asthmatic subjects (Levy et al. 1993; Chavis et al. 2000; Bonnans et al. 2002), as well as normal subjects (Bonnans et al. 2002). However, the data regarding increased or decreased lipoxin production in asthmatic patients is limited and presents a contradictory picture. LXA4 production, as measured by ELISA in supernatants of induced sputum, was significantly decreased in moderate and severe asthmatic compared with mild asthmatic subjects (Vachier et al. 2005). In contrast, levels of 15-epi-LXA4 from the same cohort were significantly higher in the severe asthmatics compared with normal controls. A study later the same year, using a novel fluorescencebased system to measure lipoxin synthesis in whole blood, found that severe asthmatic patients underproduced both 15-HETE and LXA4 (Levy et al. 2005). As inhaled lipoxins are inactivated rapidly, there is only limited information about their actions in humans in vivo. LXA4 has been shown to attenuate LTC4-induced bronchoconstriction in human asthmatic volunteers (Christie et al. 1992c), an effect explained partly by its antiinflammatory actions and also by its competitive binding to the CysLT1 receptor. ASRD is an eosinophil-driven, leukotriene-dependent phenotype of asthma that is often aggressive and refractory to treatment. The ingestion of aspirin is associated with a fourfold increase in already raised cysteinyl leukotriene levels and an acute exacerbation of symptoms (Christie et al. 1991a). In this cohort of patients, lipoxin synthesis from stimulated whole blood is reduced compared with aspirintolerant subjects (Sanak et al. 2000; Kowalski et al. 2003). Furthermore, patients with ASRD demonstrate upregulation

604

of CysLT1 expression on inflammatory leukocytes (Sousa et al. 2002; Corrigan et al. 2005) but have decreased levels of lipoxins to compete with cysteinyl leukotrienes for binding at their cognate receptor. Therefore diminished capacity to generate lipoxins may contribute to the uncontrolled chronic inflammation that characterizes this phenotype of asthma. Although it has been more recently suggested that diminished LXA4 synthesis is a feature of severe asthma regardless of aspirin sensitivity (Celik et al., 2007). There is some evidence that patients with aspirin sensitivity have decreased COX-2 expression and its activity is reduced (Picado et al. 1999; Pujols et al. 2004). One of the biosynthetic pathways for lipoxin generation is the aspirin-induced acetylation of COX-2 (Clària & Serhan 1995). This generates 15R-HETE, a biosynthetic precursor of the ATLs (15-epi-LXA4 and 15-epi-LXB4). 15-epiLXA4 is more potent than native LXA4 as an inhibitor of PMN trafficking and PMN-mediated inflammation in vivo (Fierro et al. 2003). It is therefore conceivable that reduced generation of 15-epi-LXA4 may contribute to the acute worsening of ASRD on aspirin ingestion. In 2002, Levy, Serhan and colleagues used a murine model of asthma to demonstrate that ovalbumin challenge induced airway biosynthesis of LXA4 and its leukocyte receptor (Levy et al. 2002). In the same model, preadministration of an LXA4 analog (designed using the 15-epi-LXA4 structure as a template) was administered at least 60 min prior to ovalbumin challenge and its administration was associated with a significant attenuation in AHR. Levy (2005) noted that with an ED50 of less than 0.05 mg/kg, the lipoxin analog compared favorably with the CysLT1 receptor antagonist montelukast (0.03 mg/kg in rats) and dexamethasone (0.5–3 mg/kg in mice). Furthermore, the LXA4 analog also blocked many of the features of allergen-induced inflammation including recruitment and activation of eosinophils and neutrophils. Decreased measurement of proinflammatory lipid mediators in BAL fluid including IL-5, IL-13, eotaxin, prostanoids, and cysteinyl leukotrienes was also noted. Using transgenic mice expressing leukocyte restricted human ALX, the authors then went on to investigate the importance of lipoxin receptors and their ligands. In the lung they found that there was significant inhibition of pulmonary inflammation and eicosanoidinitiated eosinophil tissue infiltration but AHR was not attenuated. Peters-Golden (2002) has hypothesized that this may be because the effects of the LXA4 analog on bronchial hyperreactivity are mediated entirely by its competitive interaction with CysLT1 receptors or alternatively because the analog’s inhibitory effects on AHR may depend on its interactions with ALXRs on airway smooth muscle and bronchial epithelium rather than on leukocytes. Using an ovalbumin model of allergic sensitization in rats, Bandeira-Melo et al. (2000) have demonstrated that stable analogs of LXA4 and ATLs attenuated allergic pleural eosinophil influx, while concurrently increasing circulating eosinophilia, hence inhibiting the earlier edema and neutrophilia associated with allergic reac-

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tion. This eosinophilic blockade was independent of effects on mast cell recruitment and degranulation but involved analog inhibition of both IL-5 and eotaxin generation, as well as PAF. More recently, ATL has been found to attenuate LPSinduced acute lung injury in a mouse model (Jin et al. 2007). It has been just over 20 years since lipoxins were discovered by Serhan and colleagues. Since then a body of literature has accumulated describing their vascular biology and immunomodulatory and antiproliferative effects. LXB4 remains the less understood of the two lipoxins and the location and structure of LXB4 receptors and the extent to which LXB4 pharmacology varies from LXA4 is unknown. Nonetheless the evidence collected thus far suggests a pivotal role for lipoxins in mediating airway homeostasis and a potential role as a new therapeutic strategy in the management of asthma.

Platelet-activating factor PAF is an ether-linked phospholipid (1-O-alkyl-sn-glycero-3phosphocholine) first described as a substance released from IgE-stimulated basophils that was found to aggregate rabbit platelets (Benveniste et al. 1972). In 1979, Demopoulos et al. described a semisynthetic phosphoacylglycerol, 1-O-alkyl2-acetyl-sn-glycero-3-phosphocholine (AGEPC), which was able to aggregate platelets and release serotonin and had the same physicochemical properties as PAF. In 1980, Hanahan et al. purified and characterized PAF from activated basophils and concluded that AGEPC and PAF are the same compound. PAF has long been implicated in the pathophysiologic mechanisms of asthma, because exogenous PAF closely mimics many of the clinical features of asthma, including AHR. Since its discovery over three decades ago, PAF has emerged as one of the most important lipid mediators known.

Biosynthesis PAF is not a single molecule and should be considered as a family of structurally related autacoid phopholipids synthesized by inflammatory cells that have platelet-stimulating and neutrophil-priming activity and whose main members include 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine. The most commonly recognized chemical structure of PAF is shown in Fig. 26.8. PAF is synthesized in a wide variety of inflammatory cells, including platelets, neutrophils, basophils, macrophages, and eosinophils (Barnes et al. 1989; Chung 1992). The synthesis of PAF in stimulated inflammatory cells is generally via a two-step enzymatic pathway. Cell stimulation leads to the release of alkylacylglycerophosphocholine from cell membranes, which can be converted to PAF via either the “remodeling” pathway or the de novo pathway (Fig. 26.9). In the remodeling pathway, ether-linked phospholipids that form part of the structure of membranes are structurally

H2C

O

(CH2)n

CH3

O CH3

C

O

C

CH CH3

O CH2

O

P

CH2

CH2

O–

N+

CH3 CH3

sn-1, alkyl group sn-2, acetyl group sn-3, O-phosphocholine n = 15:0 or 17:1 Fig. 26.8 Chemical structure of platelet-activating factor.

modified. The initial step is catalyzed by the action of PLA2 on 1-alkyl-2-acyl-glycerophosphocholine, a membrane phospholipid (Hanahan 1986; Sturk et al. 1989). This deacylation results in lyso-PAF formation and the release of arachidonic acid, which has PLA2 inhibitory activity, thereby reducing further PAF synthesis; alternatively, arachidonic acid may act as the substrate for subsequent eicosanoid synthesis. LysoPAF is then acetylated by a specific lyso-PAF acetyltransferase to form 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine-PAF. PAF can subsequently be remetabolized back to its precursor lyso-PAF by the action of a specific PAF acetylhydrolase. The other route for PAF biosynthesis is known as the de novo pathway and begins with an intermediate in the synthesis of ether-linked membrane phospholipids. This is acetylated by a specific acetyltransferase and the phosphate is removed from the sn-3 carbon by a phosphohydrolase to form alkylacetylglycerol, which is subsequently converted by choline phosphotransferase to PAF.

Biological activity Soon after the identification of this proinflammatory phospholipid mediator, PAF was found to have activity beyond the originally described platelet aggregation and was demonstrated to possess a unique profile of biological effects. Furthermore, it was found to be the most potent lipid mediator known, with biological responses detectable at concentrations as low as 10–14 mol/L and an ED50 value for guinea-pig platelet activation of about 3 × 10–10 mol/L. PAF causes potent airway obstruction in vivo but in vitro effects on smooth muscle preparations are negligible, suggesting that this effect may be mediated through increased vascular permeability and vasoconstrictor effects on airway smooth muscle (Denjean et al. 1983; Chung et al. 1986; Cuss et al. 1986). PAF produces a receptor-mediated increase in airway microvascular leakage with a 1000-fold greater potency than histamine (Evans et al. 1987). Further evidence supporting this dual mechanism of PAF-induced airway obstruction comes from studies demonstrating that as this effect is not totally reversed by the airway smooth muscle

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De novo pathway O

CH2

Remodeling pathway R

O

CH2

R

O O

H

P 1-O-alkyl-2-lyso-sn-glycerol-3-phosphate

O

C

R′

P

Choline

1-O-alkyl-2-acyl-sn-glycerol-3-phosphocholine

Acetyl CoA Phospholipase A2

Acetyltransferase

Arachidonate

CoASH O

CH2

R

O

CH2

R

CH3

O

H

P

Choline

O O

C

P 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphate

1-O-alkyl-2-lyso-sn-glycerol-3-phosphocholine Lyso-PAF Acetyl CoA Acetyltransferase

Phosphohydrolase

CoASH

P O

CH2

O

R

O

CH2

R

O Cholinephosphotransferase

O

C

CH3 CDP-choline

O

H

1-O-alkyl-2-acetyl-sn-glycerol

O

C

P

Choline

CMP

1-O-alkyl-2-acetyl-sn-glycerol-phosphocholine (PAF)

relaxant salbutamol (Diaz et al. 1997), it cannot be completely attributed to vasoconstriction.

Vascular permeability PAF has been shown to be one of the few endogenous mediators that can induce increased vascular permeability in the pulmonary circulation within minutes. This is partly through a direct effect on the vascular endothelium (Humphrey et al. 1984) and partly an indirect effect through leukocyte activation (Bjork & Smedegard 1983), particularly neutrophils.

606

CH3

Fig. 26.9 Biosynthetic pathways of plateletactivating factor (PAF) showing the de novo and “remodeling” routes.

PAF induces this increase in vascular permeability by simultaneous activation of two independent pathways: a COX-independent pathway requiring the sphingomyelinase metabolite ceramide (Goggel et al. 2004), whereas the other is dependent on PGE2 (Goggel et al. 2002). Agents that interfere with PAF-induced ceramide synthesis, such as glucococorticoids, attenuate pulmonary edema formation induced by PAF (Goggel et al. 2002). In a murine model, PAF-induced vascular permeability appeared to be mediated via the actions of PGE2 on EP3 receptors (Goggel et al. 2002). PGE2 has been

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shown to both increase (Goggel et al. 2002) and decrease (Gillespie et al. 1987) vascular permeability. Recently, using perfused lung isolated from rats it has been demonstrated that PAF-induced edema is mediated by activation of the IP3 pathway, influx of extracellular calcium, and subsequent activation of a myosin light chain kinase-dependent and Rhoassociated protein kinase-independent mechanism (Goggel & Uhlig 2005).

Activity on airway smooth muscle PAF inhalation induces bronchoconstriction within 2–3 min and this is followed by the rapid development of tachyphylaxis (Cuss et al. 1986). Increases in airway responsiveness to reference agonists such as histamine and methacholine were originally described in both the guinea pig (Mazzoni et al. 1985) and the dog (Chung et al. 1986). PAF produces acute bronchoconstriction when inhaled by patients with asthma (Barnes et al. 1989). The major mediator responsible for PAFinduced bronchoconstriction in animal models (both in vivo and in vitro) is TXA2 (Tokuyama et al. 1991; Olson et al. 1993) and to a lesser degree leukotrienes (Uhlig et al. 1994). In animal models, dual inhibition of COX and 5-LO has a pronounced inhibitory effect on PAF-induced vasoconstriction and bronchoconstriction but does not prevent the increase in vascular permeability (Uhlig et al. 1994). In humans, PAF has been shown to release TXA2 and leukotrienes but their roles are less defined (Taylor et al. 1991). In two small trials with eight healthy men, PAF-induced bronchoconstriction was partially attenuated by the leukotriene antagonists SKF 104353-Z (Spencer et al. 1991) and ICI 204,219 (Kidney et al. 1993). However, the COX inhibitor indomethacin did not inhibit PAF-induced bronchoconstriction in seven subjects (three controls and four with asthma).

Activity on secretions PAF stimulates fluid secretion from porcine isolated trachea via activation of PAF receptors and via a mechanism that does not depend on the release of acetylcholine, histamine, or cysteinyl leukotrienes (Steiger et al. 1987). In cultures of guinea-pig tracheal epithelial cells, PAF stimulates release of mucin-like glycoprotein. The mechanism involves binding of PAF to receptors on epithelial cell surfaces, intraepithelial stimulation of lipoxygenase metabolism of arachidonic acid to HETEs, and stimulation of secretion by these epithelialderived HETEs via an autocrine or paracrine mechanism (Adler et al. 1992). In isolated human airways, PAF enhances respiratory glycolipid and glycoprotein release in vitro; this may be partly dependent on cysteinyl leukotriene generation but occurs independently of acetylcholine release (Goswami et al. 1989).

Inflammatory cells and asthma PAF is a potent activator of inflammatory cells. The activation of inflammatory cells leads to the release of other inflam-

matory mediators, such as the products of both the COX (Chung et al. 1986) and the lipoxygenase (Voelkel et al. 1982) pathways and the release of oxygen free radicals (Rouis et al. 1988). Activated eosinophils (Cromwell et al. 1990) and mast cells (Schleimer et al. 1986) in the bronchial airway of asthmatics may be the cellular source of PAF as these cells produce PAF in vitro when stimulated. With the advent of the “united airway theory” and allergic inflammation in upper and lower airways being increasingly viewed as one disease, it is worth noting that PAF is thought to be a potent mediator of allergic rhinitis and has been recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation. Immunohistochemistry has revealed the presence of anti-PAF receptor antibody-labeled eosinophils, macrophages, neutrophils, mast cells, lymphocytes, vascular endothelial cells, epithelial cells, and submucosal glands in nasal mucosa (Shirasaki et al. 2005). PAF-induced degranulation of mast cells appears to be inhibited by stimulation of β2-adrenergic receptors. The effect of PAF on eicosanoid generation was investigated by measuring a wide range of lipoxygenase and COX pathway products and release in cultured feline tracheal epithelial cells. HPLC chromatograms revealed that PAF augmented the release of PGE2, PGF2α, 12-HETE, and arachidonic acid. Among these eicosanoids, PGE2 predominated under baseline conditions and following PAF exposure. Using radio-immunoassay, PAF was also demonstrated to increase the production of 6-keto-PGF1α, TXB2, PGD2, 5-HETE, and 15-HETE, as well as PGE2, PGF2α, and 12-HETE. The PAF-induced eicosanoid augmentation was dose-dependent and occurred within 1 hour, with a prompt decline following termination of PAF exposure. This stimulating effect of PAF on eicosanoid release was blocked by two PAF receptor antagonists, Ro 19-3704 and WEB 2086, suggesting that PAF stimulates the production of COX and lipoxygenase pathway products from airway epithelial cells via PAF receptors (Wu et al. 1995). In macrophages isolated from BAL fluid in asthmatic patients, stimulation with PAF led to enhanced 5-LO activity and increased LTB4 release compared with alveolar macrophages isolated from normal controls (Shindo et al. 1998). The in vitro actions of PAF on eosinophils include chemotaxis and adhesion (Kimani et al. 1988; Kroegel et al. 1991). Eosinophil chemotaxis appears to be inhibited by the phosphoinositide-3 inhibitors wortmannin and LY294002. However, in vitro, two phosphoinositide 3-kinase inhibitors inhibit PAF-induced eosinophil chemotaxis and PAF-induced respiratory burst but not PAF-induced LTC4 secretion (Mishra et al. 2005). PAF also acts as an eosinophil priming agent (Zoratti et al. 1992). PAF-mediated priming of eosinophils is mediated via different signaling pathways, compared with IL-5-induced priming, because it is not blocked by tyrosine kinase inhibitors (Van der Bruggen et al. 1998). Exposure to PAF induces cytoplasmic alkalinization and granule acidification in human eosinophils (Bankers-Fulbright et al. 2004). It

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has been proposed that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins (Bankers-Fulbright et al. 2004). After allergen challenge in asthmatic patients, PAF induces greater activation of circulating eosinophils, suggestive of cross-talk between PAF and other priming factors such as IL-5 and GM-CSF (Evans et al. 1996). PAF also has a greater activating effect on neutrophils from asthmatic patients compared with those from normal control subjects (Shindo et al. 1997). In vivo, PAF elicits marked eosinophilic infiltration into lung tissue after both intravenous and aerosol administration to guinea pigs (Lellouch Tubiana et al. 1988) and rabbits (Coyle et al. 1990). PAF-induced eosinophilia in guinea pigs is associated with increased bronchial responsiveness to inhaled histamine, but not methacholine. In guinea pigs, PAF-induced bronchoconstriction is abolished by treatment with the timedependent, reversible COX-1 inhibitor, indomethacin, suggesting that in vivo airway eosinophils may reduce nonspecific bronchial responsiveness through production of inhibitory or bronchoprotective prostanoids (Ishiura et al. 2005). PAF and its metabolite lyso-PAF have been demonstrated in BAL fluid from stable atopic asthmatic subjects (Stenton et al. 1990a) and in plasma from asthmatic subjects during both early (Chan-Yeung et al. 1991) and late (Nakamura et al. 1987) responses to inhaled allergen. Increased levels of PAF have also been observed following allergen challenge in asthmatic patients. In asthmatic patients, inhalation of PAF is associated with an increase in respiratory system resistance, increased urinary LTE4, decreased PaO2, and a transient decrease in blood neutrophils (within 5 min) followed by a rebound neutrophilia (15 min later), but no increase in total antioxidant capacity and levels of lipid peroxides (Echazarreta et al. 2005). PAF also elicits bronchoconstriction and airway microvascular leakage, resulting in disturbances of pulmonary gas exchange similar to those observed during naturally occurring acute asthma, and has been reported to induce bronchial hyperresponsiveness in humans (Kaye & Smith 1990), although this has not been universally shown (Lai et al. 1990, Spencer et al., 1990). Increased levels of PAF have been measured in plasma from asthmatic patients during an acute attack (Kurosawa et al. 1994) and this is associated with diminished levels of PAF acetylhydrolase (Tsukioka et al. 1996). PAF receptor mRNA has also been detected in airway smooth muscle in human peripheral lung (Shirasaki et al. 1994a). Transgenic mice that either overexpress or underexpress the PAF receptor have proven useful in further elucidating the role this mediator may play in the pathogenesis of asthma. Transgenic mice that overexpress the PAF receptor (PAFR) gene exhibit bronchial hyperreactivity and this appears to be mediated in part by a muscarinic pathway. Nagase et al. (2002b) examined airway responsiveness to methacholine

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and serotonin (5-hydroxytryptamine) in PAFR transgenic mice. The PAFR transgenic mice exhibited hyperresponsiveness to methacholine and PAF, but no significant differences in serotonin responsiveness were observed between control and PAFR transgenic mice. The administration of atropine significantly blocked PAF-induced responses in PAFR transgenic mice, suggesting that the muscarinic pathway may have a key role in AHR associated with PAFR gene overexpression. In a murine asthma model, subsequent to ovalbumin sensitization, mice that did not express PAFR had no alteration in the total number of cells or in the proportions of eosinophils, lymphocytes, macrophages/monocytes, and neutrophils in BAL fluid compared with mice expressing PAFR (Ishii et al. 2004). PAFR-negative and PAFR-positive mice did not exhibit any histologic differences in the degree of bronchial inflammation. Following antigen challenge, PAFR-negative mice developed significantly increased airway responsiveness compared with their saline-treated controls. Furthermore, their responsiveness proved to be significantly lower than that of PAFR-positive mice sensitized with ovalbumin. Thus, PAFR is critical for the development of AHR following repeated aeroallergen challenge in sensitized mice, and this develops via PAFR-dependent and -independent pathways.

Cellular release Resident pulmonary inflammatory cells such as mast cells, (Triggiani et al. 1991), eosinophils (Cromwell et al. 1990), and alveolar macrophages (Bratton et al. 1994) are capable of PAF synthesis via specific agonist–receptor interactions. Many phagocytic stimuli, such as bacteria and parasites opsonized by IgG or complement, immune complexes, complement chemotactic factors C5a and C3a, and eosinophil chemotactic factor A, can stimulate inflammatory cells to initiate PAF synthesis. Macrophages activated by opsonized zymosan and by immune complex in hypersensitivity reactions are capable of PAF synthesis. PAF is synthesized by peripheral blood monocytes in response to 4β-phorbol-12-myristate13-acetate (PMA), opsonized zymosan, and the calcium ionophore A23187, an effect regulated by protein kinase C (Elstad et al. 1991). Both unopsonized zymosan and A23187 induce a dose- and time-dependent increase in PAF synthesis by eosinophils (Burke et al. 1990). Neutrophils stimulated with opsonized zymosan, A23187, and formyl-methionyl-leucylphenylalanine generate PAF (Sisson et al. 1987). Cytokines such as IL-1 and TNF stimulate macrophages, neutrophils, and endothelial cells to generate PAF (Camussi et al. 1987; Bussolino et al. 1988). Endothelial cells also produce PAF in response to specific stimuli such as vasopressin, angiotensin II, thrombin, bradykinin, and histamine (Camussi et al. 1983; Prescott et al. 1984; McIntyre et al. 1985). Human basophils have also been shown to produce PAF in vitro after costimulation with a combination of IL-3 and thapsigargin, although this effect is blocked by inhibitors of cytoslic PLA2, suggesting this enzyme’s involvement (Lie et al. 2003). Lymphocytes

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are unable to synthesize PAF, although natural killer cells may produce PAF in response to IgG binding to Fc receptors (Malavasi et al. 1986). Antigen or IgE-stimulated mast cells and basophils generate PAF (Schulman et al. 1983), supporting a role for PAF in the pathogenesis of acute anaphylaxis. Vadas et al. have investigated this and measured serum PAF levels and PAF acetylhydrolase activity in 41 patients with anaphylaxis and in 23 control patients. Serum PAF acetylhydrolase activity was also measured in patients with peanut allergy who had fatal anaphylaxis and compared to several control groups including patients with nonfatal anaphylaxis, children who died of non-anaphylactic causes and children with lifethreatening asthma. The severity of anaphylaxis directly correlated with serum PAF levels whilst serum PAF acetylhydrolase activity was inversely correlated with the severity of anaphylaxis. PAF acetylhydrolase activity was significantly lower in patients with fatal anaphylactic reactions to peanuts than in patients in any of the control groups. These findings suggest that failure of PAF acetylhydrolase to inactivate PAF may contribute to the severity of anaphylaxis (Vadas et al. 2007).

Catabolism The major enzyme responsible for the catabolism of PAF is PAF acetylhydrolase (PAF-AH), which is unusually a secretory Ca2+-independent PLA2. PAF-AH was initially described as being abundant in human plasma but by 1987 it had been shown to be associated with low-density lipoproteins (Stafforini et al. 1987) and to a lesser extent high-density lipoproteins. PAF-AH not only degrades PAF but also degrades the oxidation products of phosphatidylcholine, some of which express PAF-like activity (Blank et al. 1981; Heery et al. 1995). Oxidization of the ample phosphatidylcholine-containing arachidonate at the sn-2 position of glycerol (found in lowdensity lipoproteins) gives rise to numerous oxidized products with short chains at the sn-2 position of glycerol. Acting as an esterase, PAF-AH cleaves the acetyl group at the sn-2 position of oxidized 1-palmitoyl-2-arachidonyl phosphatidylcholine (oxPAPC) and PAF, producing lysophosphatidylcholine and lyso-PAF and short-chain fatty acids (Subramanian et al. 1999). Several inflammatory cell types, including mast cells (Nakajima et al. 1997), macrophages (Elstad et al. 1989), and platelets (Korth et al. 1993), synthesize and excrete active PAF-AH. Furthermore, recent studies have identified an acetylhydrolase in BAL fluid that is distinct from either plasma acetylhydrolase or erythrocyte-derived acetylhydrolase (Triggiani et al. 1997). This enzyme was present in smaller amounts in BAL fluid obtained from patients with mild asthma (Triggiani et al. 1997).

PAF receptor The PAFR was the first lipid mediator receptor to be cloned. A guinea-pig lung cDNA library was screened using a phage

clone shown to induce PAF-dependent responses in Xenopus laevis oocytes (Honda et al. 1991). Confirmation of the identity of the receptor was carried out based on its pharmacologic properties when expressed in COS-7 cells or oocytes and using known PAFR agonists and antagonists. A 3020nucleotide sequence was reported and the 342-amino-acid sequence was deduced from the longest open reading frame of the cloned cDNA. Hydropathy profile analysis revealed the existence of seven hydrophobic transmembrane segments characteristic of GPCRs, suggesting that like previously described receptors such as the α1- and α2-adrenergic and muscarinic acetylcholine receptors, the PAFR belongs to the G protein-linked receptor superfamily. Human PAFR cDNA was isolated from a human leukocyte cDNA library using a 0.8-kb fragment of the guinea-pig PAFR cDNA as a probe. Both the guinea-pig and human PAFR were shown to possess seven putative transmembrane domains, contain 342 amino acid residues, and demonstrate 83% homology in amino acid sequence (Nakamura et al. 1991). Activation of the receptor yielded IP3 in transfected oocytes and COS-7 cells, and guanosine 5′-O-(2-thio)biphosphate injection into these cells inhibited a PAF-induced Cl–? current, providing evidence that PAF stimulates phosphoinositide turnover via G proteins. When expressed in CHO cells the PAFR couples with various second messenger systems, leading to phospholipase C activation, inhibition of adenylate cyclase, and activation of the MAPK cascade and arachidonate release. A subsequent study identified the PAFR cytoplasmic tail as not being required for forward signal transduction. The several phosphorylation sites on the cytoplasmic tail are postulated to play a critical role in the rapid agonist-induced desensitization so characteristic of PAF activity (Takano et al. 1994). It is now known that substitution of the Cys90, Cys95, or Cys173 residues in the PAFR with alanine or serine yields mutant receptors that do not bind PAF and are not expressed on the surface of cells but are found intracellularly (Le Gouill et al. 1997). PAF appears to act in an autocrine manner and may modulate the expression of its own receptor at a transcriptional level in human monocytes (Shirasaki et al. 1994b). The cell signaling pathways initiated by PAF interaction with its receptor are well characterized and include increases in [Ca2+]i (Mazer et al. 1991), increases in IP3 and diacylglycerol levels, and induction of cell cycle-active genes such as fos, jun, and egr-1 (Mazer et al. 1991; Schulam et al. 1991). Through its action on PAFR, PAF also activates the transcription factor AP-1 in airway epithelial cells (Le Van et al. 1998). More recently, increasing evidence is emerging that intracellular binding sites for PAF contribute to its proinflammatory actions. It has also been suggested that PAF may act via more than one receptor. Evidence from both human and animal studies suggests that there may be heterogeneity of PAF receptors (Kroegel et al. 1989; Hwang et al. 1990). For example, PF10040 can antagonize PAF-induced edema formation

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(Rossi et al. 1992) and PAF-induced bronchial hyperresponsiveness (Herd et al. 1994) but has no effect on PAF-induced bronchoconstriction (Herd et al. 1994). Furthermore, it has been demonstrated that only a small part of the total amount of PAF generated by cells is actually released, with intracellular PAF having been proposed as a signaling molecule itself (Stewart & Harris 1991). This observation raised the possiblity of a distinct intracellular PAFR. Almost 10 years later, receptors for PAF have been shown to be localized at the cell nucleus of Chinese hamster ovarian cells, cerebral microvascular endothelial cells of newborn pigs (Marrache et al. 2002), and rat hepatocytes (Miguel et al. 2001). The nucleus has been suggested as a putative organelle that both generates PAF and expresses its receptor (Marrache et al. 2002). Marrache et al., stimulated isolated nuclei in porcine brain endothelial cells with methylcarbamate-PAF and found this evoked the expression of the genes for proinflammatory iNOS and COX-2. Gene expression was associated with augmented ERK-1/2 phosphorylation and NF-κB binding to the DNA consensus sequence. COX-2 expression was inhibited by mitogen-activated protein kinase kinase/extracellular signalregulated kinase 1/2 and NF-kappaB inhibitors. These above observations have led to the suggestion that the immediate effects of PAF are mediated via cell-surface receptors whereas long-term responses are dependent on intracellular receptor effects (Zhu et al. 2006). It is now recognized that subsequent to PAF stimulation, cells become rapidly desensitized and this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization, and downregulation. PAFR degradation can occur via both the proteasome and lysosomal pathways and ligand-stimulated degradation is ubiquitin-dependent (Dupre et al. 2003).

PAFR antagonists Due to the association of PAF with diverse physiologic and pathologic processes, considerable efforts have been invested in the development of antagonists to PAFR. These have been used to characterize PAFRs on a wide variety of inflammatory cells and also to elucidate the part PAF may play in the pathogenesis of asthma. Despite considerable in vitro and in vivo data in animal and human studies suggesting that PAF is an important mediator of asthma, clinical studies with PAFR antagonists have been disappointing. • WEB 2086 is a hetrazepine compound that inhibits the allergen-induced late response in allergic sheep (Abraham et al. 1989). In human subjects, WEB 2086 prevents PAFinduced bronchoconstriction (Adamus et al. 1990) and prevents the histamine hyperresponsiveness induced by PAF in vitro (Johnson et al. 1990). In a study of eight mild atopic asthmatic subjects, 1 week’s treatment with WEB 2086 (300 mg daily) did not attenuate the allergen-induced early or late responses or the subsequent histamine bronchial hyperre-

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sponsiveness (Freitag et al. 1993). In a subsequently reported study of 65 asthmatic subjects, 6 weeks’ treatment with WEB 2086 (120 mg daily) did not significantly reduce the requirement for inhaled corticosteroids compared with placebo (Spence et al. 1994). • The dihydropyridine compound UK74,505 is a specific and potent PAFR antagonist that shows 10-fold greater inhibition of PAF-induced aggregation of washed rabbit platelets than WEB 2086 (Alabaster et al. 1991). In a study of 12 normal subjects, inhalation of PAF, induced broncoconstriction for 60 min, neutropenia at 5 min, rebound neutrophilia at 2 hours, and stimulated the production of urinary eicosanoids (O’Connor et al. 1994). Premedication with UK74505 completely abolished the bronchoconstrictor response, neutropenia, and rebound neutrophilia, and significantly reduced urinary levels. In asthmatic patients who received a 4 week course of modipafant (the (+)-enantiomer of UK-74505) for 4 weeks, no clinical benefit was observed (Kuitert et al. 1995). • A 1-week course of the potent, long-acting PAFR antagonist foropafant (SR27417A) produced a modest reduction in the magnitude of the allergen-induced late response, although there was no effect on the early response, allergen-induced airway responsiveness, or baseline lung function (Evans et al. 1997). • Another PAF antagonist, Y24180, has also been shown to reduce airway responsiveness to inhaled methacholine in asthmatics (Hozawa et al. 1995), although these data are at variance with findings from other studies (Evans et al. 1997). • In patients with acute asthma, where endogenous release of PAF may be enhanced, the response to a potent selective PAFR antagonist (SR27471A) on pulmonary gas exchange was studied with ventilation–perfusion (VA/Q) scans (Gomez et al. 1999). SR27471A inhibited platelet agregation tests, suggesting good bioactivity in vivo. However, no differences were observed between placebo and active groups with regard to baseline FEV1, respiratory system resistance, alveolar–arterial pressure difference for oxygen (5.2 ± 0.4 kPa), PaO2, or VA/Q distributions, as expressed by the dispersion of pulmonary blood flow. This led to the conclusion that SR27417A has limited value when added to the conventional treatment of acute asthma (Gomez et al. 1999). This disappointing clinical response may reflect the unimportance of PAF in acute or chronic asthma, or reflect an impaired ability of the antagonists used to block endogenously produced PAF, which acts locally in the airways with “paracrine” effects (Barnes et al. 1988). Alternatively, as a number of assumed PAFR antagonists (WEB 2086, SM 10661, alprazolam) have now been shown to be in fact acting as inverse agonists on the human PAFR and inducing effector cascades of LTE4 and 2,3-dinor-TXB2 (Dupre et al. 2001), this may provide another explanation for the poor clinical performance of these drugs.

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Theophylline and Isoenzyme-selective Phosphodiesterase Inhibitors Mark A. Giembycz

Summary

Table 27.1 Allergic diseases where IgE-driven mechanisms are implicated.

Cyclic nucleotide phosphodiesterases (PDEs) currently account for at least 50 distinct proteins that are all able to inactivate the ubiquitous second messengers cAMP and/or cGMP. These enzymes have proven to be very “drugable” targets and compounds are on the market or in late stage clinical development for a variety of dissimilar diseases. This chapter describes the multiplicity of mammalian PDE isoenzymes, the tissue distribution of PDEs expressed in cells which participate in allergic inflammatory reactions, and the potential sites where PDE inhibitors could act to alleviate the acute and chronic manifestations of allergic disease including clinical trial data.

Asthma Atopic and contact dermatitis Rhinitis Eczema Sinusitis Hypersensitivity pneumonitis Extrinsic alveolitis Angioedema and anaphylaxis Certain forms of migrane and gastrointestinal disorders Urticaria

Introduction The term “allergy” is often used to describe all aspects of immunology. However, this definition of allergy has been refined to refer specifically to the tissue-damaging or irritant effects to the host of immunologic reactions. Allergic diseases, including all of those listed in Table 27.1, affect 20% of the population and represent a highly significant cause of morbidity and mortality. Taking allergic asthma as a specific example, a recent assessment indicates that the prevalence and severity of this disease is increasing (Braman 2006), although the number of reported cases of fatal asthma has declined from the peak reached in the mid 1980s (Sidebotham & Roche 2003). Although significant advances in our understanding of the pathogenesis of many allergic disorders have been made, the etiology of allergy is still incompletely understood. However, as described in Chapters 2, 5, 6, 7 and 8 the likely participation of IgE-driven mechanisms in many allergic diseases, including all of those cited in Table 27.1, has been identified and


634

recognized by both the World Allergy and World Health Organizations (Asher et al. 2004). Since the incidence of allergy has reached epidemic proportions, it is only too clear that drugs which can prevent the overt and covert manifestations of allergic reactions and, ideally, suppress or even prevent the process of host sensitization could have a profound clinical and economic impact in the control of these diseases. While glucocorticosteroids are currently considered the most effective antiallergic/antiinflammatory drugs currently available, they are nonselective in action and not without adverse effects. New drugs with enhanced selectivity and improved side-effect profiles are clearly required. One group of drugs which, from a theoretical perspective, may exhibit powerful antiinflammatory and immunomodulatory activity are inhibitors of certain of the cyclic nucleotide phosphodiesterase (PDE) isoenzymes that selectively degrade cyclic adenosine3′,5′-monophosphate (cAMP) and/or cyclic guanosine-3′,5′monophosphate (cGMP) (Torphy 1998; Giembycz 2005a,b; Houslay 2005; Houslay et al. 2005; Bender & Beavo 2006a; Lugnier 2006). The prototype PDE inhibitors that have been used in the treatment of asthma for many years are the alkylxanthines of which theophylline is the most widely prescribed. The main beneficial activity of theophylline was originally attributed to its weak bronchodilator action. However, evidence accumulated in the 1990s suggested that this molecule may have antiinflammatory activity at

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Theophylline and Isoenzyme-selective Phosphodiesterase Inhibitors

Table 27.2 Representative PDE4 inhibitors for inflammatory disorders.

Company

Drug

Isoenzyme selectivity

Indication

Development stage

Almirall Glenmark Pfizer ONO Otsuka Nycomed GlaxoSmithKline GlaxoSmithKline Celgene ICOS MediciNova

Arofylline Oglemilast (GRC 3886) Tofimilast (CP-325366) ONO 6126 Tetomilast (OPC 6535) Roflumilast Cilomilast (SB 207499) AWD-12-281 (GSK 842470) CC-10004 IC-485 Ibudilast

4 4 4 4 4 4 4 4 4 4 4

Bronchitis Asthma, COPD Asthma Asthma, COPD COPD Asthma, COPD COPD Asthma Asthma, psoriasis COPD Multiple sclerosis

Phase II/III Phase III Phase II Phase II Phase III Phase III Phase III Phase II Phase II Discontinued Phase II

COPD, chronic obstructive pulmonary disease. Data collated from Mealey & Bayes (2005).

sub-bronchodilator doses (Ward et al. 1993; Sullivan et al. 1994; Djukanovic et al. 1995) which fuelled the idea that theophylline and so-called “second generation” PDE inhibitors could act as bronchodilators and potential antiallergic and/or antiinflammatory agents (Banner & Page 1995; Dent & Giembycz 1996; Palfreyman & Souness 1996; Torphy 1998; Doherty 1999; Essayan 1999; Giembycz 2000). The rationale for developing new PDE inhibitors has stemmed primarily from the realization that PDEs represent a highly heterogeneous group of enzymes (11 families have thus far been identified) that are differentially expressed between different cell types and almost certainly regulate specific functional responses. Accordingly, it was rapidly appreciated that drugs which selectively suppress the activity of a particular PDE isoenzyme may result in a discrete functional alteration of cells which express that PDE variant and, theoretically, specific functional responses within the same cell. Given the prevalence of allergic diseases as a whole, it is only too apparent that the potential clinical reward of developing a class of steroid-sparing drugs of general utility in a variety of allergic disorders is enormous and explains why many of the world’s major pharmaceutical companies have an active PDE research program and have developed highly selective PDE inhibitors, many of which are currently undergoing clinical trials for asthma and other allergic and nonallergic inflammatory disorders (Table 27.2). It is the purpose of this chapter to describe the multiplicity of mammalian PDE isoenzymes, the tissue distribution of PDEs expressed in cells which participate in inflammatory reactions and the potential sites where selective PDE inhibitors could act to alleviate the acute and chronic manifestations of allergic disease including clinical trials data. For conciseness, only those PDEs that, for established or theoretical reasons, may be exploited to therapeutic advantage in inflammatory diseases are discussed in detail. Readers interested in the

more general aspects of PDE biology are directed to recent reviews on this subject (Bender & Beavo 2006a; Lugnier 2006).

Generic properties and characteristics of cyclic nucleotide phosphodiesterases Cyclic nucleotide PDEs (EC 3.1.4.17) comprise a large group of enzymes whose sole function is to hydrolyze and thereby inactivate the biologically active cyclic purines cAMP and cGMP (Fig. 27.1). PDEs which act on cyclic pyrimidine monophosphates have also been described (see Newton & Salih 1986), although investigators have tended to focus, almost exclusively, on the PDEs which hydrolyze cyclic purine nucleotides for which functionally important second messenger roles have unequivocally been established. A cyclic nucleotide PDE that hydrolyzed the 3′-ribose phosphate bond of cAMP to the catalytically inactive 5′-AMP was first identified in 1962 (Butcher & Sutherland 1962). Since then multiple families and, indeed, subfamilies of PDEs which selectively act on cAMP and/or cGMP have been identified (see Bender & Beavo 2006a; Lugnier 2006 for reviews). The PDEs that metabolize cyclic purine nucleotides comprise at least 11 distinct families and can be distinguished by a number of criteria including substrate specificity, kinetic properties, responsiveness to endogenous allosteric regulators, susceptibility to inhibition by various compounds and in primary amino acid sequence (Table 27.3). Molecular biological studies have discovered that many PDEs are separate gene products and express multivariant regulatory domains linked to highly conserved homologous catalytic sequences located near the C-terminus of the protein. Members of one family share 20–25% sequence homology with members of another family. Furthermore, at least 6 of the 11 gene families can be divided into subfamilies that are 70–90%

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Pharmacology NH2

NH2 N

N

N

N N

N

PDE

N

N

H2O; Mg2+

cAMP

O

O

OH

O O

O P

O

HO

HO

O O

O N

N H2N

H2N

N N H

cGMP

O

N

PDE O

H2O; Mg2+

OH

O

OH

5′-GMP

OH

HO

P HO

N N H

N

O

O Fig. 27.1 cAMP and cGMP hydrolysis by cyclic nucleotide phosphodiesterases.

P

O

HO

homologous and which are derived from similar but distinct genes or, in some cases, from the same gene through alternate mRNA splicing or from differences in the initiation start sites for the translation of the protein. At the end of 2006 in excess of 22 PDE genes were unequivocally identified which, in mammals, probably encode for more than 50 distinct PDE enzymes. For further information on the molecular diversity of PDE isoenzymes, interested readers are directed toward a number of comprehensive reviews (Conti & Jin 1999; Conti 2000; Soderling & Beavo 2000; Houslay 2001; Houslay & Adams 2003; Bender & Beavo 2006a; Lugnier 2006). A diagrammatic representation of the primary structure of cyclic purine PDEs is shown in Fig. 27.2. All mammalian PDEs share essentially the same structure: there is a central cAMP cGMP

5′′-AMP 5′-GMP

Allosteric/small molecule Binding site (e.g. cGMP, Ca2+/calmodulin) Phosphorylation sites (e.g. PKA at S54 in PDE4D3)

O

core located close the C-terminus of the protein that shares 25–35% homology between isoenzyme families and which features a highly conserved domain of some 270 amino acids. Studies performed in a number of laboratories indicate that this conserved region within this central core of all PDEs represents the catalytic site (Charbonneau et al. 1986). The central core of mammalian PDEs is linked via so-called hinge regions to carboxy- and amino-terminal extensions (Fig. 27.2) which show relatively little sequence homology between PDE families (Conti & Jin 1999). This finding has led to the view that the nonconserved domains subserve regulatory functions of the protein (Conti & Jin 1999). Indeed, it is now appreciated that the N-termini of PDEs can feature binding sites for calmodulin (PDE1) and cGMP (PDEs 2, 5, 6,

Phosphorylation sites (e.g. ERK-2 at S579 in PDE4D3 and S651 in PDE4D5)

Catalytic domain Dimerization domains

Targeting domains (e.g. TAPAS-1 in PDE4A1) NH 2

636

5′-AMP

OH

HO

P

OH

CO

OH

Fig. 27.2 Generic structure of mammalian cyclic purine PDEs. Limited proteolysis and protein sequencing suggest that all mammalian PDEs so far examined contain a conserved central core that features the catalytic site. This region of PDE proteins is flanked, through putative hinge regions, by highly heterologous C- and N-termini which are believed to express regulatory domains, dimerization motifs, phosphorylation sites and sequence information which determine the subcellular localization of PDEs. See text for further details. (See CD-ROM for color version.)

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Table 27.3 Properties and selective inhibitors of cyclic nucleotide phosphodiesterases. Km (mmol/L) PDE family

cAMP

cGMP

Selective inhibitors

References

1

Ca2+/calmodulin-stimulated

0.3–124

0.6–6

SCH 57801 KS-505a IC-224

Ahn et al. (1997) Ichimura et al. (1996) Snyder et al. (2005)

2

cGMP-stimulated

28–100

10–28

ENHA Oxindole BAY 60-7550 IC933 PDP

Podzuweit et al. (1995) Chambers et al. (2006) Boess et al. (2004) Snyder et al. (2005) Seybold et al. (2005)

3

cGMP-inhibited

0.02–0.3

0.1–0.5

Siguazodan SK&F 95654 Cilostamide Cilostazol

Murray et al. (1990) Murray et al. (1992) Hidaka et al. (1979) Tanaka et al. (1988)

4

cAMP-specific

1–10

> 50

Rolipram Ro 20-1724 Cilomilast Roflumilast Piclamilast

Reeves et al. (1987); Schwabe et al. (1976) Sheppard & Tsien (1975) Barnette et al. (1998) Hatzelmann & Schudt (2001) Souness et al. (1995)

5

cGMP-binding

280

3–7

Zaprinast Sildenafil Vardenafil Tadalafil

Lugnier et al. (1986) Boolell et al. (1996) Saenz et al. (2001) Padma-Nathan et al. (2001)

6

Photoreceptor

600–700

15–17

None reported

7

Rolipram-insensitive, cAMP-specific

0.01–0.5

NA

BRL 50481 PF 332040 IC242

8

Rolipram-insensitive, cAMP-specific

0.06

NA

None reported

9

Zaprinast-insensitive, cGMP-specific

228

0.7–0.17

BAY 73-6691

10

AMP-inhibited (?)

0.22–1.1

13–14

None reported

11

Dual-selective

1–2

2–3

None reported

Smith et al. (2004) Giembycz & Smith (2006a); Jones et al. (2007) Lee et al. (2002)

Wunder et al. (2005)

NA, reliable data not available.

10 and 11), so-called GAF domains (Martinez et al. 2002a); membrane-association domains (PDE3); subcellular targeting sequences (PDE4, PDE7); phosphorylation sites (most PDEs) that control catalytic activity; and PAS domains (PDE8), involved in ligand recognition and protein–protein interactions. Similarly, sequences at the C-terminus express dimerization motifs (most PDEs exist as dimers (Charbonneau et al. 1991; Kovala et al. 1997; Martinez et al. 2002b; Richter & Conti 2002; Zoraghi et al. 2005) and/or are substrates for phosphorylation (Hoffmann et al. 1998b; MacKenzie et al. 2000). Readers should note that GAF and PAS (domains) are acronyms of the names of the first three groups of proteins identified to contain them, i.e., cGMP-regulated cyclic nucleotide phosphodiesterase, cyanobacterial Adenylyl cyclase and E. coli transcription factor, Fh1A (formate-hydrogen lyase),

and Period circadian protein, Ah receptor nuclear translocator protein and Single minded protein, respectively.

Standardized nomenclature A standardized GenBank nomenclature for existing and newly discovered PDEs was introduced in 1994 (Beavo et al. 1994). This taxonomy is based on traditional biochemical criteria and on protein sequencing and analyses of partial and full length cDNA clones. Consider two hypothetical PDEs, CPPDE4D4B and HSPDE11A1A (Fig. 27.3). The first two letters refer to the species (CP for Cavia porcellus, HS for Homo sapiens). The next three letters plus an Arabic numeral correspond to the PDE gene family (i.e., PDE, PDE11) while the

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Pharmacology

PDE family

Gene

Splice variant

Report

HSPDE4D4B CPPDE11A1A Green Purple Red Orange Yellow

Species (first 2 letters) PDE gene family (3 capital letters + 1 or 2 Arabic numerals) PDE gene within family (capital letter) Splice variant (Arabic numeral) Report (capital letter)

Fig. 27.3 GenBank nomenclature for the classification of cyclic purine PDEs. The first two letters (green) represent the species, the next three letters plus an arabic numeral denote the PDE family (purple), the next letter and numeral refer to the gene product within the PDE family (red) and the spliced variant (if appropriate; orange) respectively and the last letter indicates the report (yellow). See text for further details. (See CD-ROM for color version.)

next letter and Arabic numeral (i.e., D4, A1) denote the gene product and spliced variant (if appropriate). This nomenclature is used throughout this chapter.

Phosphodiesterase 1 PDE1 is a generic term that describes a family of hydrolytic enzymes that are active only in the presence of calmodulin and micromolar concentrations of cytosolic free Ca2+ (Conti & Jin 1999; Goraya & Cooper 2005). This Ca2+/calmodulin dependency, originally documented in the early 1970s (Cheung 1970, 1971; Kakiuchi & Yamazaki 1970), is a characteristic of all members of this enzyme family and results in a 5–20-fold increased in catalytic activity depending on the particular isoform studied (Sharma & Wang 1986a,b). Detailed kinetic analyses indicate that calmodulin modulates PDE1 activity allosterically by substantially increasing the Vmax of the reaction while reducing, relatively modestly, the affinity (Km) of the substrate for the enzyme (Sharma & Wang 1986a,b). Evidence for PDE1 multiplicity was originally provided by immunologic studies (Hansen and Beavo 1982), which effectively discriminated two bovine isoforms: a 61-kDa enzyme in brain and a 59-kDa variant in heart. Sequence analysis has revealed that the 59-kDa and 61-kDa isoenzymes (BTPDE1A1 and BTPDE1A2 respectively) are alternatively spliced variants of the same gene (Charbonneau et al. 1991; Novack et al. 1991). Similar studies, subsequently reported by Sharma et al. (1984) identified a 63-kDa isoenzyme in bovine brain that is immunologically distinct from BTPDE1A2 and represents a different gene product (BTPDE1B1). This enzyme was cloned in 1992 from bovine and rat brain cDNA libraries (Repaske et al. 1992). It is now known that PDE1 comprises a large family of closely related proteins that are encoded by at least three genes (PDE1A, PDE1B, PDE1C) with further diversity arising from mRNA splicing due to differential exon usage (Snyder et al. 1999; Michibata et al. 2001; Fidock et al. 2002; Goraya

638

& Cooper 2005). In humans 13 PDE1A cDNAs have been described (Loughney et al. 1996; Snyder et al. 1999; Michibata et al. 2001) together with two splice variants for both PDE1B (PDE1B1, PDE1B2) and PDE1C (PDE1C1, PDE1C3) (Loughney et al. 1996; Fidock et al. 2002). All PDE1A and PDE1B isoforms as well as PDE1C1 preferentially hydrolyze cGMP, whereas PDE1C3 degrades cAMP and cGMP with equal efficiency (Loughney et al. 1996; Snyder et al. 1999).

Tissue distribution and selective inhibitors Of those proinflammatory and immunocompetent cells implicated in human inflammatory and allergic diseases, only alveolar macrophages (Tenor et al. 1995a), epithelial cells (Fuhrmann et al. 1999; Rousseau et al. 1994; Wright et al. 1998) and airway smooth muscle (Torphy et al. 1993a; Souness & Giembycz 1994) express appreciable PDE1 constitutively. Indeed, depending on species, PDE1 can account in airways smooth muscle for ~90% of the cyclic nucleotide hydrolysing activity (Souness & Giembycz 1994). In addition, trace amounts of this isoenzyme are present in other cells including CD4+ and CD8+ T lymphocytes (Tenor et al. 1995b) where they may have a role in regulating mitogenesis (see below). Although PDE1 is ubiquitously expressed, establishing functional roles for these isoenzymes has been greatly hindered by a lack of selective pharmacologic tools. Vinpocetine and 8-methoxymethyl-3-isobutyl-1-methylxanthine are often used for this purpose but, in fact, these compounds are nonselective and, minimally, will inhibit PDE5 (Ahn et al. 1989) and block certain Na+ and K+ channels (Bonoczk et al. 2000) at concentrations similar to those required for PDE inhibition (Bukanova & Solntseva 1998). However, in 1997 chemists from Schering-Plough described a number of selective and potent PDE1 inhibitors based on the tetracyclic guanine template (Ahn et al. 1997). Two of these compounds (1 and 2 in Fig. 27.4) are at least 280 times more selective for PDE1 over the other major PDEs and are suitable for in vitro pharmacologic testing. In addition, it was reported in the early 1990s that a heterocyclic molecule isolated from the bacterium Streptomyces argenteolus is a potent and highly selective inhibitor of PDE1 (Nakanishi et al. 1992; Kase et al. 1993; Nagashima et al. 1993; Ichimura et al. 1996). This compound, designated KS505a, is somewhat unique as it selectively inhibits PDE1A2. Indeed, KS505a is approximately 80-fold more potent against PDE1A2 from bovine brain (IC50 ∼ 170 nmol/L) than of PDE1A1 from bovine heart (Nakanishi et al. 1992; Kase et al. 1993; Ichimura et al. 1996). These are fascinating findings. Not only do they identify KS505a as a selective inhibitor of PDE1, but demonstrate that compounds can discriminate between two highly homologous proteins that are derived from the same gene by alternate splicing. Perhaps the most exciting prospect that arises from these data is the likelihood that third generation PDE inhibitors can be synthesized which would allow the selective targeting of specific enzymes encoded by the same gene.

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O

O H 3C

N

Fig. 27.4 Chemical structures of two novel and selective inhibitors of PDE1. See text and Ahn et al. (1997) for further details.

H3C

N

N N

N H

N

N N

1

Therapeutic indications Mitogenesis The functional roles in mammals of PDE1 are largely unknown. However, evidence has emerged since the early 1990s that certain PDE1 isoforms may regulate cell division in both lymphocytes and smooth muscle. The human lymphoblastoid RPMI-8392 B-cell line, which was established from a patient with acute lymphocytic leukemia, expresses two major PDEs: PDE1 (probably PDE1B) and PDE4 (Epstein et al. 1987; Jiang et al. 1996). In contrast, normal quiescent human B lymphocytes lack PDE1 (Epstein et al. 1987; Gantner et al. 1998). However, following exposure of normal B lymphocytes to the mitogen phytohemagglutinin (PHA), significant PDE1 activity was detected together with mRNA transcripts for PDE1B (Hurwitz et al. 1990; Jiang et al. 1996). The induced enzyme eluted from anion-exchange columns as a 63-kDa protein that was indistinguishable from PDE1B purified from bovine brain (Hurwitz et al. 1990; Bentley et al. 1992). It is known that mitogens effect transcription of PDE1B (Spence et al. 1995, 1997) and it is possible that induction of this gene occurs when lymphocytes switch from a quiescent to a proliferative phenotype. As cGMP suppresses lymphocyte proliferation (Fischer et al. 2001), the induction of PDE1B by PHA could, by lowering the basal cGMP content, represent a physiologic response to permit B-cell mitogenesis. It is clear from these data that inhibition of PDE1 could prevent B-cell expansion and so compromise their ability to synthesize allergen-specific IgE. To date, the possibility that PDE1 may also regulate T-cell proliferation in an analogous manner to B lymphocytes has not been examined. Nevertheless, PDE1B1 mRNA levels are elevated in human T lymphocytes exposed to PHA and antiCD3/antiCD28 (Kanda & Watanabe 2001), which has been linked to the expression of interleukin (IL)-13, and so this possibility seems likely. Thus, PDE1 inhibition could have a profound effect on aspects of T-cell function upon which the induction and perpetuation of allergic reactions depend. Evidence has been published that the proliferation of human vascular smooth muscle cells is also controlled by PDE1 (Rybalkin et al. 1997, 2002). A discussion of these data

N H

2 SCH 57801

here is merited as one or more PDE1 isoenzymes could serve the same function in airway myocytes and so impact on the remodeling of the airways that is a characteristic of subjects with asthma (Huber & Koessler 1922; Dunnill et al. 1969; Heard & Hossain 1973; Ebina et al. 1993; Jeffery 2001; Benayoun et al. 2003; Hirst et al. 2004; Woodruff et al. 2004). The salient observation made by Rybalkin and colleagues was that PDE1C was markedly induced in proliferating but not quiescent smooth muscle cells derived from human aorta (Rybalkin et al. 1997). Subsequently, it was established that induction of PDE1C correlated with cell cycle progression and that inhibition of this enzyme with either 8-methoxymethyl3-isobutyl-1-methylxanthine (a modestly-selective PDE1 inhibitor) or antisense oligonucleotides directed against PDE1C, significantly reduced mitogenesis (Rybalkin et al. 2002). As inhibitors of PDE5 (which selectively hydrolyze cGMP) were not antimitogenic in the same system, it was concluded that proliferation was due to the induction of PDE1C and involved degradation of cAMP rather than cGMP (Rybalkin et al. 2002). Collectively, these data, obtained in B cells and vascular myocytes, provide compelling support for the hypothesis that the appearance of a PDE1 isoenzyme in dividing cells, that is normally absent or expressed at a relatively low level in the quiescent phenotype, represents a normal physiologic, mitogen-induced response that would otherwise be suppressed by the relatively high resting cyclic nucleotide levels.

Monocyte differentiation Human blood monocytes have the ability to differentiate into a variety of cell types that is governed by cytokines and other stimuli. With respect to the immune system, monocytes may differentiate into dendritic cells, which are professional antigen-presenting cells, or various types of tissue macrophages that have distinct characteristics. In all cases, the phenotypical changes are accompanied by a marked remodelling of the cell’s PDE profile. Thus, exposure of human peripheral blood monocytes (CD14+/CD1a−) to GMCSF and IL-4 (in serum) results, over a period of several days, in the formation of dendritic cells (CD14−/CD1a+) and this

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process is associated with a marked upregulation of PDEs 1 and 3 and a decline in PDE4 (Gantner et al. 1999). A similar pattern of PDE remodeling is seen when monocytes are aged for several days in serum alone, or granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence of IL-10, which results in the generation of cells with macrophagelike phenotypes (Gantner et al. 1997; Bender et al. 2004). Molecular studies have found that human blood monocytes express predominantly PDE1B2 mRNA and protein and that the abundance of this transcript and enzyme is markedly higher in GM-CSF-induced macrophage-like cells (Bender et al. 2004, 2005). PDE1B is also induced in monocytes treated with M-CSF, which again results in the formation of cells with a macrophage-like phenotype (Bender et al. 2004). The functional significance of PDE1B2 upregulation has been studied using gene silencing technologies using HL-60 cells differentiated into macrophage-like cells by phorbol esters (Bender & Beavo 2006b). The results from those studies suggest that PDE1B2 preferentially regulates the cGMP content in these cells but this does not affect many of the markers that are typical of differentiation such as adherence and the expression of CD71 and CD87. However, selective knockout of PDE1B2 mRNA alters certain characteristics of the final macrophage phenotype. In particular, CD11b expression, cell spreading and phagocytosis are all augmented (Bender & Beavo 2006b). The clinical impact of inhibiting PDE1 in cells of the monocyte/macrophage/dendritic cell lineage is unclear but enhanced phagocytic capacity could be of benefit.

Phosphodiesterase 2 The first description of, what is now termed, a PDE2 isoenzyme was reported in 1971 following the observation that cAMP hydrolysis in rat liver supernatant was stimulated by micromolar concentrations of cGMP (Beavo et al. 1971). Since then, many reports have documented the presence of cGMP-stimulated PDEs in a number of cells and tissues including those relevant to the pathogenesis of allergic reactions (Table 27.3). PDE2 isoenzymes do not discriminate between cAMP (Km 28–100 μmol/L) and cGMP (Km 10– 28 μmol/L) to any great extent, exhibit positive homotropic cooperative behavior with respect to both substrates and feature two high-affinity (Kd ∼ 0.1 μmol/L), noncatalytic dimerization/allosteric binding sites for cGMP (Moss et al. 1977; Martins et al. 1982; Yamamoto et al. 1983; Stroop & Beavo 1992; Martinez et al. 2002b). An interesting property of these enzymes is that low concentrations (1– 5 μmol/L) of cGMP enhance the degradation of cAMP approximately sixfold by a mechanism that requires occupancy of the noncatalytic cGMP-binding domain (Yamamoto et al. 1983; Stroop & Beavo 1992). In contrast, cAMP does not detectably stimulate PDE2-catalyzed cGMP hydrolysis (Stroop & Beavo 1992). A single gene has thus far been identified which encodes PDE2. However, transcription of this gene can produce at

640

least three spliced variants (PDE2A1, PDE2A2 and PDE2A3) that occurs at the 5′-end of the sequence (Trong et al. 1990; Sonnenburg et al. 1991; Yang et al. 1994; Rosman et al. 1997). It is noteworthy that only a single human PDE2 splice variant has so far been identified, which is highly homologous to bovine PDE2A3. Hence it has been named HSPDE2A3 (Rosman et al. 1997). These proteins share identical C-termini but differ in their N-termini, which dictate whether these enzymes are soluble or membrane-associated. PDE2s can be cytosolic or located to specific membrane structures including the Golgi apparatus, plasma membrane, endoplasmic reticulum and nuclear envelop where they presumably subserve distinct functions (Lugnier et al. 1999a,b; Geoffroy et al. 1999). Like many cyclic nucleotide PDEs, native PDE2s are homodimeric with a molecular mass of about 210 kDa (Stroop & Beavo 1992).

Tissue distribution and selective inhibitors Northern blot analysis has identified PDE2A mRNA in trachea and lung (Sonnenburg et al. 1991) and abundant PDE2-like enzyme activity has also been reported in these tissues from several species including human (Torphy et al. 1993a; Souness & Giembycz 1994). PDE2 is found in platelets (Simpson et al. 1988) and epithelial cells (Rousseau et al. 1994), although expression may be species dependent as it is seemingly absent from human airway epithelia (Wright et al. 1998; Fuhrmann et al. 1999). Trace amounts of PDE2 have been identified in T lymphocytes and alveolar macrophages (Tenor et al. 1995a,b; Bender et al. 2004) (Table 27.4). A study published in 1999 reported the expression profile of mRNA and protein for PDE2A3 in endothelial cells lining a variety of human vessels (Sadhu et al. 1999). Interestingly, Western blot analysis identified PDE2A3 only in certain populations of vascular endothelial cells, in particular the vasa

Table 27.4 Established PDE isoenzyme profiles in human proinflammatory and immune cells implicated in allergic disorders. Immune/proinflammatory cell

PDE profile

Mast cell Alveolar macrophage T lymphocyte B lymphocyte Eosinophil Neutrophil Basophil Monocyte Platelet Epithelial cell Endothelial cell Dendritic cell Airway myocyte Vascular myocyte

3, 4, 5, 7 1, 3, 4, 5, 7 1, 2, 3, 4, 5, 7, 8(?) 3, 4, 7 4 4, 7 4, 7 1, 3, 4, 7 1, 2, 3, 5 1, 2, 3, 4, 5, 7, 8 2, 3, 4, 5, 7 1, 3, 4, 7 1, 2, 3, 4, 5, 7, 8 1, 2, 3, 4, 5, 7

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O N N

NH2

N N

N

3 EHNA

N

S

CH3

H 3C

O O

N

N

NH

OH

H3C

HN

O

N

N

CH3

H3C

OH O

N H3C

CH3 4 Oxindole

O 5 BAY 60-7550

Fig. 27.5 Chemical structure of three selective inhibitors of PDE2. See text for further details.

vasorum and those of microvessels, whereas it was absent or very lowly expressed in large vessels such as the pulmonary artery, aorta and renal arteries (Sadhu et al. 1999). The subcellular localization of PDE2 in human endothelial cells requires clarification although in most cells it appears, by immunocytochemistry, to be cytosolic or perinuclear (Sadhu et al. 1999). Erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) was the first small molecule to be described with selective PDE2 inhibitory activity (Podzuweit et al. 1993, 1995) (see 3 in Fig. 27.5). The compound has an IC50 value of 0.5–1 μmol/L against PDE2 and is approximately 100-fold less potent against the other PDEs (Podzuweit et al. 1995). However, one drawback of ENHA is its ability to potently inhibit adenosine deaminase, which can compromise its use in certain tissues (Schaeffer & Schwender 1974). Two new PDE2 inhibitors were described in 2004, an oxindole (4), reported by Pfizer (Chambers et al. 2006), and the Bayer compound BAY 607550 (5) (Boess et al. 2004) (Fig. 27.5). The oxindole has an IC50 against human recombinant PDE2 of 40 nmol/L, over an order of magnitude more potent than EHNA. Moreover, the compound is > 290-fold selective over representatives from all other enzyme families and, in the rat, has favorable ADME, physicochemical and pharmacokinetic properties (Chambers et al. 2006). Several comprehensive screens have found that the oxindole has negligible off-target activity being devoid of significant interaction with a large panel of receptors, ion channels and enzymes. The most potent PDE2 inhibitor reported to date is BAY 60-7550 (IC50 4.7 nmol/L against human PDE2A) (Boess et al. 2004). Its selectivity relative to other PDEs ranges from 50-fold (PDE1) to > 800-fold (PDEs 3B, 7B, 8A, 9A, 11A) (Boess et al. 2004). BAY 60-7550 is bioavailable in rats and mice (Boess et al. 2004).

Therapeutic indications Despite being PDE2 are still relatively rich and/or cGMP

discovered in 1971, the functional roles of largely undetermined. In endothelial cells, a source of this enzyme, an elevation of cAMP can improve barrier function (Suttorp et al.

1993, 1996a,b; Westendorp et al. 1994; Seeger et al. 1995; Lum et al. 1999), inhibit mitogenesis (D’Angelo et al. 1997) and modulate (usual reduce) the elicited expression of certain adhesion molecules (Pober et al. 1993; Ghersa et al. 1994; Morandini et al. 1996). However, the principle PDEs implicated in the regulation of these responses is only now being addressed. In 2005, direct evidence was provided that PDE2 can regulate the integrity of the endothelium. In an elegant study Seybold and colleagues (2005) found that exposure of human umbilical vein endothelial cells (HUVECs) to tumor necrosis factor (TNF)-α resulted in a marked, time-dependent induction of PDE2A3 mRNA and protein that was associated with a decline in intracellular cyclic nucleotide levels and an increase in endothelial cell permeability. A disruption of endothelial cell integrity was also evoked in the same cells transfected with an expression vector encoding HSPDE2A3. Of significance was the additional finding that a selective PDE2 inhibitor, PNP, blocked the increase in permeability induced by thrombin in HUVECs transfected with the PDE2A3 expression vector, and this effect was reproduced in a murine model of endothelial barrier dysfunction (Seybold et al. 2005). From an allergy perspective, destabilization of the microvascular endothelium would be predicted in diseases such as asthma where TNF-α is known to be elevated (Broide et al. 1992; Ying et al. 1991) and this may facilitate edema formation and pulmonary leukocyte recruitment. A detrimental action on barrier function may not be restricted to TNF-α as many cytokines relevant to the pathogenesis of airway inflammation activate protein kinase C (PKC) (Kontny et al. 2000), which can also upregulate PDE2 (Geoffroy et al. 1999). Moreover, certain growth factor such as vascular endothelial growth factor also promote PDE2 gene expression (Favot et al. 2004) and, like TNF-α, is elevated in the asthmatic airway (Lee & Lee 2001; Chetta et al. 2005). Thus, if PDE2 also negatively regulates adhesion molecule expression and endothelial cell proliferation (Favot et al. 2003), inhibition of this enzyme could exert marked therapeutic benefit by suppressing the chronic inflammation and, potentially, airway remodeling that characterize asthma.

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Phosphodiesterase 3 Three genetically distinct cAMP-specific PDE families have been described and characterized (Bender & Beavo 2006a; Lugnier 2006). One of these, PDE3, is selectively inhibited by micromolar concentrations of cGMP and was originally classified as the cGMP-inhibited, cAMP PDE. The first clear demonstration of a cGMP-inhibited cAMP PDE was reported in 1982 (Weber & Appleman 1982). Since then, a PDE3 isoenzyme has been highly purified and characterized from a number of tissues including human platelets (Grant & Colman 1984), bovine heart (Harrison et al. 1986), rat liver (Pyne et al. 1987) and rat adipocytes (Degerman et al. 1987). Generally, members of this isoenzyme family hydrolyze cAMP and cGMP with high affinity but the apparent Vmax when cGMP is substrate is only about 10% of that achieved with cAMP (Grant & Colman 1984; Manganiello et al. 1992). For this reason, cGMP behaves as a potent and competitive inhibitor of cAMP hydrolysis (KI ∼ 0.1 μmol/L). It is noteworthy, however, that not all PDE3 preparations hydrolyze cGMP with a low Vmax (Maurice & Haslam 1990; Torphy & Cieslinski 1990; Manganiello et al. 1992; Torphy et al. 1993a). This is especially true for the PDE3 in human trachealis where less than a twofold difference in Vmax has been reported (Torphy et al. 1993a). The potential inhibitory action of cGMP on PDE3 activity may have significant physiologic implications in cells implicated in the pathogenesis of allergic diseases. Indeed, in PDE3expressing cells stimulants of guanylyl cyclase may inhibit cAMP hydrolysis and so potentiate cAMP-mediated functional responses. Evidence to support this possibility is available from studies performed with human platelets (Levin et al. 1982; Maurice & Haslam 1990), vascular smooth muscle (Shimokawa et al. 1988; Maurice et al. 1991), thymocytes (Marcoz et al. 1993) and airways smooth muscle (Turner, N.C. et al. 1994). In the latter tissue, sodium nitroprusside (SNP) markedly potentiated relaxation of guinea-pig trachea by the PDE4 inhibitor, rolipram, a finding consistent with the ability of SNP to increase cAMP in this tissue. In mammals, PDE3 isoenzymes are encoded by two genes: PDE3A and PDE3B. However, for only the former isoenzyme has evidence of multiplicity been convincingly provided (three variants have been described to date). These proteins have been denoted PDE3A1, PDE3A2 and PDE3A3 and arise from different start codon usage (Choi et al. 2001; Wechsler et al. 2002). PDE3A and PDE3B have a high degree of identity at the amino acid level (> 80% for most of the catalytic region), display similar kinetic properties and are both upregulated by phosphorylation catalyzed by PKA and PKB (Shakur et al. 2001). Unlike other PDE families, PDE3s contain a 44-amino acid insert within their catalytic domain of uncertain function although this additional sequence may contribute to both catalytic activity and inhibitor recognition (Manganiello & Degerman 1999). Full-length PDE3A (i.e., PDE3A1) and PDE3B feature one

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large (193 amino acids) and one small (50 amino acids) Nterminal hydrophobic membrane association region, NHR1 and NHR2, that are important in determining the subcellular localization of the protein (Shakur et al. 2000). Indeed, heterologous expression of these enzymes in Sf9 cells yields proteins that are entirely particulate (i.e., membraneassociated) (Kenan et al. 2000). In contrast, PDE3A2 and PDE3A3 in which the first and second NHRs are truncated are enzymes that are 50% cytosolic and totally cytosolic respectively (Kenan et al. 2000; Wechsler et al. 2002; Maurice et al. 2003).

Tissue distribution and selective inhibitors PDE3 isoenzymes are ubiquitously expressed in immune and proinflammatory cells (Table 27.4) including the platelet (Hidaka & Endo 1984; Macphee et al. 1986), basophil (Peachell et al. 1992), mast cell (Bergstrand et al. 1978; Weston et al. 1997), alveolar macrophage (Tenor et al. 1995a), T lymphocyte (Tenor et al. 1995b; Giembycz et al. 1996) and epithelial and endothelial cell (Lugnier & Schini 1990; Souness et al. 1990; Rousseau et al. 1994; Wright et al. 1998; Fuhrmann et al. 1999). PDE3 is not apparently present in eosinophils (Dent et al. 1991, 1994; Hatzelmann et al. 1995) or neutrophils (Nielson et al. 1990; Wright et al. 1990; Schudt et al. 1991a). The coexpression of PDE3A and PDE3B across tissues has not been systematically studied. However, where coincident expression has been found, PDE3A usually predominates (Manganiello & Degerman 1999; Maurice et al. 2003). Selective PDE3 inhibitors were developed in the 1980s for the treatment of dilated cardiomyopathy with the hope that a greater therapeutic index could be achieved over cardiac glycosides (Movsesian 2003). Accordingly, many potent and structurally distinct inhibitors have been synthesized some of which are currently used in the clinic for cardiovascular disease. In addition, many of these compounds have also been studied in nonvascular settings including asthma.

Therapeutic indications PDE3 has been considered a target for the treatment of asthma since selective inhibitors promote bronchodilatation in humans (Leeman et al. 1987; Fujimura et al. 1995; Bardin et al. 1998; Myou et al. 1999). Compounds of this class may also have desirable effects on the function of certain proinflammatory and immune cells although this is likely to be of relevance only during concurrent PDE4 inhibition (see below for further details). Thus, the concept of hybrid inhibitors that block PDE3 and PDE4 has found favor with some pharmaceutical companies as such compounds are predicted to have both bronchodilator and antiinflammatory activity.

Phosphodiesterase 4 A cAMP-specific PDE that was inhibited by the alkoxybenzylsubstituted imidazolidone, Ro 20-1724, but largely unaffected by cGMP was originally purified from canine kidney

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(Thompson et al. 1979; Epstein et al. 1982). It is now appreciated that this was the first report of a member of a large family of similar proteins collectively known as PDE4 isoenzymes. Since that original description, PDE4s have been purified and partially characterized from a number of sources including human monocytes (Torphy et al. 1993b) and human leukocytes (Truong & Muller 1994). Human recombinant proteins have also been heterologously expressed and purified to facilitate the identification of selective inhibitors by high throughput screening (Salanova et al. 1998). Without apparent exception, PDE4 isoenzymes are acid proteins (pI 4–6) and preferentially or even exclusively hydrolyze cAMP (Table 27.3). In 1989 Davis et al. (1989) isolated and cloned the Drosophila melanogaster “dunce” gene and found that it encoded a PDE of the PDE4 family. In the same year four rat cDNA homologs (Colicelli et al. 1989; Swinnen et al. 1989a,b) of the “dunce” cAMP PDE were identified, establishing a molecular basis for the observed heterogeneity of gene products within the PDE4 family. These clones represent transcripts of four different genes and are now named RNPDE4A, RNPDE4B, RNPDE4C and RNPDE4D according to the nomenclature proposed by Beavo and colleagues (1994). Four human genes (A–D) have also been identified and many of their products studied in some detail (see Bender & Beavo 2006a; Lugnier 2006). An astonishing finding that emerged from the molecular cloning of PDE4 isoenzymes is the presence of mRNA transcripts of different sizes for each of the four variants (greater than 26 have been unequivocally identified so far) that are differentially expressed between tissues (Houslay & Adams 2003; Bender & Beavo 2006a; Lugnier 2006). The basis for this profound heterogeneity of PDE4 isoenzymes is attributable to both alternative mRNA splicing and to the fact that the PDE4 genes express multiple promoter regions and, therefore, provide several potential start codons for the translation of the protein. Proteins encoded by each PDE4 gene may be either of the “short” or “long” form (see Bender & Beavo 2006a). Structurally, long PDE4 isoforms have an N-terminal domain, two socalled upstream conserved regions (UCR1 and UCR2; thought to mediate dimerization of long PDE4s (Richter & Conti 2002)), a highly conserved catalytic center and a unique C-terminus; in contrast, short PDE4 variants are truncated proteins that lack UCR1 (Houslay et al. 1998).

Tissue distribution and selective inhibitors Given the ubiquitous expression of PDE4 in cells implicated in the pathogenesis of allergic disease (Table 27.4), it is perhaps not surprising that many of the major pharmaceutical companies have invested heavily in PDE research over the last two decades, which has produced a variety of potent and selective inhibitors with which probe the pharmacologic consequences in vitro and in vivo of PDE4 inhibition (6–14 in Fig. 27.6; Table 27.3).

Therapeutic indications PDE4 inhibitors have been tested for efficacy in a number of immunologic and immunodeficiency conditions including asthma, chronic obstructive pulmonary disease (COPD), Crohn disease, myasthenia gravis, atopic dermatitis, psoriasis, systemic lupus erythematosis, rheumatoid arthritis, diabetes and multiple sclerosis (Sommer et al. 1997; Dyke & Montana 2002; Banner & Trevethick 2004; Aricha et al. 2006; Moore et al. 2006). Asthma and COPD are indications where development of PDE4 inhibitors is most advanced (Table 27.2). The rationale for developing compounds that attenuate PDE4 activity is based on three critical findings: (i) PDE4 is abundant and the major regulator of cAMP metabolism in almost every pro-inflammatory and immune cell; (ii) PDE4 inhibitors, of varied structural classes, suppress a myriad of in vitro responses such as cytokine generation, NADPH oxidase activity, degranulation, IgE production, proliferation, lipid mediator and histamine generation, and chemotaxis; and (iii) PDE4 inhibitors are efficacious in animal models of inflammation (Torphy 1998). If these observations hold in humans then, conceptually, PDE4 inhibitors should show a pleiotropic inhibitor profile of activity on proinflammatory and immune cell function. A further prediction is that inhibition of PDE4 should potentiate the effects of endogenous antiinflammatory agents that increase cAMP such as catecholamines, prostaglandin E2 and prostacyclin. Taken together, the preclinical pharmacology of these compounds provided an exciting rational basis for the development of novel antiinflammatory pharmaceuticals that may display steroid-like activity without the associated side-effects.

Regulation of PDE4 PDE4 is subject to both short and long-term regulation by agents that elevate cAMP. This finding has led to the proposal (see Giembycz 1996) that upregulation of PDE4 in allergic asthma could contribute to the tolerance of airway smooth muscle cells to the bronchodilator and protective effects of β2-adrenoceptor agonists (see Cheung et al. 1992; O’Connor et al. 1992; Cockcroft et al. 1993; Bhagat et al. 1995; Hancox et al. 1999, 2002; Jones et al. 2001; Wraight et al. 2003; Haney & Hancox 2005). Indeed, a metaanalysis of 22 independent randomized, placebo-controlled studies conducted between 1989 and 2001 lead the authors to conclude that regular use of β2-adrenoceptor agonists for at least one week in patients with asthma results in tolerance to the drug’s bronchodilator activity and that this may be associated with poorer asthma control (Salpeter et al. 2004). It is pertinent to mention that the mechanism of tolerance is not resticted to β2-adrenoceptor desensitization. It is likely that in many subjects poor asthma control reduces the efficacy of β2-adrenoceptor agonists. For example, mucus impaction may limit the delivery of drug to the desired sites of action and so compromise efficacy by physical means. Upregulation of PDE4 can occur through either posttranslational modification (e.g., phosphorylation) of existing

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H 3C

O

OH H N

O

HN

N

N

O

N

N

N O

N

N

N

S

N

O H3C

Cl 6 Arofylline

CH3 7 Tofimilast

8 Cilomilast OH HN

N

O

O HO

O

Cl O

CH3

O

O

N

O NH

N

N

O

Cl

CH3

N

S

O F

9 Tetomilast

F

CH3

10 Roflumilast

11 ONO 6126

F

N

N

CH3

N

O

Cl

N CH3

NH

N

HO O

O

CH3 O

CH3

12 AWD-12-281 (GSK 842470)

O

O

O

H 3C

Cl

CH3

O CH3 13 Ibudilast

CH3 14 IC-485

Fig. 27.6 Chemical structure of selective inhibitors of PDE4 that have been evaluated in the clinic for allergic diseases. See text for further details.

enzyme or gene induction. Indeed, the catalytic activity of long PDE4 isoforms is upregulated following phosphorylation by PKA of S54 within UCR1 (Sette et al. 1994; Sette & Conti 1996; MacKenzie et al. 2002). Similarly, the induction of certain PDE4 variants can be induced in a PKA-dependent manner that involves the phosphorylation of CREB (D’Sa et al. 2002; Wang et al. 2003). With respect to β2-adrenoceptors in the lung, it is the PDE4 isoenzyme family that is a primary regulator of cAMP metabolism (Torphy 1998). In this paradigm, tolerance to β2-adrenoceptor agonists is directly related to an increase in PDE activity. This effect would theoretically compromise cell signaling through all Gs-coupled receptors leading to heterologous desensitization of susceptible cells to cAMP-dependent events. It is hypothesized that this could occur as a direct consequence of regular treatment of asthmatic subjects with β2-adrenoceptor agonists (Giembycz 1996).

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While generally ignored, the concept of increased cAMP PDE activity as a mechanism of reducing the sensitivity of cells to hormones and other agonists that interact with Gscoupled receptors is not new. In fact, evidence that this phenomenon accounts for much of the reduced responsiveness that cells exhibit to chronic hormone exposure was provided in 1978 (Barber et al. 1978) and has since been documented in vitro in many cells implicated in the pathogenesis of asthma such as T lymphocytes, neutrophils, monocytes, macrophages, platelets and airway smooth muscle (Barber et al. 1978; Conti et al. 1986; Ashby 1989; Torphy et al. 1992, 1995; BousquetMelou et al. 1995; Verghese et al. 1995; Seybold et al. 1998; Mehats et al. 1999, 2001; Dasi et al. 2000; Ortiz et al. 2000; Persani et al. 2000). An important issue that arises from the aforementioned discussion is whether induction and/or phosphorylation of PDE4 can be demonstrated in immune/proinflammatory cells

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and in vivo in response to β2-adrenoceptor agonists. Although limited data are available, the answer to both parts of this question is yes. Torphy and colleagues (1995) demonstrated that the β2-adrenoceptor agonist, salbutamol, and the selective PDE4 inhibitor, rolipram when given in combination to the human monocytic cell line, U-937, increased PDE4 activity a time-dependent manner. Significantly, this effect required new protein synthesis indicating that the increase in enzyme activity was attributable to the induction of one or more PDE4 isogenes. Reverse transcription-PCR and Western analyses performed by the same authors demonstrated subsequently that salbutamol and rolipram increased the expression of PDE4A and PDE4B at the mRNA and protein level. A similar investigation by Verghese et al. (1995) essentially confirmed these observations. Thus, exposure of human peripheral blood monocytes and Mono Mac 6 cells to cyclic AMP-elevating agents promoted the transcription of the PDE4A, B and D isogenes with the generation of at least three distinct mRNA transcripts and proteins. Engels et al. (1994) have also reported induction of PDE4 isogenes in U-937 and Jurkat T-cells in response to prolonged exposure to dibutyryl cAMP and, more recently, the same phenomenon was documented in guinea-pig macrophages (Kochetkova et al. 1995), human T lymphocytes (Seybold et al. 1998), human neutrophils (Ortiz et al. 2000) and human airway smooth muscle cells (Le Jeune et al. 2002). In the latter study, upregulation of the PDE4D5 splice variant was described and this may have particular significance given that this isoform interacts preferentially with β-arrestins and may play a role in β2-adrenoceptor desensitization (Perry et al. 2002; Bolger et al. 2003). A consistent and highly significant finding is that the responsiveness of many cells in which PDE4 is induced to cAMP-generating agonists is restored, at least in part, by the addition of a PDE inhibitor providing compelling evidence that upregulation of PDE is a significant contributing factor in the development of tolerance. In 2000, Finney et al. reported the upregulation of PDE4 in the lungs of rats treated chronically for 7 days with salbutamol. Thus, this phenomenon can be reproduced in vivo and may have clinical relevance in the development of tolerance following chronic use of β2-adrenoceptor agonists.

Phosphodiesterase 5 Three major cyclic nucleotide PDE families have been described which preferentially hydrolyze cGMP. Two of these, PDE6 (or photoreceptor) and PDE9, are not implicated in asthma or allergy and are not discussed here. The other family comprises a group of cGMP-binding, cGMP PDEs that have been isolated and studied primarily from peripheral tissues and are collectively known as PDE5 isoenzymes. PDE5 was first identified in lung (Hamet & Coquil 1978; Coquil et al. 1980; Francis et al. 1980) and has since been purified to homogeneity from a number of tissues (Francis & Corbin 1988). In humans and other species PDE5 is a cytosolic

enzyme that is insensitive to Ca2+ and calmodulin and is selectively inhibited by zaprinast (Lugnier et al. 1986). PDE5 exist as a homodimer, displays selectivity for cGMP over cAMP (Table 27.3) (Thomas et al. 1990; Corbin & Francis 1999) and features two N-terminal GAF domains. In contrast to PDE2, cGMP binds only to GAF-A in PDE5 (Zoraghi et al. 2005) stimulating enzyme activity about 10-fold; this implies that PDE5 is inactive when GAF-A is not occupied by cGMP (Rybalkin et al. 2003). PDE5 is phosphorylated by PKG at a serine residue close to the GAF-A domain and is believed to stabilize the cGMP–GAF-A interaction by increasing the affinity of cGMP for GAF-A and thereby provide a means for prolonged activation of PDE5 (Corbin et al. 2000; Francis et al. 2002; Shimizu-Albergine et al. 2003). Only a single PDE5 gene has thus far been identified that encodes PDE5; however at least three variants (PDE5A1, PDE5A2, PDE5A3) can be generated from differentially regulated promoters that differ at their N-termini (Kotera et al. 1999; Loughney et al. 1998; Stacey et al. 1998; Yanaka et al. 1998; Lin et al. 2000a,b). Presumably, these promoters allow for differential control of PDE5 gene expression.

Tissue distribution and selective inhibitors PDE5 isoenzymes are widely distributed. Significant amounts are present in vascular (Lin et al. 2006) and airway smooth muscle (see Souness & Giembycz 1994), T lymphocytes (Tenor et al. 1995b; Giembycz et al. 1996), platelets (Hidaka & Endo 1984) and airways epithelial cells (Rousseau et al. 1994; Wright et al. 1998; Fuhrmann et al. 1999). Traces of this enzyme have also been detected in alveolar macrophages (Tenor et al. 1995a), vascular endothelial cells (Souness et al. 1990; Suttorp et al. 1993) mast cells and basophils (Peachell et al. 1992). The serendipitous discovery of sildenafil for the treatment of erectile dysfunction has led to the description of several new and potent PDE5 inhibitors including vardenafil and tadalafil, some of which have, in humans, a significantly longer half-life than sildenafil (Gupta et al. 2005).

Therapeutic implications From a therapeutic standpoint, selective inhibitors of the PDE5 isoenzyme family are not obvious candidates for having a major impact on allergic inflammation, although a modest degree of bronchodilatation may be elicited and afford some benefit in disease states characterized by reversible airflow limitation such as allergic asthma (see Murray 1993; Souness & Giembycz 1994). However, recent data indicate that PDE5 inhibitors can indeed suppress pulmonary inflammation and airways hyperreactivity following allergen challenge, at least in animal models of asthma (Toward et al. 2004). Moreover, there is emerging evidence that PDE5 may also play a major role in the vasculature regulating mitogenesis and angiogenesis (Ladha et al. 2005). These processes are involved in the development of airways remodeling (Wilson

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& Hii 2006) and, accordingly, may provide a viable therapeutic target for selective PDE5 inhibitors or drugs that inhibit more than a single PDE isoenzyme family.

Phosphodiesterase 7 In 1993, Micheali et al. reported the development of a highly sensitive functional screen for the isolation of cDNAs that encode cAMP PDEs. Using this technology three groups of cDNAs were isolated from a human glioblastoma cDNA library. Two of those genes were closely related to the Drosophila “dunce” cAMP PDE (i.e., PDE4-like), while the third encoded an enzyme that readily degraded cAMP but with characteristics distinct from all other known PDEs. At that time, the new PDE was given the name high affinity cAMP-specific phosphodiesterase 1 (HCP-1). Primary sequence analysis established that HCP-1 shared significant homology with a stretch of 280 C-terminal amino acids that constitute the catalytic domain of other mammalian cAMP PDEs (Michaeli et al. 1993). Within this highly conserved region HCP-1 exhibited the highest and lowest degree of homology to PDE4 (35% identity; 51% similarity) and PDE2 (24% identity; 37% similarity) family members respectively. However, given that the homology in the catalytic core between PDE4 variants varies between 85 and 95% and that cAMP hydrolysis by HCP-1 was insensitive to the selective PDE4 inhibitors, Ro-20,1724 and rolipram, it was concluded that the novel enzyme represented the first member of a previously unknown cAMP PDE family and was designated PDE7 (Michaeli et al. 1993). It is noteworthy that the discovery of PDE7 coincided with a report published at the same time of an atypical cAMP hydrolytic activity (called JK-21) in several human T-cell lines including Jurkat, MOLT-4, HPB-ALL and HUT-78 (Ichimura & Kase 1993), which is now known to be a PDE7 family member (Bloom & Beavo 1996). Two genes (PDE7A, PDE7B) have been identified in the mouse, rat and human that encode PDE7 isoenzymes (Michaeli et al. 1993; Hoffmann et al. 1998a; Hetman et al. 2000; Sasaki et al. 2000, 2002). However, it is only products derived from PDE7A, which encodes three variants (PDE7A1, PDE7A2, PDE7A3), that are potential targets for antiallergic drugs. Translation in humans of PDE7A1, PDE7A2 and PDE7A3 mRNA transcripts yields proteins that are composed of 482, 456, and 424 amino acids respectively. On SDS polyacrylamide gels the three splice variants migrate as 55–57 kDa (7A1), 50–52 kDa (7A2) and 50 kDa (7A3) proteins, which is similar to the masses predicted from their amino acid composition. The N-terminus of PDE7A1 and PDE7A3 is rich in proline, serine and positively charged amino acids whereas the same region of PDE7A2 is hydrophobic (Bloom & Beavo 1996; Han et al. 1997; Glavas et al. 2001) and features potential myristoylation (G2) and palmitoylation (C8) sites that may dictate intracellular localization. Indeed, following subcellular fractionation of a variety of tissues PDE7A2 has been found

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only in the particulate fraction, consistent with the hydrophobicity of its N-terminus; in contrast PDE7A1 is predominantly cytosolic (Bloom & Beavo 1996; Han et al. 1997). The subcellular distribution of PDE7A3 has not been investigated but it shares the same N-terminus as PDE7A1 and could localize to soluble cellular structures.

Tissue distribution and selective inhibitors PDE7A is widely expressed in the lungs and across a variety of immune and proinflammatory cells (Table 27.5) (Miro et al. 2001; Smith et al. 2003; Barber et al. 2004). Accordingly, it seems likely that this enzyme family may play an important role in regulating many cAMP-dependent processes related to the immune system. Indeed, all human proinflammatory and immune cells (primary and cells lines) that have been studied express mRNA for PDE7A1 and PDE7A2 at an approximate ratio of 4 : 1. However, irrespective of the method used for detection (i.e., Western blotting, immunocytochemistry, confocal microscopy) PDE7A2 has never been found at the protein level despite unequivocal identification of PCR products corresponding to this transcript. This finding is not peculiar to PDE7A. In human mononuclear cells several of the PDE4D splice variants are expressed only at the mRNA level (Giembycz et al. 1996; Nemoz et al. 1996). Whether this is due to translational repression or a low translation rate of these particular mRNA transcripts, instability of the active enzymes or to the fact that the proteins are expressed at very low but nevertheless functional levels is unexplored. Of the immunocompetent/proinflammatory cells that have been studied, CD4+ and CD8+ T lymphocytes express relatively high levels of PDE7A1 that are readily detected by RT-PCR and Western blotting (Table 27.5) (Giembycz et al. 1996; Seybold et al. 1998; Secchiero et al. 2000; Smith et al. 2003). Human airway smooth muscle cells, blood monocytes and lung macrophages together with several cell lines including HUT-78 (T-cell) and BEAS-2B (epithelial) are also PDE7A1+ under the same experimental conditions (Table 27.5) (Smith et al. 2003). In contrast, Western blotting has failed to detect PDE7A1 in human neutrophils although the protein can clearly be labeled using immunoconfocal laser microscopy (Smith et al. 2003). This more sensitive technique has also been employed to determine the expression profile of PDE7A in cells such as macrophages present in sputum and bronchoalveolar lavage fluid. The inability to detectable PDE7A1 in neutrophils by western blotting suggests that the concentration of this enzyme is lower than that found in the other cell types examined. Although immunoconfocal laser microscopy cannot distinguish between the PDE7A splice variants, much of the staining is cytosolic indicating that PDE7A1 (and/or PDE7A3 see below) may be the most abundant isoform expressed. Very little is known about PDE7A3. Studies by (Glavas et al. 2001) identified this splice variant in human CD4+ T lymphocytes after costimulation with antiCD3/CD28 anti-

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Table 27.5 Expression of PDE7A1 and PDE7A2 in human proinflammatory and immune cells. mRNA

Protein

Cell type

HSPDE7A1

HSPDE7A2

HSPDE7A1

HSPDE7A2

Human primary cells CD4+ T lymphocyte CD8+ T lymphocyte B lymphocyte Neutrophil Alveolar macrophage Monocyte Eosinophil Bronchial epithelial cell Lung mast cell Lung basophil Airway smooth muscle cell Vascular smooth muscle cell Vascular endothelial cell

+ + +* + + + ND +* +* +* + + +

+ +

+ + + +* +* + + ND − − + + ND

− − ND

− − ND

Human cell lines HUT-78 (T cell) Jurkat (T cell) 16-HBE14 (epithelial cell) NCIH292 (epithelial cell) BEAS-2B (epithelial cell) U-937 (monocyte) MRC-5 (fibroblast) AML14.3D10 (eosinophil) Jiyoye (B cell)

+ + + +* + + + + +

+ + + ND + + + + −

− − − ND − − − − ND

+ + + ND

+ + + + + + + + + + ND

− −

+, enzyme detected; −, enzyme not detected; ND, not determined; *, isoenzyme not determined. Data taken from Smith et al. (2003).

bodies but its distribution in other cells is unknown. Given that PDE7A3 and PDE7A1 are probably regulated by the same promoter (Torras-Llort & Azorin 2003) it is possible that the expression pattern of both transcripts is similar. PDE7A encodes cAMP-specific PDEs that are insensitive (IC50 > 100 μmol/L) to cGMP and standard inhibitors of PDE2, PDE3, PDE4 and PDE5 (Michaeli et al. 1993; Hetman et al. 2000; Richter et al. 2002; Smith et al. 2004), although it is inhibited by the generally nonselective compound 3-isobutyl1-methylxanthine (IBMX) with an IC50 of 5–53 μmol/L adopted across experiments (Wang et al. 2000; Lee et al. 2002; Richter et al. 2002; Smith et al. 2004). Despite the discovery of PDE7A in 1993, there were until 2001 surprisingly few reports of selective inhibitors. However, examples of compounds with PDE7 inhibitory activity have emerged and some of these have acceptable selectivity for both in vitro and in vivo studies (reviewed in Vergne et al. 2005; Giembycz & Smith 2006a,b). For example, Smith et al. (2004) reported on a sulphonamide derivative, BRL 50481 (see 16 in Fig. 27.7) that is > 200-fold selective for hrPDE7A1 expressed in baculovirus-infected Sf9 cells over PDEs 1 to 5. BRL 50481 is

a purely competitive inhibitor (with respect to substrate) of the enzyme with a Ki of 180 nmol/L. Another PDE7 inhibitor based on the 1,3,4-thiadiazole template is the Pfizer compound PF 332040 (see 16 in Fig. 27.7), which is at least 600-fold selective over other PDE isoenzyme families. To date, none of the compounds described are reported to discriminate between PDE7A and PDE7B (Bernardelli et al. 2004; Lorthiois et al. 2004; Vergne et al. 2004a,b).

Me Me

O S

N Me

O H 2N

N N S

O

N+ O

N

OH

−

O

15 BRL 50481

16 PF332040

Fig. 27.7 Chemical structure of two novel and selective inhibitors of PDE7. See text and Giembycz & Smith (2006a) for further details.

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Therapeutic indications The advent of selective PDE7 inhibitors has allowed an assessment of the functional role of PDE7. In the context of allergy, excitement in this area initially was fuelled by a report published in 1999 that IL-2 production by, and the subsequent proliferation of, antiCD3/antiCD28-stimulated human T lymphocytes was associated with induction of PDE7A1. Significantly, delivery to these cells of antisense oligonucleotides directed against PDE7A mRNA transcripts prevented these responses in a PKA-sensitive manner (Li et al. 1999; Glavas et al. 2001). The results described by Li et al. (1999) are supported by studies conducted in mice deficient in the PDE7A gene (Michaeli 2004). Thus, the ability of antiCD3 antibodies to promote the proliferation of splenocytes and purified T lymphocytes was reduced in PDE7A–/– mice by 66% and 60% respectively relative to their wild-type counterparts; in contrast, cell proliferation evoked by a combination of ionomycin and phorbol ester was unaffected by PDE7A gene deletion (Michaeli 2004). Similarly, the number of IFNγ +/ CD3+ T cells harvested from mice lacking the PDE7A gene and their ability to release interferon (IFN)-γ in response to antiCD3 antibodies both in vitro and ex vivo was significantly lower when compared to wild-type mice. Taken together these data implicate PDE7A in the regulation of murine T-cell proliferation and Th1 cytokine generation. However, subsequent investigations with small molecule inhibitors that are selective for PDE7A have not corroborated these findings. Thus, BRL 50481 (15 in Fig. 27.7) had no effect on IL-15-induced proliferation of human CD8+ T lymphocytes (Smith et al. 2004). Similarly, in another study where PDE7A–/– mice were used, T-cell proliferation and Th1 (IL-2, IFN-γ, TNF-α) and Th2 (IL-4, IL-5, IL-13) cytokine production evoked by ligation of CD3/CD28 was preserved (Yang et al. 2003). The same result was found using wild type mice treated with BMS-586353, a highly potent PDE7 inhibitor (Yang et al. 2003). It is unclear why the results of Smith, Yang and their respective colleagues do not concur with data reported by Li et al. (1999) or Michaeli (2004) but they are

O2N

O

NH

unlikely to be species related or to a redundant mechanism in mice that compensates for the deficiency in PDE7A. Two additional possibilities may account for some of the discrepancies. In the study reported by Smith et al. (2004), CD8+ T lymphocytes were isolated from other leukocytes by negative immunoselection using a mixture of antibodies against CD11b, CD16, CD19, CD36, CD56 and CD4. Although the same methodology was used by Li et al. (1999) antibodies against CD25 and HLA-DR were also employed, which will remove all activated and proliferating T cells. Thus, it is possible that naive T cells are regulated differently by PDE7A when compared to their activated and proliferating counterparts. Alternatively, the use of naked antisense oligonucleotides, as used by Li et al. (1999), may not have targeted specifically the mRNA of interest or, alternatively, evoked toxic effects that were sequence nonspecific (Stein 2001). The divergence in data between the two studies conducted with PDE7 –/– mice currently is inexplicable. Of potential interest is the report by Smith and colleagues in 2004 that the culture of human monocytes in RPMI-1640 for 24 hours resulted in upregulation of PDE7A1 and conferred functional sensitivity to BRL 50481, i.e., lipopolysaccharide (LPS)-induced TNF-α release was significantly inhibited (Smith et al. 2004). Moreover, in monocytes in which PDE7A1 was upregulated, the inhibition of TNF-α release evoked by rolipram and other cAMP-elevating agents was enhanced in a purely additive manner. These data imply that PDE7A inhibitors alone may regulate the responsiveness of monocytes and possibility other proinflammatory and immune cells under circumstances when PDE7A is highly expressed such as in chronic inflammation. In this respect many chemokines and cytokines relevant to the pathogenesis of chronic inflammatory diseases signal, in part, through a PKC-dependent mechanism (Kontny et al. 2000; New & Wong 2003; Johnson et al. 2004) and it is known that the human PDE7A1 promoter is activated by phorbol esters (Torras-Llort & Azorin 2003). Although inhibition of PDE7A has no demonstrable antiinflammatory activity under normal conditions, PDE7A–/– mice respond to immunization (with keyhole limpet hemocyanin) with a significantly enhanced antibody response when compared to wild-type animals. Thus, PDE7A may play a central role in cAMP/PKA signaling processes that are unrelated to T-cell activation (Yang et al. 2003) such as B-cell function.

Phosphodiesterase 8 F N NH O 17 Fig. 27.8 Chemical structure of a selective inhibitor of PDE3B. See text and Edmondson et al. (2003) for further details.

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The PDE8 isoenzyme family is composed of two genes, PDE8A and PDE8B, that encode predominantly cytosolic proteins (Gamanuma et al. 2003) that hydrolyze, almost exclusively, cAMP (Fisher et al. 1998; Hayashi et al. 1998) (Table 27.3). Multiple isoforms can be derived from PDE8A and PDE8B (5 and 4 variants respectively) that arise through alternative splicing and/or start codon usage (Wang et al. 2001; Hayashi et al. 2002). Putative regulatory domains of unknown func-

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tion have been identified towards the N-terminus of these enzymes including a “receiver” and a PAS domain. In other proteins PAS domains represent sites for ligand binding and protein–protein interaction but, thus far, no ligands have been identified that interact with the PAS domain on PDE8. In the context of allergy, PDE8A1 has been identified in human primary T-lymphocytes and T-cell lines (Glavas et al. 2001) and evidence, albeit circumstantial in nature, suggests that inhibition of PDE8 can attenuate T-cell chemotaxis (Dong et al. 2006). However, no selective PDE8 inhibitors have been described with which to more formally address this possibility.

Theophylline, hybrid and nonselective PDE inhibitors The nonselective PDE inhibitor, theophylline, was originally identified by Kossel in Berlin in 1888 and was synthesized 12 years later by Boehringer. However, more than two decades elapsed before the bronchodilator activity of theophylline was realized (Schultze-Werninghaus & Meier-Sydow 1982) and a further 56 years before a possible mechanism of action was suggested (Butcher & Sutherland 1962). Since the early 1950s, theophylline and some related alkylxanthines have been used widely in the treatment of asthma (and latterly, COPD) although recent international guidelines recommend that these drugs be relegated to third-line therapy for both indications. The primary use of theophylline is as a bronchodilator and its mechanism of action is due primarily to inhibition of PDE3 in airways smooth muscle cells (Cortijo et al. 1993). Indeed, in vitro studies have found that the EC50 of theophylline for the relaxation of human isolated tracheal smooth muscle is ∼ 70 μmol/L (Finney et al. 1985; Goldie et al. 1986; Cortijo et al. 1993), which equates to a plasma concentration of 32 μg/mL assuming that 60% of the drug is bound to plasma proteins (Guillot et al. 1984). Theophylline has also been reported to suppress a number of inflammatory indices associated with respiratory diseases (Pauwels et al. 1985; Mapp et al. 1987; Ward et al. 1993; Sullivan et al. 1994; Jaffar et al. 1996; Kraft et al. 1996; Lim et al. 2001; Culpitt et al. 2002) at low doses (5–10 μg/mL) that produce only a modest but nevertheless physiologically significant inhibition of PDE (see Dent & Rabe 1996 and Clinical Pharmacology). Based on these data it is tempting to speculate that second generation, nonxanthine PDE inhibitors that are devoid of activity at adenosine receptors with broader PDE specificity may have superior therapeutic activity over theophylline. Improved activity may be realized for several reasons. First, it is well established that inhibitors of PDE4 can act synergistically in many proinflammatory and immune cells with compounds that block PDE3 (Robicsek et al. 1991; Schudt et al. 1995; Giembycz et al. 1996) or PDE7 (Smith et al. 2004).

Indeed, in the context of asthma, T lymphocytes, macrophages, monocytes, endothelial and endothelial cells are sources of PDE3, PDE4 and PDE7 (Torphy 1998; Smith et al. 2003). Functionally, in vitro studies have shown that while PDE3 inhibitors generally have little or no effect themselves on T-cell proliferation or on IL-2 generation, they significantly enhance the antimitogenic effect of PDE4 inhibitors (Robicsek et al. 1991; Giembycz et al. 1996). Similar results have been reported for TNF-α release from human alveolar macrophages (Schudt et al. 1995). Concurrent inhibition of PDE4 and PDE7 may also promote a superior antiinflammatory effect than a PDE4 inhibitor alone. Thus, while inhibition of PDE7A with BRL 50481 does not attenuate the proliferation of T cells per se, it significantly augments the antimitogenic and cAMP-elevating activity of rolipram (Smith et al. 2004). The suppression by PDE4 inhibitors of TNF-α release from LPS-stimulated human blood monocytes and lung macrophages is also significantly enhanced by BRL 50481 (Smith et al. 2004). Taken together, these in vitro data imply that a nonselective compound may be more efficacious than a PDE4 inhibitor administered as a monotherapy. Moreover, because of synergy, lower doses of hybrid inhibitors may be efficacious such that adverse events attributed to the inhibition of PDE4 in nontarget tissues are reduced (see section on Adverse Events). Second, regardless of synergy, concurrent inhibition of multiple PDEs would be expected to exert clinically relevant effects (not induced by PDE4 inhibition alone) on other processes that contribute to the pathogenesis of asthma including airways/vascular remodeling (PDE1 and PDE5 regulated), endothelial cell permeability (PDE2 regulated), mast cell stabilization (PDE3 mediated) and airways smooth muscle tone (PDE3 regulated).

Antiinflammatory effects of PDE inhibitors in vivo The actions of theophylline and isoenzyme-selective PDE inhibitors upon the acute (IgE-mediated) or chronic (proinflammatory/immunocompetent cell-mediated) consequences of allergen provocation in vivo have, in general, been documented in some detail and include studies of passive cutaneous anaphylaxis (PCA), cell infiltration into sites of inflammation and microvascular leakage.

IgE-mediated processes It seems unlikely that PDE inhibitors of any isoenzyme family reduce IgE levels in vivo. In humans, it is reported that ibudilast, which is an essentially nonselective PDE inhibitor (Souness et al. 1994) is without effect on IgE levels in asthmatic individuals (Kawasaki et al. 1992). Similarly, the elevated level of total serum IgE in a murine model of dermatitis caused by the repeated application of 2,4,6-trinitro-1chlorobenzene is unaffected by oral administration of the PDE4 inhibitor rolipram (Harada et al. 2006). In contrast, PDE3 and PDE4 inhibitors exhibit efficacy at reducing PCA reactions in rats, mice and guinea-pigs (Davies

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& Evans 1973; Broughton et al. 1975). Furthermore, rolipram, but neither zaprinast nor SK&F 94120, is effective at reducing the infiltration of indium-labeled eosinophils into the skin of guinea-pigs following a PCA reaction (Teixeira et al. 1994). Collectively, these data imply that PDE4 inhibitors can suppress the degranulation of IgE-bearing leukocytes and, therefore, allergen-induced mediator release. Further support for this proposal derives from studies in sensitized guineapigs where rolipram, administered intravenously, inhibits antigen- but not leukotriene (LT)D4-induced bronchoconstriction (Howell et al. 1993). Thus, rolipram preferentially exerts an inhibitory influence at the level of mast cells and basophils rather than exerting a direct antispasmogenic action at the level of airways smooth muscle. In contrast, PDE3 inhibitors including CI-928 inhibit both allergen- and LTD4induced bronchoconstriction under identical conditions indicating a direct smooth muscle effect of these compounds (Howell et al. 1993).

after chronic (6 days) dosing (Sanjar et al. 1990a,b). Studies conducted more recently with rolipram and second generation PDE4 inhibitors including cilomilast, roflumilast and AWD 12-281 have corroborated these findings both in the airways an in allergic cutaneous reactions (Underwood et al. 1993, 1994, 1998; Griswold et al. 1998; Bundschuh et al. 2001; Baumer et al. 2002, 2003; Kuss et al. 2003; Draheim et al. 2004; Hoppmann et al. 2005). Similarly, treatment of sensitized cynomolgus monkeys with rolipram abrogates the pulmonary neutrophilia and eosinophilia, and airways hyperresponsiveness after multiple exposures to the antigen but is without effect on the immediate increase in airways resistance that follows acute antigen provocation (Turner, C.R. et al. 1994). Thus, these data are consistent with the findings of that PDE4 inhibitors may be antiinflammatory and act primarily to prevent the activation of immune cells in the lung rather than by exerting a antispasmogenic or spasmolytic effect the level of airways smooth muscle (Howell et al. 1993).

Proinflammatory cell infiltration Intravenous injection of a low dose of theophylline in to a guinea-pig model of allergic asthma immediately prior to antigen prevents both the immediate bronchoconstriction and the late-phase reaction (LPR) (Andersson et al. 1985). Qualitatively identical results were obtained in an allergic sheep and rabbit model (Ali et al. 1992). Independent experiments conducted with sensitized guinea-pigs and rats have demonstrated that theophylline suppresses pulmonary eosinophil and, in the case of the rat, also neutrophil recruitment which is believed to be intimately associated with the development of the LPR (Sanjar et al. 1990b; Gristwood et al. 1991; Tarayre et al. 1991a,b, 1992; Lagente et al. 1994; Manzini et al. 1993). In many of these studies, however, theophylline was administered acutely as a single dose that is significantly higher than that used therapeutically indicating that the observed effects may not be relevant to the clinical situation. In contrast, chronic treatment of guinea-pigs for seven days with theophylline effectively prevents plateletactivating factor (PAF)- and allergen-induced eosinophil recruitment at therapeutically-relevant doses (Sanjar et al. 1990a,b). A large number of investigations (too many to describe individually herein) have evaluated the effect of PDE inhibitors upon the infiltration of proinflammatory cells into the airways lumen, skin and eye of guinea-pigs, rats and monkeys in responses to a variety of dissimilar stimuli including allergen. For example, pretreatment of sensitized guinea-pigs with zardaverine, a mixed PDE3/4 inhibitor, markedly suppressed allergen-induced infiltration of eosinophils, macrophages and neutrophils into the bronchoalveolar lavage (BAL) fluid to a level achieved with dexamethasone (Schudt et al. 1991b). Qualitatively identical data have been reported for the PDE3/4 inhibitor, benafentrine, on PAF- and allergeninduced pulmonary eosinophil recruitment in guinea-pigs

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Microvascular leakage and edema It is well recognized that the microcirculation plays an important role in inflammatory reactions. Under normal conditions, the endothelium lining the postcapillary microvenules in the skin and bronchial circulation (capillaries in the pulmonary vasculature) is largely impermeable to blood cells and to macromolecules but following a proinflammatory insult, localized arteriolar vasodilatation occurs with a consequent increase in blood flow. This effect is induced by the liberation of proinflammatory mediators such as histamine and tachykinins. An increase in capillary and microvenular pressure then ensues together with the liberation of other mediators such LTD4, which contract directly the microvenular endothelial cells. Together these effects, by increasing microvenular permeability, permit the loss of plasmaproteins from the vascular compartment. Furthermore, the resulting increase in osmotic pressure due to loss of solute from the circulation leads to marked fluid exudation and to oedema. In guinea-pigs, theophylline reduces plasma exudation in to the trachea, bronchi and BAL fluid but its efficacy may depend upon the nature and strength of the leak-evoking stimulus (Boschetto et al. 1989; Erjefalt & Persson 1991; Raeburn & Karlsson 1993; Raeburn & Woodman 1994). With respect to selective drugs intravenous, oral and intratracheal administration of representative inhibitors of the PDE4 and PDE5 isoenzyme families markedly attenuate PAF-induced microvascular leakage in both small and large airways, and into the BAL fluid of anesthetized guinea-pigs (Ortiz et al. 1992; Raeburn & Karlsson 1993; Raeburn et al. 1994). The finding that rolipram and zaprinast are active when given directly in to the airways indicates an important local action in the lung and highlights that systemic administration is not necessary for these compounds to exert an antiinflammatory influence. This is an important observation

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since the administration of PDE inhibitors by the inhaled route should reduce untoward side effects while maintaining efficacy. Drugs which inhibit PDE1 and PDE3 do not inhibit PAF-induced microvascular leakage in guinea-pig airways (Ortiz et al. 1992; Raeburn & Karlsson 1993). This latter finding is curious given that a PDE3 isoenzyme has been identified in endothelial cells, and that a selective PDE3 inhibitor, motapizone, blocks the increase in permeability elicited by H2O2 in vitro (Suttorp et al. 1993). It is likely, however, that this discrepancy is due to a difference in species, the leak-evoking stimulus or in vessel type. In keeping with this latter possibility, Teixeira et al. (1994) have reported that rolipram does not inhibit edema formation in guinea-pig skin in response to histamine, zymosan-activated plasma and PAF (cf. guinea-pig lung). It is tempting to speculate that the complement of PDE isoenzymes and/or the regulation of endothelial cell contractility by cyclic nucleotides varies significantly between vessels of the pulmonary and systemic vascular beds. Svensjo et al. (1993) reported that PDE4 and PDE3/4 inhibitors attenuate the increase in microvascular permeability evoked by bradykinin in the hamster cheek pouch. Qualitatively identical results are obtained for the effect of rolipram and denbufylline upon arachidonate-induced edema in rodents (Crummey et al. 1987).

Clinical pharmacology Theophylline Until relatively recently, the therapeutic efficacy of theophylline in asthma was attributed to its weak bronchodilator activity. However, while theophylline is an effective bronchodilator, there is now increasing evidence that this drug may also exert an immunomodulatory action at plasma concentrations that do not affect airway smooth muscle tone (reviewed in Spina 2003). Several lines of investigation have lead to this conclusion. In essentially all studies, theophylline protects against the LPR following allergen provocation implying that the emigration of proinflammatory and immunocompetent cells from the circulation in to the lung and/or their subsequent activation is suppressed. Indeed, suppression of the LPR at nonbronchodilator doses (< 10 mg/L) of theophylline is associated with a reduction in the typical increase in CD4+ and CD8+ T lymphocytes. Theophylline also suppresses the activity of other proinflammatory and immune cells ex vivo including neutrophils and macrophages that is positively correlated with the concentration of theophylline measured in the BAL fluid. Similar experiments have demonstrated that the number of activated eosinophils and CD4+ T cells are reduced in allergic subjects given low dose theophylline and that eosinophil accumulation in bronchial tissue in patients with atopic asthma is also reduced. All these changes are mirrored by improvements in lung function.

PDE4 inhibitors PDE4 inhibitors have been shown to be effective in patients with exercise-induced asthma. Nieman et al. (1998) have reported the results of a randomized, placebo-controlled, double blind crossover trial with cilomilast in 27 patients with exercise-induced asthma. Subjects were randomized to receive cilomilast or placebo for 7 days followed by a 7 day washout and then the alternative treatment for 7 days. The primary efficacy variable was the maximum percentage decrease (MPD) in forced expiratory volume in 1 s (FEV1) in response to exercise. In the placebo group the mean fall in FEV1 after exercise was 32.9%, which was significantly greater than the deterioration in lung function seen when the same subjects that received a single dose of cilomilast (23.6% reduction in FEV1). The improvement in lung function was incremental such that after 7 days of therapy the MPD in FEV1 was further reduced to 21.8% (Nieman et al. 1998). There were also improvements in the MPD in peak expiratory flow rate (PEFR), time to recovery after exercise, and percent protection against exercise-induced bronchoconstriction (Nieman et al. 1998). Similar data have been obtained with roflumilast. Sixteen patients with exercise-induced asthma were recruited in to a placebo-controlled, randomized, double-blind, two-period crossover study where placebo or roflumilast (500 μg o.d.) was administered in random order for 28 days (Timmer et al. 2002). FEV1 was measured before and repeatedly up to 12 min after the end of the exercise challenge. Blood was taken for determination of LPS-induced TNF-α released ex vivo as a surrogate marker of inflammatory cell activation. At the end of the study the mean fall in FEV1 in response to exercise was significantly reduced (by 41%) when compared to placebo (Timmer et al. 2002). Similarly, the median TNF-α concentration was reduced by 21% during roflumilast treatment whereas it remained constant under placebo (Timmer et al. 2002), indicating that an antiinflammatory plasma concentration of the drug had been achieved. Other trials of cilomilast in asthma have yielded equivocal results. In one of these, the results of a multicenter, placebocontrolled, double-blind, randomized parallel group study with cilomilast (5, 10 and 15 mg b.i.d. for 6 weeks) involving 283 patients taking inhaled corticosteroids concurrently was reported (Compton et al. 1999). All patients had an FEV1 of approximately 66% of predicted, expressed a 12% or greater responsiveness to salbutamol and had asthma that was inadequately controlled with inhaled corticosteroids (Compton et al. 1999). Two hundred and sixty six patients completed the study. At the highest tolerated dose (15 mg b.i.d.) cilomilast as well as placebo increased FEV1 from week 1 onwards and this effect was greater with the active treatment group (Compton et al. 1999). However, the improvement in lung function failed to reach statistical significance at any time except at week two when the mean difference in trough FEV1 was 210 mL greater than in the placebo treatment group

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(Compton et al. 1999). Improvements, relative to placebo, in forced expiratory flow at 25–75% of forced vital capacity (FEF25 –75) and domiciliary PEFR were also detected but, again, statistical significance was not achieved. However, in the physicians’ global assessment 59% of patients taking cilomilast (15 mg b.i.d.) were rated as “markedly improved” compared to 39% of patients given placebo, which was significant. Similarly, in the patients’ global assessment 69% of patients in the active treatment group (15 mg b.i.d.) indicated that they were “markedly improved” compared to 41% of patients that received placebo (Compton et al. 1999). An international (Germany, UK, France and South Africa), multicenter phase IIb, double-blind, parallel group 12 month efficacy, safety and tolerability study of cilomilast (10 and 15 mg b.i.d.) has also been evaluated in 211 asthmatic patients between 19 and 70 years of age, which was an extension of three double-blind randomized phase II studies of 4–6 weeks’ duration (Compton et al. 2000a,b). One hundred and fifty eight patients received cilomilast and the remainder were given placebo. Inclusion criteria included a history of episodic wheezing for at least 6 months, an FEV1 ≥ 45% and ≤ 90% of predicted for height, sex and weight, and responsiveness to salbutamol (≥ 12%) at time of screening. Clinically relevant and statistically significant improvements above placebo were seen in forced vital capacity (FVC) and PEFR, which were sustained from week one to the end of month 12. A consistent improvement in FEV1 was also noted but this did not reach statistical significance. Diary asthma symptom scores also indicated a reduction in cough, wheeze, and breathlessness/chest tightness in the active treatment group when compared to placebo (Compton et al. 2000a). More encouraging data have been obtained with roflumilast. In particular, a trial in 23 subjects with mild asthma showed that roflumilast (250 or 500 μg od) given for 7–10 days reduced the early and late asthmatic reactions evoked by allergen challenge (van Schalkwyk et al. 2005). These are potentially exciting data as the suppression of LPR (43% in the 500 μg treatment group) indicates that the drug may have exerted a clinically relevant antiinflammatory effect. Indeed, in many clinical trials of oral PDE4 inhibitors, a maximum level of lung function improvement is achieved within the first 2–4 weeks of treatment implicating a nonbronchodilator mode of action. Further studies have also found that roflumilast improved FEV1 in subjects with mild to moderate asthma (Bateman et al. 2006) and also reduced allergen-induced AHR to histamine (Louw et al. 2006). One study has also found that roflumilast is as effective as an inhaled corticosteroid at improving lung function (Bousquet et al. 2006). In this study 499 asthmatic patients were randomized to receive either roflumilast (500 μg od) or beclomethasone dipropionate (200 μg b.i.d.) through a metered-dose inhaler for 12 weeks. At the end of the study, both treatment groups had significant and comparable improvements in FEV1, FVC and PEFR; asthma symptoms and β2-agonist use were also reduced.

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Finally, roflumilast has been shown to be effective in the treatment of allergic rhinitis (Schmidt et al. 2001), which supports the idea that PDE4 inhibitors may have utility in treating a variety of allergic (and nonallergic) inflammatory disorders.

Theophylline and PDE4 inhibitors: a comparison of their safety, tolerability, drug metabolism and pharmacokinetics Table 27.6 shows a comparison of the drug metabolism, pharmacokinetics (DMPK) and clinical safety of theophylline and PDE4 inhibitors for which information is available. Perhaps the most striking difference is in the pharmacokinetics, which has implications for patient compliance and the extent to which the plasma concentration requires monitoring. At bronchodilator doses, intra- and inter-subject variability to theophylline together with a low therapeutic ratio poses a significant clinical problem requiring careful titration with routine plasma monitoring to avoid serious cardiac and CNS side effects (Vignola 2004). This is a particular problem with smokers as polycyclic aromatic hydrocarbons present in vapor phase of cigarette smoke are known to induce drug-metabolizing enzymes including CYP1A1 and CYP1A2 (Campbell et al. 1987; Shimada et al. 1989; Vistisen et al. 1992). As theophylline is principally metabolized by CYP1A2 (Fuhr et al. 1992; Sarkar et al. 1992), dose adjustments are often necessary to compensate for the increased clearance in cigarette smokers (Hunt et al. 1976; Jusko et al. 1978; Jusko 1979). Age is another factor that has a marked effect on the pharmacokinetics of theophylline. Indeed, the clearance of theophylline decreases 15–28% in the elderly when compared to young adults, which probably reflects a decrease in the elimination of theophylline by CYP1A2 (Antal et al. 1981; Shin et al. 1988; Ohnishi et al. 2003). In contrast, the pharmacokinetics of cilomilast and roflumilast are linear providing dose-proportional systemic exposure that is essentially unaffected by age and cigarette smoking status indicating that no dose adjustments will be necessary in elderly smoking subjects with COPD (see Giembycz 2001, 2002b; Vignola 2004). Despite producing bronchodilatation, theophylline is prone to cause adverse events. These are particularly pronounced when dosed to give plasma concentrations of 20 mg/L or greater, although these unwanted actions can be offset by gradually titrating the dose of theophylline until therapeutic levels are achieved (Barnes 2003). In contrast, a major benefit of the PDE4 inhibitors in clinical trials is their superior safety and tolerability profile over theophylline. Although nausea and vomiting are not uncommon with PDE4 inhibitors, this is usually of moderate severity and is reported to be selflimiting (Compton et al. 2001). Moreover, PDE4 inhibitors are generally well tolerated in both short- and long-term dosing trials with a low incidence of adverse events; they have no action at adenosine receptors and, with the exception

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Table 27.6 Comparison of the mechanism of action, DMPK, safety and tolerability of theophylline and PDE4 inhibitors. Theophylline

PDE4 inhibitors*

Unclear (inhibition of PI3K-d; histone acetylation status) PDE inhibition only at high (> 20 mg/L) doses

Selective inhibition of PDE4

Nonlinear Significant intersubject variability affected by age, smoking status, and concomitant medication necessitating plasma monitoring

Linear providing dose proportional systemic exposure Low intersubject variability: no plasma monitoring required

Absorption

Variable, depends on formulation

Oral formulations (o.d. or b.i.d) tmax ∼ 1–2 hours

Bioavailability

Variable, depends on formulation

> 80%, unaffected by food or antacids

Half-life

7–9 hours

7–16 hours depending on inhibitor

Volume of distribution

500 mL/kg Plasma protein binding ∼ 56%

Low High

Clearance

∼ 400 mL/kg per hour Affected by genetic factors, cigarette smoking, coexisting pathology, and drugs that affect hepatic metabolism

Low

Metabolism

∼ 90% metabolized by liver (CYP1A2)

Negligible first-pass hepatic metabolism

Drug interactions

High potential for drug interactions including propafenone, mexiletine, enoxacin, ciprofloxacin, cimetidine, propranolol, oral contraceptives, erythromycin, rifampicin, phenytoin, carbamazepine, phenobarbital, isoproterenol, tobacco smoke

Low potential for drug interactions Can be taken with other drugs prescribed for asthma and COPD

Excretion

10% excreted unchanged via the kidneys

Depends on inhibitor

Dosing adjustment

May be required in cigarette smokers, the elderly, individuals with liver disease, and subjects taking concomitant medication Contraindicated in individuals with heart disease, seizure disorders, and gastroesophageal reflux

None except in individuals with moderate hepatic and severe renal impairment Contraindicated in subjects with severe hepatic impairment

Clinical efficacy

Effective in a subset of patients with COPD Nonbronchodilator doses effective in asthma; steroid sparing

Phase III clinical trials ongoing

Safety and tolerability

Serious cardiovascular and CNS side effects Gastrointestinal irritation, nausea, insomnia in 10–15% of patients

No cardiovascular or CNS side effects Headache, nausea, vomiting, arteriopathy(?)

Mechanism of action

DMPK Pharmacokinetics

* Details refer to cilomilast and roflumilast, which are in late Phase III clinical trials. COPD, chronic obstructive pulmonary disease; PI3K, phosphatidylinositol 3-kinase.

of headache, are devoid of adverse cardiovascular activity (see Giembycz 2001, 2002b). Nevertheless, despite these improvements over theophylline, there are still major safety concerns with current second generations PDE4 inhibitors at the doses believed necessary to impart therapeutic benefit (see Adverse events, below). Another significant clinical problem is that theophylline has a high potential for drug interactions (see Giembycz 2001, 2002b). Thus, in addition, to CYP1A2, theophylline is

also metabolized, albeit to a lesser extent, by CYP2E1 and CYP3A4. Accordingly, many drug interactions may occur including all of those indicated in Table 27.6. In contrast, PDE4 inhibitors have in general a far reduced propensity for drug interactions. With respect to cilomilast, none of the metabolic pathways involve, to any great extent, cytochrome P450 enzymes (CYP1A2, CYP2D6, CYP3A4) most susceptible to competitive inhibition by other drugs. Indeed, the only P450 enzyme implicated (CYP2C8), has few other substrates

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or inhibitors. Moreover, cilomilast does not inhibit any important hepatic cytochrome P450 enzymes in vitro. These data are supported by the finding that, at steady state, cilomilast has no clinically meaningful effect on the pharmacokinetics of digoxin, theophylline or prednisolone. Conversely, neither theophylline nor Maalox Plus, an antacid commonly used in the elderly that contains salts of calcium, magnesium and/or aluminum that can alter the absorption or bioavailability of some drugs, had any significantly influence on the pharmacokinetics of cilomilast. Similar results have been reported for roflumilast. Thus, taken together these data demonstrate that the two most clinically advanced PDE4 inhibitors are not contraindicated with commonly prescribed medications for asthma and can be safely coadministered with these drugs.

Adverse events of theophylline and PDE inhibitors Theophylline Despite demonstrable therapeutic benefit in asthma, theophylline has a low therapeutic ratio. This undesirable property is a major cause for concern as adverse events tend to occur when the plasma concentration exceeds 20 μg/mL (110 μmol/L). The most common adverse events include nausea, vomiting, gastrointestinal irritation and headache (probably due to PDE4 inhibition, see below). Other, more serious side effects such as CNS stimulation, diuresis and cardiac arrhythmias occur at higher doses and may be due to the ability of theophylline at act as an adenosine A1 receptor antagonist (Fredholm 1980; Barnes 2003). Higher plasma concentrations still may precipitate convulsions and even death (Barnes 2003).

PDE4 inhibitors Despite some encouraging data from Phase III efficacy studies in asthma and the superior DMPK over theophylline, roflumilast and other PDE4 inhibitors are still hampered by a low therapeutic ratio. This limitation became clear early on in the development of these compounds with nausea, diarrhea, abdominal pain, vomiting and dyspepsia being the most common adverse events reported (reviewed in Giembycz 2001, 2002b) (see Fig. 27.2). For example, the number of subjects failing to complete all controlled trials of cilomilast conducted by GSK due to an adverse event was positively dose-related with gastrointestinal disturbances being the most prevalent (GlaxoSmithKline 2003a). Unfortunately, these unwanted actions, which are mediated both locally (i.e., in the gastrointestinal tract) and centrally, can be accounted for by the ubiquitous distribution of PDE4 isoforms across many tissues, and represent an extension of the pharmacology of PDE4 inhibitors that is typical of first generation compounds such as rolipram.

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Documentation of serious toxicities resulting from the administration of PDE4 inhibitors is relatively sparse (cf. PDE3 inhibitors: Hanton et al. 1995; Zhang et al. 2002). However, the most worrying potential toxicity generic to PDE4 inhibitors is arteritis. This condition is characterized by inflammation, hemorrhage and necrosis of blood vessels, and is believed to be irreversible in animals. Mechanistically, arteritis is thought to result from hemodynamic changes produced by excessive and prolonged vasodilatation of specific vascular beds, although the means by which PDE4 inhibitors cause certain vessels to become targets of inflammation is unknown. In nonhuman primates, studies with PDE4 inhibitors generally have not identified pathologies, including arteritis, similar to those reported in other species used for toxicology (Larson et al. 1996; Robertson et al. 2001), and this has lead to a view that arteriopathies maybe nonprimate-specific. Indeed, rats and dogs may have an increased susceptibility to drug-induced vascular lesions because of the common occurrence of arteriopathies in these species (Bishop 1989; Ruben et al. 1989). Consistent with this hypothesis, cilomilast is reported not to produce vascular lesions in primates unlike comparable studies performed in rodents where medial necrosis of mesenteric arteries is reproducibly precipitated (GlaxoSmithKline 2003c). However, a recent comprehensive toxicologic study found that a PDE4 inhibitor, SCH 351591, produced, in cynomolgus monkeys, acute to chronic inflammation of small to medium sized arteries in many tissues and organs (Losco et al. 2004). These findings of arteriopathy in primates, which were previously thought to be resistant to toxicity, have serious implications for human risk, and it is noteworthy that Merck in 2003 abandoned development of their lead PDE4 inhibitor (licensed from Celltech Group) due to an incidence of colitis, raising the possibility that it was secondary to arteritis (Data Monitor 2003). Moreover, as asthma is a chronic disease requiring, in many subjects with moderate to severe disease, long-term therapy, a wide margin of safety will be needed because toxicity cannot be adequately monitored. The major problem for the physician is that presentation of mesenteric ischemia is vague in humans and diagnostic tools are poor. Indeed, attempts by the pharmaceutical industry to develop biomarkers of arteritis to assist the development of PDE4 inhibitors have, to date, been unsuccessful. However, perhaps some comfort can be derived from the knowledge that no clinically relevant effects have been produced in patients treated for many years with bronchodilator doses of theophylline (which produce medial necrosis of mesenteric vessels in rats) (Collins et al. 1988; Nyska et al. 1998) as well as more selective PDE4 inhibitors including rolipram and denbufylline (GlaxoSmithKline 2003c). Other adverse events generic to PDE4 inhibitors and of potential concern based on margin of safety calculations are testicular toxicity, manifested as degeneration of the epithelium lining the seminiferous tubules, hypertrophy and

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hyperplasia of the adrenal cortex, focal myocardial necrosis, erosion of the gastrointestinal mucosa and squamous cell hyperplasia of the nonglandular stomach, which is indicative of an irritant action of cilomilast on the gut (GlaxoSmithKline 2003a–c). However, it is currently believed that these findings do not have clinical relevance. Indeed, there is no evidence from the Phase II or Phase III trials conducted to date that cilomilast at a dose 15 mg (b.i.d.) produces these adverse effects in humans.

PDE3 inhibitors Although the clinical rationale is clear for developing hybrid PDE3/PDE4 inhibitors, there are also major safety concerns with this approach, not least the potential of these compounds to cause arteriopathies. There is an extensive literature on PDE3 inhibitor-induced arteriopathy in laboratory animals with the splanchnic vessels and coronary arteries of rats and dogs respectively being the most susceptible to lesions (Joseph 2000). Whether clinically meaningful bronchodilatation can ultimately be achieved in humans with an acceptable degree of vasodilatation is largely unexplored but coincident headache is not uncommon with current PDE3 or PDE3/PDE4 inhibitors at bronchodilator doses (Wilsmhurst & Webb-Peploe 1983; Yamashita et al. 1990; Brunnee et al. 1992; Fujimura et al. 1995). Conceivably, airway-selective compounds could be realized through formulation, route of administration or by the exploiting the fact that PDE3 and PDE4 inhibitors may interact synergistically to reduce human airways smooth muscle tone (Torphy et al. 1993a). Selective targeting of either PDE3A or PDE3B could also offer a more novel approach to reduce cardiovascular toxicity. Indeed, when PDE3 heterogeneity was first appreciated, the terms cardiac-type and adipocyte-type PDE3 were used to describe PDE3A and PDE3B respectively. This crude taxonomy is based on the finding that PDE3A is expressed primarily in cardiac and vascular myocytes whereas adipocytes, among other cells, are rich in PDE3B. While differential expression of PDE3 gene expression might lead one to hope that the cardiovascular and bronchodilator actions of PDE3 inhibitors can be separated pharmacologically, human cardiac myocytes and airways smooth muscle cells express mRNA for both isoforms. This raises the question of whether PDE3A and PDE3B mediate nonoverlapping functions in the same tissue. While this issue is unexplored, studies in PDE4A-, PDE4B- and PDE4D-deficient mice have found there to be no functional redundancy suggesting that PDE3A and PDE3B likely mediated discrete responses. Thus, it is possible that the cardiovascular actions of PDE3 inhibitors can be dissociated from their bronchodilator and antiinflammatory activities. Fortuitously, subtype selective compounds are beginning to emerge such as vinylogous amide pyrazolones (17 in Fig. 27.8), which preferentially target PDE3B, that could be employed to assess this possibility (Edmondson et al. 2003).

PDE7 inhibition Although very little is known of the adverse effects that may be produced by inhibiting PDE7 in nontarget tissues, this enzyme family is expressed widely throughout the CNS and could therefore compromise the beneficial activity of selective inhibitors that are able to penetrate the blood–brain barrier (Hoffmann et al. 1998a; Miro et al. 2001; Perez-Torres et al. 2003). In addition, compounds that inhibit both PDE7A and PDE7B, which probably include all of those thus far described, may be of particular concern as high levels of PDE7B mRNA are expressed in the area postrema (and adjacent nucleus tractus solitarius) of the rat brain (Reyes-Irisarri et al. 2005), which lies outside the blood–brain barrier (Miro et al. 2001). If the same holds true in the human brain, then additional caution is warranted as these structures are intimately involved in regulating emesis (Miller & Leslie 1994; Miller 1999) and are targets for the vomiting-inducing effects of PDE4 inhibitors (Robichaud et al. 1999). Thus, PDE7 inhibitors that do not discriminate between the two gene families may evoke the same or similar adverse events that have plagued the development of PDE4 inhibitors. This may be a particular problem with dual PDE4/PDE7 inhibitors if simultaneous inhibition of these enzymes in the area postrema results in a synergistic activation of those mechanisms that promote vomiting.

Concluding remarks Many of the major pharmaceutical companies in the world have developed potent and novel “second generation” PDE4 inhibitors for the treatment of a number of allergic and nonallergic inflammatory disorders (see Table 27.2). There is abundant predictive evidence from preclinical studies that these new PDE4 inhibitors should demonstrate global antiinflammatory activity with an improved and acceptable therapeutic ratio over so-called first-generation compounds (Souness & Rao 1997; Torphy 1998; Torphy et al. 1999; Giembycz 2001). That said, one must be cautious of this interpretation since only the acute effects of PDE4 inhibitors in animals have been demonstrated and whether they translate into a useful therapy in chronic human allergic diseases remains to be determined. Indeed, despite initial optimism, the disappointing results of a number of Phase III studies indicate that dose-limiting adverse events are a major cause for concern and probably reflect an interaction of the compound with PDE4 expressed in “nontarget” tissues. An additional contributing factor may lie in the knowledge that all of the PDE4 inhibitors currently in development will inhibit PDE4D, which may promote emesis (Giembycz 2002a; Robichaud et al. 2002) but not suppress inflammation (Hansen et al. 2000). Nevertheless, by 2010 it is likely that results from clinical trials will reveal whether the optimism in the therapeutic potential of this class of compounds was justified.

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Adrenergic Agonists and Antagonists Tony R. Bai

Summary Adrenergic agonists and antagonists act as ligands at either α or β adrenoceptor subtypes to mimic or antagonize the action of circulating epinephrine or neuronally released norepinephrine. Most cells possess adrenoceptors of varying subtype; the effects of ligand binding can be diametrically opposite in different cell systems, specificity being determined by the second messenger pathway coupled to the adrenoceptor. Adrenergic agonists are usually administered by inhalation or parenterally, and antagonists typically orally. In the field of allergy and immunology, the key benefit of α-adrenoceptor ligands is vasoconstriction induced by the agonism of medications such as epinephrine, pseudoephedrine (acting on both α1 and α2 adrenoceptors) or xylometazoline (α1 selective). The primary role of β-adrenoceptor ligands is the β2-agonist action of agents such as isoprenaline or salbutamol, causing smooth muscle relaxation, and hence inducing bronchodilation, although β2 agonist-mediated inhibition of mast cell mediator release is one mechanism of action of epinephrine in reducing symptoms of anaphylaxis such as itch and edema. A similar mechanism is involved in the inhibition of the early airway response to inhaled allergen. The β2-selective agonists are preferred as bronchodilators. Both short-acting (1–4 hours) and long-acting β2-selective bronchodilators (8–12 hours) are widely available. These medications vary in both adrenoceptor selectively, rapidity of action, and efficacy (maximal response). They should generally not be used in asthma more than two to three times per week without concomitant antiinflammatory therapy. β2-Adrenoceptor desensitization (i.e., waning of the response in the face of continuous or repeated agonist exposure) can occur rapidly; the receptor becoming uncoupled from its stimulatory G protein that usually is responsible for initiating downstream intracellular effects. The β2 adrenoceptor can also be internalized (downregulation) as a response to prolonged agonist stimulation. Uncoupling can be enhanced by proinflammatory mediators and downregulation reversed to some extent by corticosteroids.


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The use of β-adrenoceptor antagonists predictably increases the severity of anaphylaxis, and nonselective β-adrenoceptor antagonists are contraindicated in asthma but generally tolerated in chronic obstructive pulmonary disease. Relatively selective β1 antagonists such as bisoprolol may be tolerated in some asthmatics as long as asthma is controlled as assessed by standard measures such as normal exhaled nitric oxide fractional concentrations and lack of induced sputum esosinophila.

Introduction Ligands acting at adrenergic receptors are among the most commonly used medications in clinical immunology and allergy. Long before Langley (1905) and Dale (1906) developed the concept that the specific biological effects of hormones, neurotransmitters and drugs result from high-affinity stereospecific interactions with tissues, the English physician Henry Salter reported in 1859 what is probably the first account in modern times of the therapeutic effects of activation of adrenergic receptors when he wrote that “asthma is immediately cured in situations of either sudden alarm or violent fleeting excitements.” Endogenous levels of circulating catecholamines, particularly epinephrine (adrenaline), influence airway caliber in asthmatic patients, and it is likely that Salter was describing sympathoadrenal release of epinephrine following emotional triggers. Around 1900, the vasodilator hypothesis of asthma had considerable support in both Germany and the USA. This hypothesis stated that airway obstruction was caused by swelling of the bronchial mucosa secondary to vasodilatation. The other major hypothesis at that time was that asthma was due to “the spasm of the circular muscles of the bronchi.” Thus, in 1900, Solis-Cohen, encouraged by reports that adrenal extracts caused vasoconstriction, gave large oral doses of desiccated adrenal glands to asthmatic subjects with success, which he interpreted to be consistent with his view that asthma was a “vasomotor ataxia of the relaxing variety” (Solis-Cohen, 1990). However, it is unlikely the epinephrine

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content of the adrenal could have survived the oral route as an active drug and, indeed, the slow onset of action of the extract treatment reported in his paper is now thought to be more likely the demonstration of the beneficial effects of glucocorticosteroids (Persson 1989). Soon after this, epinephrine became available as a pure substance and in 1903 Bullowa and Kaplan successfully gave injections of it to asthmatic patients. They too thought this success was consistent with the vascular hypothesis of asthma but in 1907 epinephrine was shown to relax airway smooth muscle (Kahn 1907). Although it is possible in some asthmatics that the α-adrenoceptor agonist (α-agonist) effect of epinephrine contributes to increased airway caliber, it is probable that the β-adrenoceptor agonist (β-agonist) effect dominates (see later). In 1924, ephedrine was introduced to Western medicine, although the plant from which it is derived has been used for more than 5000 years in China for respiratory and other allergic conditions. Ephedrine, an α agonist with a weak β-agonist activity, and epinephrine were widely used over the ensuing decades in the treatment of asthma, rhinitis and anaphylaxis. Konzett (1941) isolated isoproterenol (isoprenaline), the first β agonist devoid of α-adrenergic effect. Subsequently, Ahlquist (1948) used isoproterenol to partition sympathomimetic effects into α (mainly excitatory) and β (mainly inhibitory) based on physiologic responses in isolated tissues.

Adrenergic Agonists and Antagonists

Table 28.1 Tissue distribution of adrenoceptor subtypes. Airways Lung parenchyma Nose Heart Blood vessels Inflammatory cells Gut, kidney, liver, pancreas, spleen Uterus Adipose tissue Noradrenergic and cholinergic nerve terminals Brain

a1, b2 b1 a1, a2, b2 a1, a2, b1, b2 a1, b2 a2, b2 a1, a2, b2 b2 b3 a2, b2 a1, a2, b1, b2

erally, but not invariably, β1 responses appear to be initiated by the neurotransmitter norepinephrine in innervated tissues, whereas β2 responses are triggered by the circulating hormone epinephrine (O’Donnell 1991). Subsequently, a third subtype of β adrenocepter, β3, was defined (Emorine et al. 1989). The β adrenoceptors are, in general, a low abundance receptor (500–5000/cell), although on airway smooth muscle they number 30 000–40 000/cell. Tissue distributions of both α and β adrenoceptors are summarized in Table 28.1. Heart In the ventricle 40% and in the atrium up to 55% of β adrenoceptors are of the β2 subtype (Bristow & Ginsburg 1986). The remainder are β1 subtype.

Adrenoceptor localization The mammalian G protein-coupled receptors (GPCRs) can be divided in three main classes according to sequence homology: class I or rhodopsin-like (which is the largest subfamily), class II or secretin-like, and class III or glutamatemetabotropic-like. Within class I, the adrenoceptors mediate the functional effects of epinephrine and norepinephrine. The adrenoceptor family includes nine different gene products: three β (β1, β2, β3), three α2 (α2A, α2B, α2C) and three α1 (α1A, α1B, α1D) receptor subtypes. The receptor population mediating α effects is characterized by a rank order of potency of epinephrine > norepinephrine > isoproterenol and β effects isoproterenol > epinephrine > norepinephrine (Table 28.1).

The b adrenoceptors Evaluation of a large volume of data generated in the study of β-adrenergic pharmacology enabled Lands et al. (1967) to suggest a further division of the β-adrenoceptor response into subtypes termed β1 and β2. Again this distinction was based on the relative potency of the naturally occurring catecholamines, epinephrine and norepinephrine. The β1 responses are equally sensitive to these two agonists; β2 responses are more potently stimulated by epinephrine. Gen-

Vasculature β2 Adrenoceptors predominate on vascular smooth muscle. Nose A homogeneous population of β2 adrenoceptors has been noted in several studies (van Megen et al. 1991). Lung Organ bath and autoradiographic studies (Fig. 28.1) have demonstrated that the airway smooth muscle relaxant effect of β agonists is largely via β2 adrenoceptors directly on the muscle surface (Nadel & Barnes 1984; Carstairs et al. 1985; Bai et al. 1992). This is not unexpected, given that β1 adrenoceptors are found at sites of sympathetic innervation responding to norepinephrine release and there is no direct sympathetic innervation of human airway smooth muscle (Daniel et al. 1986). Similarly, the adrenoceptors on mucous and serous glands and inflammatory cells are largely of the β2 type (Basbaum et al. 1990). β2 Adrenoceptors also predominate on bronchial epithelium, type I and II pneumocytes, and pulmonary vascular smooth muscle so that they make up 70% of the β adrenoceptors in the human lung, the other 30% being β1 on alveolar walls. The density of β2 adrenoceptors increases from the large to small airways and is much greater

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(a)

(b)

(c)

(d) Fig. 28.1 Distribution of b adrenoceptors in human normal and asthmatic bronchi. (a, b) Dark-field photomicrographs of sections showing the distribution of autoradiographic grains after incubation with 25 pmol/L 125I-iodocyanopindolol. (c, d) Bright-field photomicrographs of adjacent sections showing the epithelium (Ep), smooth muscle (SM) and submucosal glands (G) after staining with 1% cresyl fast violet. The epithelium is partially shed in the asthmatic sections. (From Bai et al. 1992, with permission.)

on alveolar walls than other structures in the lung (Carstairs et al. 1985). Other sites Functional and gene expression studies suggest adipocytes contain β1 and β3 adrenoceptors.

of α1 and α2 subtypes are summarized in Table 28.2. In the nasal vasculature, both α1 and α2 adrenoceptors are present (Lacroix, 1989; van Megen et al. 1991). In the nasal capacitance vessels, α2 adrenoceptors dominate over α1 adrenoceptors.

Adrenoceptor biology The a adrenoceptors At least seven α adrenoceptor subtypes (α1A–D and α2A–C) were initially proposed based on pharmacologic experiments and gene cloning. However, as α1A and α1C subtypes have very similar pharmacologic properties (Hieble & Bond 1994), only six subtypes are now accepted (Cotecchia 2007). The α1 subtypes are located on smooth muscle membranes of most sympathetically innervated tissues, mediating contraction. α1 Adrenoceptors are found on arterioles in skin, mucosa, viscera, and resistance vessels in the kidney as well as in all veins. The α2 adrenoceptors are primarily presynaptic, inhibiting traffic through autonomic ganglia and other nerve terminals. The localization and pharmacologic properties

670

Molecular structure Prior to 1974, the adrenergic receptors were known only indirectly as entities that responded to drugs in a selective manner to mediate a variety of physiologically important responses. Then a variety of high-affinity 125I-labeled radioligands selective for these receptors were developed that led to experiments utilizing direct binding assays to establish the biochemical properties of the receptor proteins. These techniques, when coupled with efficient methods for detergent solubilization, formed the basis of receptor purification using affinity chromatography, and when coupled with autoradiographic methods lead to the cellular localization and quanti-

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b2AR agonist

Table 28.2 a2 Receptor subtypes.

b2AR

Subtype

Localization

Pharmacologic properties

a1A

Brain, vas deferens, kidney, heart, spleen

Norepinephrine > epinephrine > phenylephrine

a1B

Lung, brain, heart, liver, kidney, spleen

Oxymetazoline > epinephrine > norepinephrine

a1D

Vascular smooth muscle

Norepinephrine > epinephrine > phenylephrine

a2A

Brain and platelets

p-Aminoclonidine > epinephrine

a2B

Kidney, neonatal lung

Clonidine > norephinephrine > oxymetazoline

a2C

Kidney

Oxymetazoline > norepinephrine

fication of adrenergic receptors on thin sections of tissues (Stadel & Lefkowitz 1991). The availability of substantial amounts of purified β2 adrenoceptor allowed determination of the molecular mass and amino acid sequence of part of the receptor. This was the first adrenergic receptor isolated. This new information in turn led to the production of polynucleotide probes and eventually to cloning of the receptor gene and determination of the complete primary sequence of the receptor protein (Strader et al. 1989; Fraser & Venter 1990). The β2 adrenoceptor gene maps to chromosome 5 and encodes a protein of 413 amino acids, only 54% of which are shared with β1 adrenoceptors. α Adrenoceptors were cloned using similar techniques and show many homologies to β adrenoceptors (Fraser & Venter 1990). Current models of the adrenoceptors show seven transmembrane segments connected by alternating intracellular and extracellular loops (Fig. 28.2). Homology among all the members of the seven transmembrane region (serpentine) receptor family is greatest in the transmembrane-spanning domains. Genetic and biochemical manipulation of the β2 adrenoceptor has identified that the ligand-binding domain is a pocket buried within the membrane bilayer, with agonists interacting with amino acid residues within transmembrane helices III, V and VI (Johnson 2006). Four residues are of critical importance, namely the aspartate residue 113, positioned on the third domain, two serine residues, 204 and 207, on the fifth domain, and asparagine 293 in the sixth domain. Aspartate binds to the nitrogen of the β2-agonist molecule while the two serine residues interact with the hydroxyl groups on the phenyl rings. Antagonists do not bind with the same amino acids. Thus, β antagonists bind to aspartate 113 and a residue in the seventh domain rather than with serine residues in the fifth domain (Tota et al. 1991; Liggett 2002). Most β2 adrenoceptors exist as homodimers for at least part of the time, homodimers being essential for transport to the cell surface from the endoplasmic reticulum (Salahpour et al.

G AC

ATP

cAMP

Bronchodilatation Fig. 28.2 Simplified model of b2-adrenoceptor (b2-AR) signaling. Following agonist binding the Gs protein couples with, and stimulates, adenylate cyclase (AC) to catalyze the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). (See CD-ROM for color version.)

2004), but the importance of cell-surface dimerization in determining high-affinity binding remains controversial. The receptors do oscillate between an activated and inactivated state, and are at equilibrium under resting conditions (Liggett 2002). In the past decade it has become apparent that some ligands act as “inverse agonists,” decreasing the number of spontaneously active receptors (Bond & Ijzerman 2006). Studies of the regulation of adrenergic receptor gene transcription are incomplete. In both cell culture and homogenized human lung (Mak et al. 1995) as well as in nasal mucosa in vivo (Baraniuk et al. 1997), glucocorticoids increase β2adrenoceptor mRNA levels and receptor protein by increasing the rate of gene transcription and isoproterenol decreases mRNA levels by decreasing stability of the mature mRNA. Hamid et al. (1991) have reported the distribution of β2adrenoceptor mRNA in human lung by in situ hybridization and correlated this with receptor autoradiographic distribution. They report qualitative differences between the densities of labeling with the two techniques in different cell types. Pulmonary vascular and airway smooth muscle showed a high intensity of mRNA hybridization but only a low density of adrenoceptors and the converse was reported for the alveolar epithelium. These differences may be due to either a rapid rate of β2-adrenoceptor synthesis or high stability of mRNA in the airways and may explain contribute to the difficulty in demonstrating desensitization in airway smooth muscle (see later). G proteins are membrane-associated heterotrimers composed of α, β and γ subunits. Interaction with a receptor causes the release of GDP from the α subunit of the G protein, allowing GTP to bind and leading to the dissociation of the activated α subunit from the receptor and from the βγ complex (Fig. 28.2). Various G proteins activate or inhibit different effector enzymes, modulating the levels of intracellular second messengers. In the case of the β2 adrenoceptor, which

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is coupled to the stimulatory guanine-nucleotide binding protein, Gs, binding of an agonist to the receptor catalyzes the release of GDP from the α subunit of the G protein (αs), allowing the binding of GTP; this in turn leads to the direct activation of adenylate cyclase by αs-GTP. Adenylate cyclase catalyses the formation of the classical second messenger cyclic AMP so that levels of cAMP up to 400-fold over basal can occur within minutes of agonist exposure (Johnson, 2006). Upon removal of agonist, the activation of adenylate cyclase persists until the intrinsic GTPase activity of αs hydrolyses the bound nucleotide. Sequencing of ADRB2 has identified at least 58 single nucleotide polymorphisms (SNPs) within the coding and promoter region (Weiss et al. 2006). To date, the most frequently studied ADRB2 coding SNPs are characterized by substitutions of glycine for arginine at position 16 (Arg16Gly) and glutamine to glutamic acid at position 27 (Gln27Glu), both of which occur commonly in the population (minor allele frequency approximately 0.4– 0.5 in whites, with some differences in allelic frequency in blacks).

Mechanisms of adrenoceptor agonist action The contractile state of smooth muscle, the primary therapeutic target of most adrenergic agonists, is determined by cytoplasmic calcium concentrations. The cell membrane is negative with respect to the extracellular space and more positive potentials (depolarization) opens voltage-gated calcium channels, causing calcium influx to increase cytoplasmic calcium and trigger contraction. The effect of adrenergic agonists on smooth muscle are complex and vary from one site to the next. For example, α agonists relax intestinal smooth muscle and contract vascular smooth muscle by different and now well understood mechanisms involving coupling of G proteins with different second messenger pathways. Thus, the contractile response to α1 agonists is determined by activation of G protein l-nked adrenoceptors that activate phospholipase C, releasing inositol 1,4,5-trisphosphate (IP3) and increasing intracellular calcium (Hague et al. 2006). α2 Agonists inhibit adenylate cyclase activity and decrease cAMP levels. A number of other mechanisms, apart from increases in cyclic AMP, have been shown to be involved in the smooth muscle relaxation induced by β agonists (Table 28.3). Relaxation is primarily determined by generation of cAMP and activation of cAMP-dependent kinases which have several actions including shifting myosin light chain kinase to a less active form. The rise in cAMP also leads to calcium reuptake into the sarcoplasmic reticulum and organelles and calcium extrusion from the cell. cAMP also causes suppression of IP3 formation. β Agonists also reduce acetylcholine release from smooth muscle cholinergic nerve terminals and thus inhibit contraction (Bai et al. 1989). Finally, activation of β2 adrenoceptors stimulates a calcium-activated potassium channel in the cell membrane which leads to hyperpolarization of the membrane and cell relaxation (Johnson 2006).

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Table 28.3 Mechanisms of smooth muscle relaxation by b2 agonists. Stimulation of cAMP and subsequent reduction of myosin light chain kinase activity cAMP inhibition of phospholipase C with reduction of IP3 formation Stimulation of calcium extrusion pumps Direct interaction of Gs with membrane potassium channels Inhibition of acetylcholine release from cholinergic nerve terminals

b-Adrenoceptor agonists Structure and metabolism The structures of commonly used agonists and the natural catecholamines from which they are derived are shown in Fig. 28.3. The term “catecholamine” refers to all compounds with a catechol nucleus (benzene plus two adjacent hydroxyl groups) and an amine group. The three principal naturally occurring catecholamines are dopamine (dihydroxyphenylethylamine) and the metabolic products of dopamine, norepinephrine, and epinephrine. Monoamine oxidase, predominantly an intraneuronal enzyme, and catechol-Omethyltransferase, predominantly an extraneuronal enzyme, are the two enzymes primarily responsible for degradation of catecholamines. Ligand receptor interactions are stereospecific. All the commonly used β agonists exist in racemic mixtures of optical isomers referred to as R and S enantiomers. The agonist activity lies predominantly in the R enantiomer. The in vitro effects of the R and S enantiomers of some but not all β2 agonists have been shown to be different (Volcheck et al. 2005). There has been speculation that the S enantiomers possess adverse effects in clinical usage but this remains unclear. There are now preparations of R enantiomers of salbutamol alone available for commercial usage. However the clinical significance of such differences remains uncertain. Isoproterenol is a catecholamine like epinephrine and this class of compounds is both chemically and metabolically unstable. The resorcinol analog metaproterenol (orciprenaline) is structurally closely related to isoproterenol, and thus shares the nonselective actions of isoproterenol but is more stable. Both salbutamol and terbutaline, the first of the current generation of “short-acting” relatively β2 adrenoceptor-specific agonists used in the treatment of asthma were synthesized and characterized before Lands subtyped β adrenoceptors. Since the 1960s, a number of other β2 agonists have been developed as therapeutic agents. The key substitutions to the β-phenylethylamine parent have been to the catechol ring, or related structure, to make the compounds resistant to metabolism by endogenous methyltransferases and monoamine oxidase, and addition of an ethanolamine side chain of varying length (Fig. 28.3). Such alterations prolong half-life and increase the selectivity of these agents for β2 adrenoceptors. A short-acting β2-selective compound still metabolized by endogenous enzymes is sometimes useful in clinical research;

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b-agonists

Catecholamines HO

HO

OH

HO

CH

NHCH3

CH2

HO

OH

HO

CH

CH3

CH

CH3

HO

CH3 CH3

OH

NHCH

Isoprenaline

NHC

CH2

Terbutaline

HO

CH3 CH2

CH3

CH

Epinephrine HO

OH

CH2

NHCH CH2

Fenoterol

OH

a-agonists CH

CH2

OH

CH3

NH

HOCH2

CH3

OH

HO

CH

Ephedrine

OCH3

HOCH2 CH2

OH

CH3

NHC

NH2

CH3 CH3

OH

HO

CH

CH2

NH

(CH2)6

O

(CH2)4

Salmeterol

Methoxamine O

HO CH

CH2

OH Fig. 28.3 Representative catecholamines and selective a- and b-adrenoceptor agonists.

CH2

Salbutamol

OCH3 CH

CH3

NH

CH3

HCNH HO

OH CH

Phenylephrine

rimiterol is one such compound, although only modestly β2 selective. The prototypic short-acting β2 agonists salbutamol and terbutaline, despite the subsequent development of many other compounds such as fenoterol, clenbuterol and procaterol (Table 28.4), remains the most widely prescribed of this class of drug. The major shortcoming of these medications is duration of action and much effort has been expended to prolong duration beyond 4 hours. Agents such as pirbuterol and clenbuterol have been reported to possess a significantly longer duration of action but this is marginal at best and, in the case of clenbuterol, only after oral administration. A more recently developed compound, bambuterol, does have a more extended duration of action but is a prodrug of terbutaline and is effective only after oral administration, the least preferred route of administration because of systemic side effects. Two compounds in common clinical use, formoterol and salmeterol, have been shown to have a therapeutically significant increased duration of action by inhalation. Even

CH3 CH2

NHCH

Formoterol

CH2

OCH3

Table 28.4 b Agonists in clinical use. Short-acting (b2 selective) Salbutamol Terbutaline Fenoterol Metaproterenol (orciprenaline) Clenbuterol Pirbuterol Bitolterol Procaterol Long-acting (b2 selective) Salmeterol Formoterol Catecholamines Epinephrine (adrenaline) Isoproterenol (isoprenaline) Isoetharine

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longer-acting agents such as indacaterol, which increase forced expiratory volume in 1 s (FEV1) > 15% 24 hours after delivery, are in phase III trials. Formoterol was developed by investigating β-agonist analogs with increasing affinity for the β2 adrenoceptor itself. In contrast, salmeterol was designed to introduce large lipophilic N-substituents into saligenin ethanolamines to facilitate binding in hydrophobic regions of the cell membrane or to nonpolar amino acid residues in the β2-adrenoceptor protein (Johnson 2006). These differences in design lead to important differences between these two long-acting β2-adrenoceptor agonists. Both are moderately lipophilic compared to salbutamol and terbutaline which are hydrophilic in nature; this property leads to greater persistence in the cell membrane and may explain some but not all of the prolonged duration of action. The duration of action of short-acting β2 agonists is determined in part by the rate of diffusion of these hydrophilic compounds away from the receptor site. In contrast, salmeterol seems bound to the cell membrane for prolonged periods and offset of action may be determined by internalization of the bound receptor–drug complex. All the commonly used β2 agonists are either excreted unchanged in the urine or excreted in a conjugated fashion. For example, salbutamol and terbutaline are susceptible to 4-O ′-sulfate conjugation in intestinal wall and liver (when administered by the oral route) (Morgan et al. 1986). Following aerosol administration, the significant proportion that impacts in the oropharynx and is swallowed is also conjugated in the intestinal wall. Following intravenous administration, more of the free drug is excreted compared with the oral and aerosolized route. Fenoterol is also susceptible to 5-O′sulfation. Salmeterol is extensively metabolized by hydroxylation and formoterol is excreted unchanged or subject to glucuronide conjugation.

Selectivity, affinity, and efficacy The basis of β1/β2 adrenoceptor selectivity may be in differ-

ences in the amino acid sequences of the two adrenoceptors (Tota et al. 1991). Selectivity is determined by comparing the potency of β agonists on tissue preparations containing primarily β1 adrenoceptors (e.g., atrial inotropic responses) versus preparations containing primarily β2 adrenoceptors (e.g., bronchial relaxant responses). In this situation, agents are ranked compared to the effect of a completely nonselective β-adrenoceptor agonist, usually isoproterenol (Table 28.5). The ratio of the relative potencies at β2 vs. β1 sites gives the selectivity ratio. Using these criteria, the long-acting agent, salmeterol, is the most β2-selective agent in common use and fenoterol is the least selective. Formoterol is not very selective using these approaches. The potency of a given agonist is usually measured as the concentration of the drug required to cause 50% of maximum response to that agonist. Potency is a function of both receptor affinity and receptor efficacy and of tissue-related factors such as receptor density and efficiency of G-protein coupling. Affinity describes the degree of attraction of a ligand for a receptor, as determined by binding studies. A radiolabeled version of agonist is used in increasing concentrations until the maximum is reached for bound label compared to labeling in the presence of a high concentration of unlabeled agonist. Efficacy describes the ability of an agonist to induce a response in a particular tissue. β Agonists and β antagonists may both share similar affinities for β adrenoceptors but have different efficacies; a full agonist will have a high efficacy while a pure antagonist will have low or zero efficacy. The majority of short-acting β2-selective agonists have intermittent efficacy and potency compared to isoproterenol (Table 28.5). β2 Agonist efficacy is usually assessed by examining relaxation responses of contracted preparations of airway smooth muscle. Again, one compares the maximum relaxant response with isoproterenol. This value is called the intrinsic activity and is a ratio of the maximum response of a given β agonist to the maximum response of isoproterenol. Using these criteria,

Table 28.5 Potency, selectivity, and intrinsic activity of commonly used b-adrenoceptor agonists.

Agonist

Potency b1

Potency b2

Selectivity ratio (b2 : b1)

Airway smooth muscle intrinsic activity

Isoproterenol Salbutamol Terbutaline Fenoterol Salmeterol Formoterol

1.0 0.0004 0.003 0.005 0.001 0.05

1.0 0.48 0.08 0.9 8.5 25

1.0 1375 267 180 85 000 100

1.0 0.91 0.83 0.99 0.70 0.94

Data are relative to response to isoproterenol. Intrinsic activity is a measure of efficacy (the ratio of the maximum response to a given b agonist to the response to isoproterenol). Data compiled from O’Donnell & Wanstead (1979); Decker et al. (1982); Bai et al. (1989); Johnson (1991).

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none of the synthetic β agonists have higher intrinsic activity than isoproterenol. Agents with equivalent efficacy are procaterol and formoterol whereas most saligenins and resorcinols are of moderate efficacy (65– 85% of isoproterenol). The efficacy of β agonists at extrapulmonary sites may be of clinical relevance. Fenoterol and formoterol have the same efficacy as isoproterenol at cardiac β1 adrenoceptors, despite being less potent, whereas salbutamol and salmeterol have very low efficacy. In contrast to efficacy, lung β2-adrenoceptor potency can be greater than isoproterenol. For example, salmeterol is five times more potent than isoproterenol, formoterol 25-fold more potent, and procaterol 24-fold more potent. Neither intermediate intrinsic activity (efficacy) nor intermediate potency negates the clinical value of a given β agonist as a bronchodilator drug. Rather, the adverse consequences of prolonged use of these drugs may be influenced by whether they are partial or full agonists. The rate of desensitization is one phenomenon that could be influenced by full versus partial agonist activity (see later).

Physiologic effects Lung cells Although in vivo the most obvious and therapeutic pulmonary effect of β-adrenergic stimulation is bronchodilation mediated by airway smooth muscle relaxation, a number of other effects also occur (Table 28.6). β Agonists promote secretion from serous cells and, to a lesser extent, mucous cells, in mucous glands. Serous cell stimulation produces antibacterial proteins such as lysosomes and lactoferrin. This effect has been demonstrated convincingly in vitro only, using human tracheal explants at relatively high concentrations of β agonists (Basbaum et al. 1990) However, theoretical calculations of luminal β2 agonist concentrations following inhalation indicate such levels may be achieved in vivo. Furthermore, β2 agonists increase chloride iron transport through apical membranes of epithelial cells via an increase in cyclic AMP. Sodium follows passively via paracellular channels and water by osmosis. The net effect is to increase periciliary fluid (Wanner 1988). The combined effects of stimulation Table 28.6 Physiologic effects of b2-adrenoceptor stimulation in human lung. Airway smooth muscle relaxation Prejunctional inhibition of acetylcholine release from parasympathetic neurons in airway smooth muscle Stimulation of mucus and serous cell secretion Stimulation of chloride ion secretion across the apical membrane of airway epithelial cells Increase in ciliary beat frequency Stimulation of surfactant secretion from alveolar type II cells Inhibition of mediator release from lung mast cells and neutrophils ? Reduction in microvascular permeability (animal models) ? Increase in bronchial blood flow (animal models)


of mucous glands and chloride channels, together with an increase in ciliary beat frequency (Wanner 1988), is to increase mucocilary clearance. Increased clearance has been demonstrated in vivo using radio tracer methods although the clinical relevance of this enhancement in patients with asthma is unknown. β2 Agonists also stimulate the secretion of surfactant from alveolar type II cells in vitro, although the magnitude of the effect is modest (Mason & Williams 1991). In animal models, inflammatory mediators increase microvascular permeability by contracting postcapillary venular endothelial cells so that spaces form between the cells. In such models, β2 agonists relax endothelial cells and therefore reduce permeability (Baluk & McDonald 1994). However, β2 agonists also increase bronchial blood flow by acting as vasodilators of bronchial arterioles (Kelly et al. 1992). The net effect of these two opposing effects on exudation or transudation of fluid into the lumen and wall of inflamed human airways is unclear. A report that nebulized epinephrine was no more effective in producing bronchodilatation in acute asthma than a nebulized β agonist, which lacks an α-adrenergic vasoconstrictor effect, suggests that potential alterations in bronchial blood flow induced by β2 agonists do not adversely affect fluid shifts across the lumen wall (Coupe et al. 1987). Moreover, the lack of additional benefit by epinephrine suggests that the potential decrease in lumen area produced by mucosal bronchodilation induced by β agonists is not an important component of airflow resistance in asthma. β Adrenoceptors are present in peribronchial parasympathetic ganglia, which receive direct sympathetic innervation (Widdicombe 2003). β2 Adrenoceptors are also present on cholinergic nerve terminals in airway smooth muscle and act here to inhibit acetylcholine release (prejunctional inhibition), thereby reducing the cholinergic component of bronchoconstriction. It is possible that β antagonists such as propranolol induce asthma exacerbations not only by reducing the tonic bronchodilator effect of circulating epinephrine on airway smooth muscle in maintaining airway patency, but also by blocking the effect of epinephrine on cholinergic nerve terminals leading to the exuberant release of acetylcholine. The observation by Grieco and Pierson (1971) that cholinergic antagonists partially reverse propranolol-induced bronchoconstriction provides some support for this hypothesis.

Inflammatory cells β2 Adrenoceptors are present on a variety of inflammatory cells including lymphocytes, granulocytes, mast cells, and macrophages. Stimulation of β receptors on immunocompetent cells primarily results in antiinflammatory effects. Circulating lymphocytes and neutrophils have low numbers of adrenoceptors which appear to be relatively poorly coupled to second messenger pathways in that they are easily downregulated (Insel 1991). Human neutrophils possess approximately 900–1800 β2 adrenoceptors per intact cell and mediator

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Cardiovascular β Agonists increase the force and rate of cardiac contraction and thus cause an increase in systolic blood pressure. Increases in inotropic reponses are predominantly mediated via β1 adrenoceptors, although β2 adrenoceptors also contribute. In contrast, chronotropic responses are predominantly β2 mediated (Fig. 28.4). β2 Agonists are also vasodilators, via β2 adrenoceptors on vascular smooth muscle, which leads to a slight fall in diastolic blood pressure. The role of baroreceptormediated reflex withdrawl of cardiac vagal tone in response to peripheral vasodilatation, in determining heart rate increases seems less important than direct cardiac effects of β agonists. β Agonists cause a dose-dependent increase in QTc interval, which has been reported to be correlated with

676

the degree of hypokalemia induced by these agents (Fig. 28.4) (Crane et al. 1989). However, isoproterenol, which causes minimal hypokalemia, prolongs QTc interval to a similar degree as salbutamol, suggesting a direct cardiac β adrenoceptor-mediated effect (Lipworth & McDevitt 1992).

DHR (beats/min)

30 25 20 15 10 5 0 100

200

500

1000

2000

4000

Dose salbutamol (mg)

ΔK (mmol/L)

release is inhibited in a dose-dependent manner by isoproterenol. Triggering of β2-receptors on neutrophils also results in inhibition of oxygen release and a reduction of the adhesion of neutrophils to the vascular endothelium and airway epithelial cell (Maris et al. 2005). Studies employing circulating lymphocytes or neutrophils as a marker of pulmonary β-adrenoceptor function can therefore be misleading (see below). However, one report showed a strong relationship between β2-adrenoceptor densities on circulating mononuclear leukocytes and in lung tissue obtained at thoracotomy (Liggett et al. 1988). β Agonists modulate lymphocyte cytokine production. Type 2 T cells freshly isolated from peripheral blood mononuclear cells directly respond to β agonists to activate protein kinase A and inhibit CD3-stimulated interleukin (IL)-13 production (Loza et al. 2006). Human alveolar macrophages contain 5000 β2 adrenoceptors per cell (Liggett 1988). There is controversy as to whether β2 agonists prevent mediator release from activated human alveolar macrophages; short-acting agents may not (Fuller et al. 1988) as opposed to longer-acting agents (Maris et al. 2005). There is evidence that β agonists reduce the release of histamine from mast cells. β Agonist-mediated inhibition of mast cell mediator release is probably one mechanism of action of epinephrine in reducing symptoms of anaphylaxis such as itch and edema. In addition, inhibition of mast cell mediator release is part of the mechanism of action of β agonists in abating the early response to allergic bronchial challenge. β Agonists are also functional antagonists of the airway smooth muscle contraction induced by release of mediators. β Agonists may also inhibit mediator release from basophils (Weston & Peachell 1998). Mediator release from the human eosinophil, although possessing a greater density and affinity of β2 adrenoceptors (5000 sites per cell) than neutrophils, was not shown to be inhibited by isoproterenol (Yukawa et al. 1990), but more recent studies show inhibitory effects on IL-5 induced superoxide production, an effect of the R but not the S enatiomer of salbutamol (Volcheck et al. 2005).

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Fig. 28.4 Changes in heart rate (upper panel), plasma potassium (middle panel), and FEV1 (lower panel) in response to cumulative doubling doses of inhaled salbutamol from pressurized metered-dose inhalers in 12 mild asthmatic patients. Subjects were studied after 2 weeks’ pretreatment with placebo (circles), low-dose regular salbutamol (800 mg/day) (triangles) or high-dose regular salbutamol (4000 mg/day) (squares). Randomized crossover design. Note that significant tolerance to the systemic but not bronchodilator effects of the b agonist develop with regular use. (Redrawn from Lipworth et al. 1989, with permission.)

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Pharmacokinetics of b-adrenoceptor agonists

Salbutamol 200 mg

Oral administration

Parenteral administration Subcutaneous, intramuscular, or intravenous injection of terbutaline, salbutamol or epinephrine provides almost immediate action and assured delivery. Following subcutaneous terbutaline 0.5 mg, significant levels are present within a few minutes and peak at 20 min (van den Berg et al. 1984). Epinephrine has a slightly shorter duration of action although a slow-release form is available in some countries. In an intensive care setting, intravenous infusion is sometimes used when patients are moribund or responding poorly to intermittent administration of β agonists by other routes. Both intravenous terbutaline and salbutamol have been quite widely used in this context, with a loading dose followed by a continuous infusion of 10– 20% of the loading dose. Intravenous isoproterenol has little advantage apart from quicker offset of action, and has greater side effects.

Aerosol administration This is the preferred method of administration of all β agonists as there is an effect on airway caliber within seconds, with the effect of short-acting β agonists such as salbutamol reaching 80% of maximum in 5 min (Figs 28.4 and 28.5). Compared to parenteral or oral administration, following aerosolization a given degree of bronchodilatation is achieved with significantly less adverse effects such as tremor or palpitations. Shortacting β2 agonists achieve peak effects within 30–60 min and bronchodilatation slowly reduces over a variable time after this, in part dependent on the severity of asthma, but airway caliber is back to baseline within 4–5 hours. There are no important clinical differences between commonly available short-acting β2 agonists in terms of bronchodilatation or duration of action. The effects of catecholamine aerosols such as rimiterol or epinephrine peak earlier and bronchodilatation persists for only 30 min to 2 hours. Serum levels are very low after inhaled administration and do not correlate

Salmeterol 50 mg Salmeterol 100 mg Salmeterol 200 mg

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Per cent of max PEF1

After administration of an oral or parenteral β agonist, the bronchodilator effect is closely dependent on serum levels (Morgan et al. 1986). Orally administered β agonists are incompletely absorbed and the greater proportion metabolized by sulfate conjugation in the gut epithelium and liver. For example, oral terbutaline absorption varies from 30 to 65%, and of this only 25% remains unconjugated in the urine. Net biovavailability is thus 10–15%. Protein binding of terbutaline varies from 14 to 25%. Salbutamol is more completely absorbed with peak levels within 1 hour of administration when the subject is fasting; 60% of the absorbed dose is conjugated. Sustained release preparations of salbutamol and terbutaline are available in various formations in some countries, and may be useful in asthmatics with marked nocturnal symptoms, although long-acting inhaled β2 agonists have superseded such preparations.

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Time (hours) Fig. 28.5 Time-course of bronchodilator response to standard shortand long-acting b agonists. Peak expiratory flow (PEF) after inhalation of salbutamol (200 mg) or salmeterol (50, 100 or 200 mg). (From Ullman & Svedmyr 1988, with permision.)

with bronchodilatation. The effects of different aerosol delivery devices on intrapulmonary β agonist deposition is discussed in a subsequent chapter. Systemic effects of inhaled β2 agonists are in part dependent on airway caliber, which influences peripheral deposition such that normal individuals have more systemic effects than asthmatics (Mortimer et al. 2006). Formoterol is long acting only by inhalation, reflecting unusually prolonged retention in the airway wall. Systemic effects are less prolonged (Lotvall 2002). In contrast to salmeterol, which is “dose loaded” (i.e., available only as a dose that has a maximal effect), formoterol is available in a range of doses and shows dose-dependent increases in both maximal effect and duration of action in clinical use.

Adverse effects Desensitization β-Adrenoceptor desensitization, i.e., waning of the response in the face of continuous or repeated agonist exposure, can occur by several mechanisms (McGraw et al. 2003; Johnson, 2006). Rapid desensitization is mediated by an alteration in the function of the β adrenoceptor in that it becomes uncoupled from the stimulatory G protein, Gs. This uncoupling phenomenon involves phosphorylation of receptor proteins in the third transmembrane intracellular loop and the terminal intracellular segment by at least two different kinases, protein kinase A and a β adrenoceptor-associated kinase (βARK) or related kinases, which are activated under different desensitization conditions. Phosphorylation leads to binding of β-arrestin and thus decreased efficiency of

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coupling of the β adrenoceptor to Gs, leading to decreased adenylyl cyclase activity and hence decreased cAMP levels. Desensitization can also occur by intracellular sequestration of the receptor complex or by downregulation, which refers to agonist-induced decrease in receptor number. Downregulation occurs on prolonged exposure to agonists and results in degradation of the receptor, presumably via a lysozymal pathway. Both uncoupling and sequestration (internalization) occur within minutes of exposure to micromolar concentration of β-adrenoceptor agonists and the process is essentially complete within 30 min. Downregulation is evident after only several hours of exposure. It has been proposed that the rapid desensitization mechanisms involving phosphorylation of the β adrenoceptor (uncoupling) may be operative mainly for nonneural β2 adrenoceptors that respond to circulating concentrations of epinephrine which are in the nanomolar range (Stadel & Lefkowitz 1991). Downregulation is also mediated by a decrease in β-adrenoceptor mRNA, caused by a decrease in mRNA stability rather than a decreased rate of transcription. Airway smooth muscle contains much less βARK and related kinase activity compared with mast cells for example, and this may explain discrepancies in the ability to induce desensitisation in inflammatory cells versus smooth muscle preparations. Relatively large numbers of β adrenoceptors on human airway smooth muscle or rapid turnover of adrenoceptors may explain why this tissue is relatively resistant to desensitization (McGraw & Liggett 1997). Phosphorylation and therefore uncoupling of the β adrenoceptor can also be induced by stimulation of adjacent GPCRs (receptor cross-talk) such as cholinergic muscarinic M3 receptors (Grandordy et al. 1994) and prostaglandin EP1 receptors (McGraw et al. 2006). Activation of muscarinic receptors leads to stimulation of phosphatidylinositol pathways with secondary activation of protein kinase C by diacylglycerol which, in turn, can phosphorylate and uncouple the β adrenoceptor (Malbon 1989). Prostaglandin EP1 receptors modulate β2 adrenoceptors directly by receptor heterodimerization, and thus PGE2, which has variable effects on smooth muscle, reduces the smooth muscle relaxation effect of β agonist via uncoupling of β2 adrenoceptors from Gs (McGraw et al. 2006). As discussed above, glucorticosteroids have been demonstrated in vitro to reverse desensitization and this is probably due to increased β2-adrenoceptor gene transcription (Mak et al. 1995), and possibly increased coupling, and may be an important mechanism of action of glucocorticosteroids in the treatment of asthma. There have been many clinical studies of desensitization following regular short-acting β-agonist use (reviewed by Broadley 2006). There is evidence in some studies of a small decrease in peak bronchodilator effect and duration of action in stable mild asthma but not in peak bronchodilator effect in more severe asthmatic patients (Lipworth et al. 1989) (see Fig. 28.4). Genetic polymorphisms in the β2

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adrenoceptor influence this effect (see later). Oral β agonists are more likely to induce tolerance. In contrast to studies in asthmatics, normal subjects readily demonstrate desensitization both in the lung and in nonpulmonary β-adrenergic systems. Small increases in airway responsiveness have been detected following cessation of regular short-acting β agonists. One explanation of these findings is desensitization of airway smooth muscle β adrenoceptors (Vathenen et al. 1988). There is no evidence that long-term use of long-acting β2 agonists such as salmeterol or formoterol leads to clinically relevant tolerance to their bronchodilating effects. However, regular treatment of patients with asthma with long-acting β2 agonists leads to tolerance to the protective effect against exercise induced bronchoconstriction (Storms et al. 2004). Differences in the effects of regular long- versus short-acting β2 agonists, even when used with corticosteroids, are evident, supporting the as-needed rather than regular use of shortacting β2 agonists (Frey et al. 2005). In this study, despite improved basal airflow rates with either short- or long-acting β agonist, regular treatment with salmeterol reduced risk of asthma attacks compared with regular salbutamol. Overall, the importance of desensitization as a clinically relevant effect of β-agonist treatment remains unclear.

Tremor Tremor, due to activation of β2 adrenoceptors in skeletal muscle, occurs in up to 20% of patients at initiation of β2agonist therapy. Tremor usually abates with regular use due to the development of desensitization.

Cardiac effects Palpitations are reported by up to 5% of asthmatics at initiation of therapy, more so with agents which are full rather than partial agonists at β2 adrenoceptors. Desensitization develops with regular therapy (see Fig. 28.4). Despite concern that tachyarrhythmias could develop secondary to effects of β agonists on QTc interval and potassium in hypoxemic patients, serious cardiovascular events are extremely rare. However, caution should be exercised in individuals with unstable ischemic heart disease receiving high doses of nebulized β agonists as angina has been precipitated in this situation.

Hypokalemia and other metabolic effects Hypokalemia is seen following both inhaled or systemic administration of β agonists due to stimulation of Na+/K+ATPase activity and stimulation of insulin secretion (see Fig. 28.4). When high cumulative inhaled doses of salbutamol and fenoterol (1200 μg) were given to normal subjects, decreases in K+ of 0.67 and 1.13 mmol/L, respectively, were observed. Again desensitization is observed to this effect with chronic use of β agonists (see Fig. 28.4). Glycolysis also occurs secondary to β2-adrenoreceptor activation; the changes

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induced are small and of uncertain significance in patients with diabetes mellitus. Lipolysis is activated by β agonists via β1, β2, and possibly β3 adrenoceptors and results in the mobilization of free fatty acids from adipose tissue.

Hypoxemia All β agonists, including epinephrine, can reduce arterial oxygen tension. These changes are apparent 5 min after administration of inhaled β agonists and return to normal values by 30 min. These changes are secondary to an increase in pulmonary blood flow in poorly ventilated regions of the lung, hence worsening ventilation–perfusion inequality. The increase in blood flow may be secondary to pulmonary vasodilatation via stimulation of β2 adrenoceptors on vascular smooth muscle in the lung and also to increases in cardiac output following cardiac adrenoceptor stimulation. The reduction in arterial oxygen tension is small and unlikely to be clinically significant.

Pharmacogenetics Associations between β2-adrenoceptor SNPs and therapeutic outcomes suggest that genetic variation can influence the response to a variety of β2 agonists; however, the clinical significance of these observations is not yet clear (Tattersfield & Harrison 2006). Carefully designed studies have shown individual β2adrenergic receptor genotype can predict lung function in response to β2 agonists. With daily salbutamol therapy, morning peak expiratory flow progressively decreases in patients homozygous for an arginine SNP at amino acid position 16 of the coding region of the β2 adrenoceptor, but not in Gly16 homozygotes (Israel et al. 2004). Other work has shown an increased number of asthma exacerbations in individuals homozygous for the Arg16 allele who were given long-term salbutamol treatment, but not in those homozygous for the Gly16 variant (Taylor et al. 2000). A potentially critical factor in unravelling pharmacogenetic associations centers on the pharmacologic properties of the β2 agonists administered. The predominance of literature has centered on the short-acting β2 agonist, salbutamol, but because pharmacologic properties of β2 agonists vary, conclusions drawn from clinical studies that investigate associations between lung function and ADRB2 pharmacogenetics may not be comparable between short- and long-acting β2 agonists. In a single 24-week crossover study comparing use of short-acting β2 agonists with salmeterol evaluating patients by Arg16Gly genotype, lower exacerbation rates were reported for Arg/Arg patients receiving salmeterol compared with regular or as-needed albuterol use (Taylor et al. 2000). In this study the authors also demonstrated that while there was reduced peak flow over time with albuterol in the Arg/Arg genotype, there was no diminution of pulmonary function with the Arg/Arg genotype in patients receiving salmeterol. In another study, Arg16Gly effects were associated with lower


morning peak expiratory flow in response to salmeterol compared with placebo, regardless of baseline inhaled corticosteroids (ICS) therapy (Wechsler et al. 2006), but there was no difference in exacerbation rates based on genotype. Only limited pharmacogenetic information is available for β2 agonists when coadministered with ICS. No pharmacogenetic associations predictive of clinical response to salmeterol, salmeterol coadministered with fluticasone proprionate (FP), or formoterol coadministered with budesonide have been identified in exploratory retrospective analyses performed to date. In one such study, the ADRB2 Arg16Gly SNP was evaluated in subjects with persistent asthma (N = 183). Following 12 weeks of chronic dosing with salmeterol administered with FP in a single device, no differences in morning peak expiratory flow were observed across Arg16Gly genotypes (Dorinsky et al. 2004). Studies evaluating the ADRB2 Arg16Gly SNP and asthma deaths in whites (who are using overusing β2 agonists) have not detected a risk of this allele (Weir et al. 1998). It is clear that the predictability of β2-agonist pharmacogenetics is low, due to incomplete understanding of the haplotypic structure of the gene and incomplete understanding of the genetic variants in other genes likely to be important determinants of β2-agonist responses (Weiss et al. 2006).

b-Adrenoceptor antagonists Propranolol was the first β antagonist developed for clinical use and remains the prototypic agent (van Zwieten 1988). It is a nonselective competitive antagonist without agonist activity. Nadolol, timolol, pindolol, and sotalol are other examples of this group. Although selectivity is not absolute, metoprolol, atenolol, acebutolol, bisoprolol, and esmolol are relatively β1 adrenoceptor selective. Labetalol represents a class of drugs that act as competitive antagonists at both α1 and β adrenoceptors, with greater affinity for the latter as well as some partial agonist activity at β2 adrenoceptors. Pindolol and acebutolol also have partial agonist activity (intrinsic sympathomimetic activity) at β1 adrenoceptors. Esmolol is a very short-acting β antagonist useful intravenously when short-duration β blockade is desired. Some, indeed most, antagonists, for example metoprolol, exhibit inverse agonism, as defined above, and this may be part of their therapeutic benefit in diseases such as heart failure (Bond & Ijzerman 2006) versus “neutral antagonists” such as bucindolol. None of these antagonists can be safely used in asthmatics, even by topical application, although most patients with chronic obstructive pulmonary disease (COPD) do tolerate these agents (Salpeter et al. 2005), reflecting the differences in the pathogenesis of the two conditions. It is likely, although unproven, that if asthma control is assured with antiinflammatory treatment and careful follow-up, that β1-selective antagonists are tolerated in asthma. The use of β antagonists,

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predictably, also increases the severity and resistance to treatment of anaphylaxis (Schellenberg et al. 1991).

ergic and cholinergic neurons. They inhibit norepinephrine release and they also inhibit, in some circumstances, acetylcholine release.

a-Adrenoceptor agonists a-Adrenoceptor antagonists Most of these compounds are drugs with mixed effects, i.e., they both displace norepinephrine from storage sites within the neuron and have direct α-adrenoceptor-stimulating effects. As for β agonists, the receptor subtype specificity and potency is determined by chemical structure (see Fig. 28.1). Ephedrine is the classic compound in this category. Ephedrine is an alkaloid derived from ma-huang (Ephedra equisetina) and exists in four enantiomers: (+) and (−) ephedrine and (+) and (−) pseudoephedrine. The most potent form in relation to sympathomimetic activity is (−)-ephedrine; this compound is used clinically, as is (−)-pseudoephedrine. Ephedrine produces a prompt rise in blood pressure, causes coronary vessel vasodilatation, and increases heart rate. The other commonly used oral α agonist is phenylpropanolamine. Ephedrine and phenylpropanolamine possess both α1- and α2-agonist activity and are both commonly used as nasal and sinus decongestants. In the nasal vasculature, stimulation of the α1 and α2 adrenoceptors on resistance blood vessels regulates blood flow. In the capacitance vessels, regulating blood volume changes (Lacroix & Lundberg 1989), the α2-adrenoceptor response predominates over α1 adrenoceptors. Phenylpropanolamine and pseudoephedrine are well absorbed with a half-life of 4 hours. Slow-release preparations enable twice daily dosage. Direct effects of ephedrine on airway β2 adrenoceptors result in bronchodilation. Both α1 and α2 agonists reduce vascular engorgement and therefore improve nasal patency (Bende & Loth 1986). There may be advantages to using selective α1 agonists as α2 agonists reduce mucosal blood flow by 30– 40%, which may impair long-term mucosal (i.e., nasal epithelium) health. Phenylephrine is a selective α1 agonist used clinically that is chemically closely related to epinephrine but is less potent and has a longer duration of action. It has little effect on α2 adrenoceptors. It is a useful agent as a topical nasal decongestant but is poorly absorbed orally. Methoxamine is also an effective α1 agonist, used sometimes to reduce airway narrowing when the latter is due to the dilatation of bronchial mucosal vessels secondary to left heart failure (“cardiac asthma”). Selective α1 agonists have a short duration of action and the predominantly α2-agonist imidazole derivatives oxymetazoline and xylometazoline are useful sympathomimetic agents with a larger duration of action. Use of α agonists causes rebound vasodilatation in the nose following offset of action, and prolonged use may lead to rhinitis medicamentosa. Other selective α2 agonists include clonidine and αmethyldopa which are widely used in hypertension and in conditions of “sympathetic overactivity.” These compounds stimulate presynaptic α2 adrenoceptors present both on adren-

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Currently there are no specific indications for α-adrenergic antagonists in the management of allergic diseases. Dale (1906) reported that the pressor effects of large doses of epinephrine could be reversed to cause a fall in blood pressure by certain ergot preparations now known to have αadrenoceptor blocking properties. Large numbers of drugs with the ability to antagonize the effects of α-adrenoceptor stimulation have been synthesized. They are classified into reversible noncompetitive antagonists, for example phenoxybenzamine, and reversible competitive antagonists such as phentolamine and prazosin. Phentolamine and phenoxybenzamine are nonselective antagonists whereas prazosin selectively blocks α1 adrenoceptors. Yohimbine is a plant alkaloid with selective α2-antagonist action. Interest has continued in the possibility of using α-adrenergic antagonists in the treatment of asthma as, in some patients, the administration of an α agonist such as methoxamine provokes airway narrowing (Black et al. 1984). There is also the longstanding observation of increased α-adrenergic responsiveness in patients with allergic disorders. Overall, the response to αadrenergic antagonists in clinical trials has been disappointing (Barnes 1986), although the possibility of individual responses to α-adrenergic antagonists still exists.

Acknowledgment Supported by the Canadian Institutes of Health Research.

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Loza, M.J., Foster, S., Peters, S.P. & Penn, R.B. (2006) Beta-agonists modulate T-cell functions via direct actions on type 1 and type 2 cells. Blood 107, 2052– 60. McGraw, D.W. & Liggett, S.B. (1997) Heterogeneity in beta-adrenergic receptor kinase expression in the lung accounts for cell-specific desensitization of the beta2-adrenergic receptor. J Biol Chem 272, 7338–44. McGraw, D.W., Almoosa, K.F., Paul, R.J., Kobilka, B.K. & Liggett, S.B. (2003) Antithetic regulation by beta-adrenergic receptors of Gq receptor signaling via phospholipase C underlies the airway beta-agonist paradox. J Clin Invest 112, 619– 26. McGraw, D.W., Mihlbachler, K.A., Schwarb, M.R. et al. (2006) Airway smooth muscle prostaglandin-EP1 receptors directly modulate beta2-adrenergic receptors within a unique heterodimeric complex. J Clin Invest 116, 1400–9. Mak, J.C., Nishikawa, M. & Barnes, P.J. (1995) Glucocorticosteroids increase beta 2-adrenergic receptor transcription in human lung. Am J Physiol 268, L41–L46. Malbon, C.C. (1989) Physiological regulation of transmembrane signaling elements. Am J Respir Cell Mol Biol 1, 449–50. Maris, N.A., De Vos, A.F., Dessing, M.C. et al. (2005) Antiinflammatory effects of salmeterol after inhalation of lipopolysaccharide by healthy volunteers. Am J Respir Crit Care Med 172, 878–84. Mason, R.J. & Williams, M.C. (1991) Alveolar type II cells. In: Crystal, R.G. & West, J.B., eds. The Lung: Scientific Foundations. Raven Press, New York, pp. 543–57. Morgan, D.J., Paull, J.D., Richmond, B.H., Wilson-Evered, E. & Ziccone, S.P. (1986) Pharmacokinetics of intravenous and oral salbutamol and its sulphate conjugate. Br J Clin Pharmacol 22, 587–93. Mortimer, K.J., Harrison, T.W., Tang, Y. et al. (2006) Plasma concentrations of inhaled corticosteroids in relation to airflow obstruction in asthma. Br J Clin Pharmacol 62, 412–19. Nadel, J.A. & Barnes, P.J. (1984) Autonomic regulation of the airways. Annu Rev Med 35, 451– 67. O’Donnell, S.R. (1991) Pharmacology of β-adrenoceptors in lung disease. In: Szabadi, E. & Bradshaw, C.M., eds. Adrenoceptors: Structure, Mechanisms, Function. Advances in Pharmacological Sciences. Birkhauser-Verlag, Basel, pp. 265– 74. O’Donnell, S.R. & Wanstall, J.C. (1979) The importance of choice of agonist in studies designed to predict β2 : β1 adrenoceptor selectivity of antagonists from pA2 values in guinea-pig trachea and atria. Naunyn-Schmiedeberg’s Arch Pharmacol 308, pp. 183–90. Persson, C.G. (1989) Glucocorticoids for asthma: early contributions. Pulm Pharmacol 2, 163– 6. Salahpour, A., Angers, S., Mercier, J.F., Lagace, M., Marullo, S. & Bouvier, M. (2004) Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting. J Biol Chem 279, 33390–7. Salpeter, S., Ormiston, T. & Salpeter, E. (2005) Cardioselective beta-blockers for chronic obstructive pulmonary disease. Cochrane Database Syst Rev CD003566. Schellenberg, R.R., Ohtaka, H., Paddon, H.B., Bramble, S.E. & Rangno, R.E. (1991) Catecholamine responses to histamine infusion in man. J Allergy Clin Immunol 87, 499– 504. Solis-Cohen, S. (1990) The use of adrenal substance in the treatment of asthma. 1900. J Asthma 27, 401– 6; discussion 399. Stadel, J.M. & Lefkowitz, R.J. (1991) Beta-adrenergic receptors: identification and characterization by radioligand binding studies.

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In: Perkins, J.P. ed. Beta-adrenergic Receptors. Humana Press, Clifton, NJ, pp. 1–41. Storms, W., Chervinsky, P., Ghannam, A.F., Bird, S., Hustad, C.M. & Edelman, J.M. (2004) A comparison of the effects of oral montelukast and inhaled salmeterol on response to rescue bronchodilation after challenge. Respir Med 98, 1051–62. Strader, C.D., Candelore, M.R., Hill, W.S., Dixon, R.A. & Sigal, I.S. (1989) A single amino acid substitution in the beta-adrenergic receptor promotes partial agonist activity from antagonists. J Biol Chem 264, 16470–7. Tattersfield, A.E. & Harrison, T.W. (2006) Beta-adrenoceptor polymorphisms: focus moves to long-acting beta-agonists. Am J Respir Crit Care Med 173, 473–4. Taylor, D.R., Hancox, R.J., McRae, W. et al. (2000) The influence of polymorphism at position 16 of the beta2-adrenoceptor on the development of tolerance to beta-agonist. J Asthma 37, 691–700. Tota, M.R., Candelore, M.R., Dixon, R.A. & Strader, C.D. (1991) Biophysical and genetic analysis of the ligand-binding site of the beta-adrenoceptor. Trends Pharmacol Sci 12, 4– 6. Ullman, A. & Svedmyr, N. (1988) Salmeterol, a new long-acting inhaled β2 adrenoceptor agonist: comparison with salbutamol in adult asthmatic patients. Thorax 43, 674. Van Den Berg, W., Leferink, J.G., Maes, R.A., Fokkens, J.K., Kreukniet, J. & Bruynzeel, P.L. (1984) The effects of oral and subcutaneous administration of terbutaline in asthmatic patients. Eur J Respir Dis 134, 181–93. Van Megen, Y.J., Klaassen, A.B., Rodrigues De Miranda, J.F., Van Ginneken, C.A. & Wentges, B.T. (1991) Alterations of adrenoceptors in the nasal mucosa of allergic patients in comparison with nonallergic individuals. J Allergy Clin Immunol 87, 530–40. Van Zwieten, P.A. (1988) Antihypertensive drugs interacting with alpha- and beta-adrenoceptors. A review of basic pharmacology. Drugs 35, 6–19. Vathenen, A.S., Knox, A.J., Higgins, B.G., Britton, J.R. & Tattersfield, A.E. (1988) Influence of inhaled terbutaline on bronchial responsiveness Lancet ii, 161. Volcheck, G.W., Kelkar, P., Bartemes, K.R., Gleich, G.J. & Kita, H. (2005) Effects of (R)- and (S)-isomers of beta-adrenergic agonists on eosinophil response to interleukin-5. Clin Exp Allergy 35, 1341–6. Wanner, A. (1988) Autonomic control of mucociliary function. In: Kaliner, M.A. & Barnes, P.J., eds. The Airways: Neural Control in Health and Disease. Marcel Dekker, New York, pp. 172–91. Wechsler, M.E., Lehman, E., Lazarus, S.C. et al. (2006) Betaadrenergic receptor polymorphisms and response to salmeterol. Am J Respir Crit Care Med 173, 519–26. Weir, T.D., Mallek, N., Sandford, A.J. et al. (1998) Beta2-adrenergic receptor haplotypes in mild, moderate and fatal/near fatal asthma. Am J Respir Crit Care Med 158, 787–91. Weiss, S.T., Litonjua, A.A., Lange, C. et al. (2006) Overview of the pharmacogenetics of asthma treatment. Pharmacogenomics J 6, 311–26. Weston, M.C. & Peachell, P.T. (1998) Regulation of human mast cell and basophil function by cAMP. Gen Pharmacol 31, 715–19. Widdicombe, J.G. (2003) Overview of neural pathways in allergy and asthma. Pulm Pharmacol Ther 16, 23–30. Yukawa, T., Ukena, D., Kroegel, C. et al. (1990) Beta 2-adrenergic receptors on eosinophils. Binding and functional studies. Am Rev Respir Dis 141, 1446–52.

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Cholinergic Antagonists Nicholas J. Gross

Summary Anticholinergic agents have important uses as bronchodilators, particularly in chronic obstructive pulmonary disease (COPD). They dilate the central airways by relaxing peribronchial smooth muscle using biochemical pathways that are distinct from other bronchodilators. Currently approved agents of this class are synthetic quaternary ammonium compounds and thus very poorly absorbed following inhalation. Ipratropium and oxitropium are relatively short-acting (4–8 hours). Tiotropium was introduced in the early 2000s and is unique in having a duration of action in excess of 24 hours, making it ideally suited for once-a-day routine use. Clinical studies show that, with daily routine use, tiotropium improves both airway function and lung hyperinflation. It also reduces symptoms and improves exercise tolerance and quality of life by clinically meaningful amounts. In patients with moderate or severe COPD, it also reduces the frequency of acute exacerbations and hospitalization by about 20–25%. All agents of this class have a wide therapeutic margin and are safe and well tolerated.

this was due to the discovery of adrenergic agents and methylxanthines, but the fact that atropine and its congeners have a narrow therapeutic margin and their use resulted in many systemic side effects. Interest in the use of anticholinergic agents as bronchodilators was revived by physiologic studies which showed that the airways, both of animals and humans, were largely under the control of the cholinergic parasympathetic nervous system. Moreover, many bronchospastic stimuli acted through cholinergic reflexes (Widdicombe 1979; Nadel 1980). Inhibition of these reflexes by anticholinergic agents prevented or relieved bronchospasm. The resulting need for anticholinergic agents that could be effective when taken by inhalation and that lacked widespread systemic effects led to the development of ipratropium and its approval specifically as a bronchodilator in the 1980s. Subsequently, longer-acting agents such as oxitropium and tiotropium have been developed and several other anticholinergic bronchodilators are in development.

Rationale for use of anticholinergic agents Introduction Cholinergic antagonists form an important class of agents in the treatment of respiratory disorders. Their use goes back millennia in several Asian countries where many alkaloids with anticholinergic properties are found and available in indigenous plants (Gandevia 1975). The leaves of stramonium and belladonna, for example, were eaten or smoked releasing atropine and related agents. These traditional herbal remedies were imported and used in Western medicine until the early part of the 20th century, together with moreor-less purified extracts of their bronchoactive components. However, their use declined in the 1920s and 1930s. Partly


Autonomic control of airways The human airways of large and intermediate size are surrounded by smooth muscle whose tone, a low level of contraction, controls the caliber of the airways. The smooth muscle is supplied by branches of the efferent autonomic system, almost all of which derive from the vagus nerve, a branch of the parasympathetic nervous system (Richardson 1982). These preganglionic cholinergic nerves enter the lung at the hila and divide with the airways, ending as varicosities in peribronchial ganglia where they synapse with short postganglionic fibers that supply cholinergic drive to the peribronchial smooth muscle. They also supply other airway structures such as mucosal cells and possible the ciliated epithelial cells although the latter is not certain. So-called cholinergic drive is limited to central and intermediate airways where cholinergic nerve endings and the relevant receptors are found and results in contraction of peribronchial smooth muscle, increased release of mucus from mucosal cells in the epithelium, and possibly increased bronchial ciliary activity.

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The low level of cholinergic bronchomotor tone that is present at rest (and whose role in health is poorly understood) can be augmented in a phasic manner via neural reflex pathways by a number of factors. Stimulation of irritant receptors and C fibers in a wide territory of the lower and upper airways, and possibly the esophagus and carotid bodies, results in the rapid onset (within seconds) of bronchoconstriction and increased mucus production in the central airways. Stimuli which can initiate these effects include mechanical irritation, a variety of irritant gases, aerosols and particles, rapid temperature changes, allergens, and several mediators such as histamine and some eicosanoids (Widdicombe 1979; Nadel 1980; reviewed by Gross & Skorodin 1984a). The bronchoconstriction resulting from these stimuli can be shown to be inhibitable or reversed at least in part by administration of atropine, supporting the notion that cholinergic mechanisms are responsible. This provides the rationale for the use of anticholinergic agents in diseases of airways obstruction. In addition there is indirect evidence that resting bronchomotor tone is increased in both asthma (Shah et al. 1990) and chronic obstructive pulmonary disease (COPD) (Gross et al. 1989a). Indeed, as discussed below (section Protection against bronchospastic stimuli), inhibiting bronchomotor tone may be the only means of producing bronchodilatation in COPD.

Muscarinic receptor subtypes Muscarinic receptors are a family of typical transmembrane protein receptors, three of which (M1, M2, and M3) are expressed in the human lung (Fig. 29.1). Our understanding of their interrelated functions is not complete (Gross & Barnes 1988; Fryer & Jacoby 1991, 1993; Coulson & Fryer 2003). The current concept is that M1 receptors are located in the peribronchial ganglia where they facilitate transmission of cholinergic traffic or possibly expand cholinergic activity to adjacent cells. M3 receptors are located on the effector cells, namely smooth muscle cells, bronchial mucosal cells, and possibly bronchial epithelial cells at which sites they mediate bronchoconstriction and other cholinergic actions. M2 recep-

Pharmacology and available agents The naturally occurring atropine-like alkaloids are all tertiary ammonium compounds, a designation which refers to the

PERIBRONCHIAL GANGLION

POSTGANGLIONIC NERVES

1

2

PREGANGLIONIC PARASYMPATHETIC NERVE N

1

684

tors are located on postganglionic nerves themselves and, in contrast, limit release of acetylcholine from those nerve terminals when stimulated by acetylcholine. Thus they have an autoreceptor action, braking or modulating cholinergic activity. The difference in activity of M1 and M3 receptors that mediate and promote bronchoconstriction on the one hand, and M2 receptors which tend to limit it on the other, has bearing on the development and clinical properties of selective anticholinergic agents as discussed in the next section. The important role of M2 receptors in limiting cholinergic activity may also be said to represent a potential vulnerability. Fryer and Jacoby have shown that M2 receptors are selectively damaged by parainfluenza virus infections (Fryer & Jacoby 1991) and by exposure to eosinophil products (Fryer & Jacoby 1993), providing an explanation for the bronchospasm commonly seen in association with these clinical situations. The distribution of muscarinic receptors in airways has been mapped by autoradiographic methods (Mak & Barnes 1990). Consistent with the location of vagal nerve endings, all three muscarinic receptor subtypes are located predominantly in the central airways. There are also some M1 receptors in the alveolar regions; their physiologic function is unknown. It should be stated that the rationale for the use of anticholinergic agents in diseases of airways is limited to their bronchodilator action by the inhibition of smooth muscle contraction. They have little or no effect on the numerous other mechanisms of airways obstruction. Although they have been shown to have some antiinflammatory actions in vitro (Morr 1979; Sato et al. 1998), the relevance of these to clinical practice is uncertain. In similar vein, experimental evidence from guinea-pig airways suggests that regular anticholinergic administration may decrease airway remodeling (Gosens et al. 2005).

SMOOTH MUSCLE

3

2

3

2

3

Fig. 29.1 Muscarinic receptor subtypes in airways. M1 receptors are localized to parasympathetic ganglia, M2 receptors (autoreceptors) are found on postganglionic cholinergic nerves, and M3 receptors on airway smooth muscle and mucus-producing cells.

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N

CH3 + CH3 N

CH3

O O Fig. 29.2 Structures of atropine sulfate, a tertiary ammonium anticholinergic, and tiotropium bromide, a quaternary ammonium anticholinergic.

Cholinergic Antagonists

C

CH2OH O

CH

ATROPINE

valence of the nitrogen atom on the tropane ring (Fig. 29.2). Consequently, atropine, scopolamine and other members of that family of alkaloids are freely soluble in aqueous media and lipids and cross mucosal surfaces and the blood–brain barrier rapidly and quantitatively. Following either inhalation or oral ingestion they are thus very widely distributed throughout the body and counteract multiple homeostatic actions of the parasympathetic system, producing dose-related systemic effects. This limits their clinical utility. Atropine inhalation, for example, causes bronchodilation in adult doses of about 1.0– 2.0 mg. At the same dose, patients are quite likely to experience dryness of the mouth, skin flushing, and mild tachycardia. At only slightly larger doses effects such as urinary retention, blurred vision, and mental changes can occur. Because of their very narrow therapeutic margin, tertiary ammonium anticholinergic agents are no longer recommended for purposes of bronchodilation. On the other hand, quaternary ammonium compounds, all of which are synthetic, have a five-valent nitrogen atom in the tropane ring which carries a charge. They are only moderately soluble, and cross lipid barriers with difficulty. They have local anticholinergic activity and will, for example, dilate the airways if inhaled or dilate the pupil when instilled in the eye. However, they are not significantly absorbed into the systemic circulation from either site even when delivered in large doses (Gross & Skorodin 1985). They can thus be regarded as topical anticholinergic agents and have a very wide therapeutic margin. This makes them well suited for use as bronchodilators. The class of quaternary anticholinergic agents includes ipratropium bromide (Atrovent), oxitropium bromide (Oxivent) and, most recently, tiotropium bromide (Spiriva). Other related quaternary agents that have been studied but not approved for routine clinical use as bronchodilators include atropine methonitrate, glycopyrrolate bromide (Robinul), and several long-acting agents in various stages of development that have only been identified by numbers as yet. Pharmacokinetic studies on the quaternary ammonium agents in humans show that, following oral or inhaled doses, the serum levels are relatively low, with peak blood levels being obtained at about 1–2 hours and half-lives of about 4 hours. However, blood level values do not reflect the bio-

O TIOTROPIUM

O

OH

C

C

S Br+ · H2O

S

Table 29.1 Dissociation half-lives (hours) of ipratropium and tiotropium on muscarinic receptor subtypes in Chinese hamster ovary cells. (From Disse et al. 1993, with permission.)

Ipratropium Tiotropium

M1

M2

M3

0.11 14.6

0.035 3.6

0.26 34.7

logical half-lives of the agents. The half-life of the agent on the muscarinic receptor is a more important determinant of the duration of activity of the agent. Table 29.1 shows the dissociation half-lifes of ipratropium at each of the relevant muscarinic receptors in comparison with those of tiotropium (Disse et al. 1993). The notable difference between the agents is the very much longer half-life of tiotropium than ipratropium on the M3 receptor. Consistent with this, the duration of bronchodilator effect of ipratropium is approximately 4–6 hours, and that of tiotropium is longer than 24 hours. (The corresponding value for oxitropium 6–8 hours, while that of glycopyrrolate is quite long, possibly also 24 hours.) Another notable feature of the data in Table 29.1 is the relatively short half-lifes of ipratropium and tiotropium on the M2 receptor. The significance of this is that these agents are functionally selective antagonists of M1 and M3 receptors. This is seen as an advantage over nonselective antimuscarinic agents such as atropine in that M3 antagonism is desirable from a therapeutic viewpoint and retention of M2 activity is also desirable. Concerning the pharmacodynamics of current antimuscarinic agents, as relatively small amounts are absorbed into the systemic circulation following inhalation or oral ingestion, exposure of bodily systems to quaternary agents is minimal, again in contrast to atropine and other tertiary ammonium compounds which are rapidly absorbed and widely distributed. Effects on the eye such as pupillary dilatation, leading to blurred vision and possibly acute glaucoma in susceptible individuals, or urinary retention in men, are very unlikely and rarely seen in practice. Furthermore, for the same reasons, very little crosses the blood–brain barrier to reach the central nervous system.

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The dose–response of anticholinergic agents given by various inhalational methods was provided in a previous review (Gross & Skorodin 1987) and is summarized only briefly here. For ipratropium by metered-dose inhaler (MDI), the optimal dose in young adults with asthma is 40–80 μg, but in older patients with COPD the optimal dose is much higher, possibly 160 μg, particularly when airways obstruction is severe (Gross et al. 1989b). That for oxitropium MDI is similar. By nebulized solution, the optimal dose of ipratropium is 500 μg in adults and 125– 250 μg in children (Gross et al. 1989b). With inhalers that employ a dry powder form without propellants the optimal dose may be a little lower; thus 10 μg of ipratropium delivered by Turbohaler was equipotent to 20 μg delivered by MDI (Bollert et al. 1997). The optimal dose of glycopyrrolate is 0.02 mg/kg. Dose-ranging studies of tiotropium dry powder administered by the Handihaler device showed little dose–response between 9 and 80 μg (Maesen et al. 1995; Littner et al. 2000). The approved dose of 18 μg once daily is also the optimal dose. Combinations of bronchodilators often result in greater bronchodilation than do single agents with less potential for adverse effects. Fixed-dose combinations of ipratropium and a short-acting β agonist, Combivent, Berodual and DuoVent, have been in wide use for both COPD and asthma since the l980s. Use of a fixed combination of ipratropium and albuterol for COPD, either in a metered-dose formulation (Petty 1994; Ikeda et al. 1995; Combivent Study Group 1997) or in a nebulized formulation, DuoNeb (Gross et al. 1998), shows that the combination results in greater bronchodilation than each of its components, although the duration of action is not significantly prolonged. The greater bronchodilation achieved by the combination was achieved without increasing the risk of side effects. Recently, combinations of tiotropium and the long-acting β agonist formoterol have been explored (Cazzola et al. 2005; Richter et al. 2006; van Noord et al. 2006). No such fixed combination is currently commercially available.

Clinical uses Protection against bronchospastic stimuli This topic is relevant mainly in asthmatic subjects. The protection afforded by anticholinergic agents against specific bronchospastic stimuli in a research setting has been reviewed (Gross & Skorodin 1984a). The degree of protection varies, being more or less complete against cholinergic agonists such as methacholine, but somewhat less against histamine, prostaglandins, nonspecific dusts and irritant aerosols, exercise, and hyperventilation due to cold dry air in asthmatic subjects (Ayala & Ahmed 1989; Azevedo et al. 1990). Ipratropium has no prophylactic effect against leukotriene-induced bronchoconstriction (Ayala et al. 1988). Ipratropium can prevent bronchospasm induced by beta-blocking agents, where

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β-adrenergic agents tend to be ineffective, and by psychogenic factors.

In stable COPD Stable COPD is the condition for which anticholinergic agents offer the most benefit and the prime indication for their use. Although patients with COPD usually do not exhibit as much response to any bronchodilator as do patients with asthma, most are capable of a clinically meaningful bronchodilator response (Gross 1986). A large number of studies have compared anticholinergic agents with other bronchodilators in patients with COPD (Thiessen & Pedersen 1982; Passamonte & Martinez 1984; Gross 1988). Most show that the anticholinergic agent is a more potent bronchodilator than other agents in COPD (Bleecker & Britt 1991; Braun et al. 1989; Tashkin et al. 1986). After large cumulative doses, an anticholinergic agent alone achieved all the available bronchodilatation in subjects with predominantly emphysema (Gross & Skorodin 1984b). In studies where bronchodilator responsiveness was compared between patients with asthma and COPD who had similar baseline airflows, patients with bronchitis had a better response to ipratropium than to adrenergic agents, the reverse being true for patients with asthma (Lefcoe et al. 1982). The possible explanation for the difference in responsiveness to an anticholinergic agent may be that in asthma, airflow obstruction has multiple mechanisms including mucosal inflammation and swelling besides smooth muscle contraction. Many of these mechanisms are at least partially modified by adrenergic agents but not by anticholinergics. In COPD, the major reversible component, and perhaps the only one, is bronchomotor tone which, being cholinergic in origin, is best reversed by an anticholinergic agent. Increases in forced expiratory volume in 1 s (FEV1) of 100–250 mL following optimal dosage of an anticholinergic agent can be seen in typical COPD patients who are in stable condition; and these changes are also typically associated with reductions in hyperinflation and increases in inspiratory capacity (Celli et al. 2003). The role of ipratropium for symptomatic relief and bronchodilation in COPD is now well established and ipratropium is currently recommended as a first-line treatment option for stable COPD in most current guidelines (Celli & MacNee 2004; GOLD 2006). The newer anticholinergic agent, tiotropium bromide, has somewhat different properties that merit separate consideration, as reviewed by Gross (2004). The prolonged half-life of tiotropium on the M3 receptor has been referred to previously. This confers the very long duration of action, more than 24 hours, on the agent. Following once-daily use, the trough FEV1, namely the FEV1 shortly before each daily administration, is consistently 100–200 mL greater than at the original baseline before the first dose (Casaburi et al. 2002; Vincken et al. 2002). The elevation in trough and postdose FEV1 seen after 6 weeks of regular once-

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Day 1

1.6


Day 8 Tiotropium day 8

Fig. 29.3 FEV1 profiles of tiotropium and ipratropium on day 1 and day 8 of a 1-year trial showing the increase in trough level seen with tiotropium. (From Vincken et al. 2002, with permission from the European Respiratory Journal.) (See CD-ROM for color version.)

Tiotropium day 1 Ipratropium day 1 Ipratropium day 8

1.4

1.3

Tiotropium (n = 329)

1.2

Ipratropium (n = 161) 1.1 –60

daily tiotropium were similar to those of twice-daily salmeterol plus fluticasone in a pilot study involving 107 patients with COPD (Bateman et al. 2006). The elevation in trough FEV1 is due to persistence of bronchodilatation from the previous doses, or carry-over of effect (Fig. 29.3). Not only is the trough FEV1 increased but so is the plateau level seen at about 3 hours after administration, albeit by a relatively small amount. This round-the-clock persistence of bronchodilatation is thought to provide better symptom control than the saw-tooth rise and fall seen in the 4–6 hours following repeated administrations of short-acting bronchodilators. This change in the FEV1 profile occurs over the first 2–4 days of regular once-daily administration of the drug (van Noord et al. 2002) and is not seen with other currently available long-acting bronchodilators. Trough forced vital capacity (FVC) rises in the same way but continues over the first 8 days of tiotropium administration. In the same way that airway function improves day-by-day following treatment initiation, it takes approximately 3 weeks to return to its original baseline following discontinuation (Littner et al. 2000). Another unique feature of tiotropium is that regular daily use has not been associated with any loss of efficacy or tachyphylaxis over a 1-year period of continuous treatment; there is even the as yet unconfirmed impression that the baseline FEV1 does not fall as much as would be expected from the usual age-related rate of FEV1 decline (Casaburi et al. 2002; Vincken et al. 2002). The latter impression is the subject of a 4-year ongoing trial (UPLIFT). (A similar long-term study with ipratropium failed to show any decrease in the agerelated decline of lung function (Anthonisen et al. 1994).) Along with the improvement in airflow, Celli et al. (2003) reported a decrease in static lung volumes consistent with a reduction in the static hyperinflation typically associated with moderate to severe COPD (Fig. 29.4). This would be expected to provide symptomatic relief from breathlessness and indeed the transition dyspnea index (TDI) of Mahler et al. (1996) was shown, in each of the 1-year studies cited above,

–5

30

60 Time (min)

120

Tiotropium

600

180

Placebo

400 200 0 mL

FEV1 (L)

1.5

–200 –400 –600 –800

FEV1

FVC

IC

FRC

SVC

Fig. 29.4 Changes in spirometry and static lung volumes following 4 weeks’ treatment of COPD patients with tiotropium or placebo. All differences between treatments were statistically significant (P < 0.01). (From Celli et al. 2003, with permission from Chest.)

to show a clinically meaningful decline in those patients who received tiotropium. Other studies show that regular tiotropium use also decreases the dynamic hyperinflation associated with moderate and severe COPD (O’Donnell et al. 2004; Maltais et al. 2005), dynamic hyperinflation being the augmentation of static hyperinflation that develops within minutes of the onset of an increase in ventilation. O’Donnell et al. (2004), for example, in a double-blind study, found that endurance time on a cycle ergometer improved approximately 21% in the COPD patients who had received tiotropium for 6 weeks as compared to the group that received placebo. Associated with this effect were reductions in residual volume and functional residual capacity, and increases in FVC and inspiratory

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Placebo: Exercise

Maximal 10

Recovery

Tiotropium: Exercise

Very, very severe 9

Recovery

8 Very severe 7

* †

6 Severe 5

†

Somewhat severe 4 *

Moderate 3 Slight 2 Very slight 1 †

Nothing at all 0 0

2

4

6

8

10

12

Exercise time (min)

capacity. Maltais et al. (2005) performed a similar doubleblind study with 261 COPD patients who received tiotropium or placebo. After 6 weeks, patients performed constant work rate cycle ergometry 2.25 and 8 hours after receiving their morning treatments. Patients in the tiotropium group were able to continue almost 3 min longer (> 30% increase in endurance time) with less dyspnea (as measured on a Borg scale) at each time point, the differences being statistically significant (Fig. 29.5). In the same study, lung volumes were measured before and during the ergometry protocol; these showed that operating lung volumes were significantly lower at baseline and at the end of exercise in the tiotropium group. Thus, after 6 weeks of regular tiotropium treatment, both static and dynamic hyperinflation were significantly reduced as was the intensity of exercise-induced dyspnea. One other clinically important outcome that has been extensively studied with regular tiotropium administration is quality of life (health status). Quality of life has been measured in each of the long-term tiotropium studies and almost uniformly shows an improvement in health status that is not only statistically significant but also clinically meaningful. The improvement may be detected at first measurement, e.g., 8 weeks into regular therapy, and persists throughout the months of study (Donohue et al. 2002). In the latter study, tiotropium was statistically better than placebo, but not better than salmeterol. Sleep disturbance is common in patients with COPD. A randomized double-blinded study involving 36 patients with moderate to severe COPD showed that ipratropium increased total sleep time, decreased the severity of nocturnal desaturation, and improved the patient’s perceptions of sleep quality (Martin et al. 1999).

Acute exacerbations of COPD Two questions are considered: whether an anticholinergic agent should be used for the management of an acute exacer-

688

14

16

18

20

Fig. 29.5 Intensity of dyspnea (Borg scale) 8 hours after tiotropium or placebo during constant work rate cycle ergometry to symptom limitation at 75% Wmax and the first 5 min of recovery on day 42. *, P = 0.05; †, P < 0.01 difference between groups. (From Maltais et al. 2005, with permission from Chest.)

bation, and whether regular use of an anticholinergic agent reduces the frequency of acute exacerbations. Regarding the first question, four studies failed to discern a difference among adrenergic agents, anticholinergic agents, or their combination (Rebuck et al. 1987; Karpel et al. 1990; Patrick et al. 1990; Koutsogiannis & Kelly 2000). Current guidelines (Celli & MacNee 2004; GOLD 2006) recommend the use of a short-acting β agonist initially, to which an anticholinergic agent may be added if the episode is severe or if the response to the adrenergic agent alone is unsatisfactory. Whether maintenance use of an anticholinergic agent reduces the frequency of acute exacerbations of COPD has been a secondary outcome in many ipratropium trials and in all the long-term tiotropium trials previously mentioned. However, it has been definitively studied (as a primary outcome) in two large trials. Niewoehner et al. (2005) performed a prospective study of 1829 US Veterans with moderateto-severe COPD. Patients were randomly assigned to either tiotropium in the approved dose or placebo once daily and were followed for 6 months. Both co-primary outcomes, the percentage of patients with an acute exacerbation and the percentage of patients requiring a COPD-related hospitalization, were reduced by about 25%. The numbers needed to treat to prevent one of these events were 23 and 40 respectively. A very similar study in Europe on 1010 patients with moderate to severe COPD has since been reported by Dusser et al. (2005) with almost identical results. In a metaanalysis that included 15 276 patients in 22 selected trials, Salpeter et al. (2006) found that anticholinergics but not β agonists significantly reduced the frequency of a severe exacerbation or death. The relative risk of a severe exacerbation with an anticholinergic agent compared with placebo was 0.67 (95% CI 0.53–0.81); that for “death related to a lower respiratory event” was 0.27 (95% CI 0.09–0.81). Thus tiotropium not only improves lung physiology in COPD, but improves a wide variety of clinically relevant

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outcomes. Accordingly, tiotropium is recommended by guidelines as an appropriate long-acting bronchodilator treatment option in patients with moderate or worse COPD. Some practical consideration concerning tiotropium use in the clinic should be mentioned. Because of its slow onset of action, tiotropium is not appropriate for use as a rescue bronchodilator but rather, if it is to be used, should be used as a regular daily maintenance treatment. The recommended dose (18 μg q.a.m.) is optimal and should not be exceeded. Tiotropium has no unfavorable interactions with other bronchoactive agents and can be used concomitantly with any other respiratory drugs, e.g., formoterol as recently described by van Noord et al. (2006). However, it is not appropriate to use tiotropium with other anticholinergic bronchodilators. There are theoretical concerns that its access to muscarinic receptors may be blocked if it is coadministered with another anticholinergic. While this has never been shown in a clinical study, nor is there evidence of any safety concern, as a practical matter ipratropium has almost no bronchodilator potency when the patient is receiving regular tiotropium (Kerstjens et al. 2004). So the use of ipratropium would be a waste. If and when patients on regular tiotropium experience “breakthrough” dyspnea, they should take a short-acting β-adrenergic agent for relief. A bronchodilator reversibility test before starting regular tiotropium administration is not predictive of a beneficial response to tiotropium and is unnecessary (Tashkin & Kesten 2003). Tiotropium is not recommended for the management of acute exacerbations of COPD; however, if the patient is already receiving tiotropium on a maintenance basis, it can be continued through the exacerbation.

Adult asthma When the first quaternary ammonium anticholinergic agent, ipratropium bromide, was being developed, it was assumed that it would be used exclusively to treat asthma. Consequently, the literature contains a substantial number of studies comparing the bronchodilator potential of anticholinergic agents with that of other bronchodilators in patients with asthma. These studies mostly date to the 1970s and 1980s. Typically, they show that ipratropium had a relatively slow onset of action by comparison with a short-acting β agonist, and that, on average, the peak improvement in airflow was not as great as that seen with the β agonist but the duration of action tended to be perhaps an hour or two longer than that of the β agonist (Ruffin et al. 1977). Thus, anticholinergic agents tended not to find much use in the treatment of stable asthma except possibly as adjunctive treatment. Nor have they received an asthma indication in the USA and some other countries. Nevertheless, it has been the clinical experience of many physicians that occasional asthma patients respond quite well to ipratropium. The reasons for this are unclear and attempts to identify subgroups of asthmatics likely to benefit from a response to anticholinergic therapy


have not been very successful. The bronchodilating effect of ipratropium may increase with age, in contrast to the decline in response to albuterol (Ullah et al. 1981). Patients with intrinsic asthma and those with longer duration of asthma may also respond better than individuals with extrinsic asthma (Jolobe 1984). However, both of these factors appear to be poor predictors of a response. An individual trial has been suggested as the best way to identify responsiveness (Brown et al. 1984). There are at present no definitive publications of the effects of tiotropium in asthma, nor is this agent approved in the USA for the treatment of asthma. Ipratropium is probably most widely used for asthmatic bronchospasm as a component of a number of combinations with β-adrenergic agents, examples being with salbutamol, as Combivent MDI, or in combination with fenoterol as Berodual MDI or DuoVent MDI. However, there are two instances where ipratropium may have a uniquely valuable role in asthma. Ipratropium is capable of achieving bronchodilatation in situations where a β-adrenergic blocking agent has been administered to an asthmatic patient who then experiences bronchospasm (Grieco & Pierson 1971). The β agonists are of course of little use in this situation. The second instance is in the treatment of psychogenic bronchospasm. McFadden et al. (1969) showed that prior administration of atropine prevented the bronchospasm induced by suggestion in susceptible subjects, ipratropium not being clinically available at that time. Additionally, Rebuck and Marcus (1979) showed that subjects whose asthma was believed to be psychogenic in origin experienced better and more prolonged bronchodilatation to ipratropium than did subjects whose asthma was believed not to be of psychogenic origin. Mention has been made above to the possible antiinflammatory effects of anticholinergic agents (Morr 1979; Sato et al. 1998; Gosens et al. 2005). A recent review of this field (Kanazawa 2006) provides further laboratory evidence pointing to possible future exploitation of anticholinergic agents in the long-term control of asthma. In acute severe asthma (status asthmaticus) there is a clearer picture of the role of anticholinergic agents, particularly ipratropium. The β-adrenergic agonists are more effective bronchodilators in the setting of acute severe asthma, and an anticholinergic agent should not be the sole initial bronchodilator. However, an anticholinergic agent may add to the bronchodilatation achieved by adrenergic monotherapy. Rebuck et al. (1987) found that the combination of 500 μg nebulized ipratropium with 1.25 mg nebulized fenoterol resulted in significantly more bronchodilatation during the first 90 min of treatment than either agent alone. Moreover, patients with more severe airway obstruction obtained the greatest benefit from the combination. Other studies have addressed this same question and a metaanalysis of 10 such studies (total 1377 patients) concluded that the addition of ipratropium reduced hospital admissions (relative risk 0.73) and increased FEV1 by 7.5% (average 100 mL, 95% CI

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50–149 mL) more than groups that received a beta adrenergic agent alone (Stoodley et al. 1999). These benefits were both statistically and clinically significant. It seems appropriate to recommend that both classes of bronchodilators be given in acute severe asthma, especially in the early hours of treatment (Brophy et al. 1998), and particularly in patients with more severe airflow obstruction. They can be given separately, or in a fixed combination (e.g., Combivent by MDI with spacer, or DuoNeb by nebulization). Conventionally, two to three doses should be given in the first hour of treatment.

Pediatric asthma Evidence in support of the use of anticholinergic agents in stable childhood asthma is sparse by comparison with that in adults, and is mostly confined to relatively small short-term clinical studies. Two consensus reports reviewed the published evidence and concluded that although ipratropium was safe for the pediatric population, its benefit as monotherapy compared with adrenergic monotherapy was slight at best (Warner et al. 1989; Hargreave et al. 1990). As in adult asthma, there is evidence that the addition of an anticholinergic may augment the bronchodilation achieved by albuterol alone in children aged 10–18 years with stable asthma (Vichyanond et al. 1990). There are no systematic studies of long-acting anticholinergic agents in childhood asthma to my knowledge. There are also scattered reports of ipratropium use in other pediatric conditions such as cystic fibrosis, viral bronchiolitis, exercise-induced bronchospasm, and bronchopulmonary dysplasia, but these do not provide strong and consistent evidence for the benefit of ipratropium over alternative bronchodilators. For acute severe asthma in children, two well-conducted trials in the 1980s showed that the addition of ipratropium accelerated the rate of improvement in airflow over albuterol alone (Beck et al. 1985; Reisman et al. 1988). More recent studies have yielded conflicting results regarding the efficacy of combination therapy over monotherapy (Schuh et al. 1995; Ducharme & Davis 1998; Qureshi et al. 1998; Zorc et al. 1999). A systematic review of this question that included 10 selected studies (Plotnick & Ducharme 1998) concluded that combination therapy which included ipratropium was safe, improved lung function, and reduced hospitalization rates, especially in children with severe asthma. Therefore, as in adults, an anticholinergic alone is not recommended in acute severe asthma but the combination of ipratropium with an adrenergic agent is safe and may be more effective than adrenergic monotherapy, particularly in severe exacerbations.

Adverse effects Atropine and its natural congeners, being well absorbed and widely distributed in the body produce numerous systemic

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side effects, which is the principal reason they are no longer used as bronchodilators. The quaternary agents that are currently used for respiratory purposes are poorly absorbed. Ipratropium bromide was carefully monitored for atropinelike adverse effects, particularly for effects on the eye (narrowangle glaucoma), urinary tract (urinary retention in males), and respiratory mucus transport. Ipratropium was found to be essentially free of such atropine-like effects after extensive investigation (Gross 1988). It can, for example, be given to patients with glaucoma without affecting intraocular tension, provided it is not sprayed directly into the eye (Watson et al. 1994). It has been found not to affect urinary flow characteristics in older men. Nor has it been found to alter the viscosity and elasticity of respiratory mucus, or mucociliary clearance, as does atropine (Pavia et al. 1979). It has negligible effects on hemodynamics, minute ventilation (Tobin et al. 1984), and the pulmonary circulation (Chapman et al. 1985). Consequently, administering ipratropium or tiotropium does not carry the risk of worsening hypoxemia (Gross & Bankwala 1987; Gross et al. 2003) as do adrenergic agents (Ashutosh et al. 1995), a theoretical consideration in exacerbations of asthma and COPD. Even massive, inadvertent overdosage of one such agent resulted in trivial effects (Gross & Skorodin 1985). In normal clinical use, the only side effects of ipratropium and tiotropium are dryness of the mouth, a typical local effect of anticholinergic agents. Sometimes a brief coughing spell occurs. Paradoxical bronchoconstriction occurs in perhaps 0.3% of patients that receive ipratropium. Paradoxical bronchoconstriction, which may also occur with other anticholinergic agents, warrants withdrawing the drug from that patient. Concerns about the high incidence of cardiovascular events in patients with COPD have led to studies of the cardiac effects of long-term bronchodilator use. Covelli et al. (2005) reported a study of 225 patients with moderately severe COPD who received tiotropium or placebo for 12 weeks. Serial cardiograms and Holter monitoring were performed. The investigators found no evidence of significant ECG changes, or changes in heart rate, rhythm, or QT intervals during the study. The frequency of cardiac events of any kind was low, serious adverse events occurring in one patient in the tiotropium group (onset of atrial fibrillation) and two patients in the placebo group. For similar purposes, a retrospective review of several long-term tiotropium trials was performed by Morganroth et al. (2004). In all, 1167 patients received tiotropium, 405 patients received salmeterol, and 846 patients received placebo for periods from 1 to 12 months of observation. In each study, serial ECGs and Holter monitoring were performed. No significant differences in any of the ECG or Holter outcomes were detected. Specifically, there were no clinically relevant arrhythmias or changes in heart rate or conduction patterns. Thus, extensive investigation and the worldwide use of ipratropium for over two decades and tiotropium for 3 years demonstrate a low incidence of untoward reactions.

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forced vital capacity in normal, asthmatic and bronchitic subjects after salbutamol and ipratropium bromide. Respiration 43, 304–16. Tobin, M.J., Hughes, J.A. & Hutchison, D.C. (1984) Effects of ipratropium bromide and fenoterol aerosols on exercise tolerance. Eur J Respir Dis 65, 441–6. Ullah, M.I., Newman, G.B. & Saunders, K.B. (1981) Influence of age on response to ipratropium and salbutamol in asthma. Thorax 36, 523– 9. Van Noord, J.A., Smeets, J.J., Custers, F.L. et al. (2002) Pharmacodynamic steady state of tiotropium in patients with chronic obstructive pulmonary disease. Eur Respir Dis 19, 639–44. Van Noord, J.A., Aumann, J.L., Janssens, E. et al. (2006) Effects of tiotropium with and without formoterol on airflow obstruction and resting hyperinflation in patients with COPD. Chest 129, 509– 17. Vichyanond, P., Sladek, W.A., Sur, S., Hill, M.R., Szefler, S.J. & Nelson, H.S. (1990) Efficacy of atropine methylnitrate alone and in combination with albuterol in children with asthma. Chest 98, 637– 42. Vincken, W., van Noord, J.A., Greenhorst, A.P. et al. (2002) Dutch/ Belgian Tiotropium Study Group. Imroved health outcomes in patients with COPD during 1 yr’s treatment with tiotropium. Eur Respir J 19, 209–16. Warner, J.O., Gotz, M., Landau, L.I., Levison, H., Milner, A.D. & Pedersen, S. (1989) Management of asthma: a consensus statement. Arch Dis Child 64, 1065–79. Watson, W.T., Shuckett, E.P., Becker, A.B. & Simons, F.E. (1994) Effect of nebulized ipratropium bromide on intraocular pressures in children. Chest 105, 1439–41. Widdicombe, J.G. (1979) The parasympathetic nervous system in airways disease. Scand J Respir Dis Suppl 103, 38–43. Zorc, J.J., Pusic, M.V., Ogborn, C.J., Lebet, R. & Duggan, A.K. (1999) Ipratropium bromide added to asthma treatment in the pediatric emergency department. Pediatrics 103, 748–52.

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Antileukotriene Agents Graeme P. Currie and Brian J. Lipworth

Summary A variety of proinflammatory cells, mediators, and cytokines orchestrate the development of airway hyperresponsiveness, which results in the episodic airflow obstruction characteristic of asthma. As a consequence, modulation of the underlying disease process with antiinflammatory agents is firmly established as being the cornerstone of successful management. Inhaled corticosteroids are the most potent antiinflammatory agents available and satisfactorily suppress underlying airway inflammation in most individuals. However, despite regular treatment with inhaled corticosteroids, patients can experience persistent symptoms and exacerbations due to unchecked airway inflammation and hyperresponsiveness. Moreover, children and the elderly may have a dislike of inhaled treatment and demonstrate difficulties in coordinating some hand-held devices, while some individuals may also express a preference for oral medication. Cysteinyl leukotrienes are potent proinflammatory and bronchoconstrictor lipid mediators that perpetuate the asthmatic inflammatory response. Accumulating data suggest that inhaled corticosteroids fail to have a significant impact on cysteinyl leukotriene levels in the airway, which in turn has prompted the development and introduction of drugs (antileukotrienes) that attenuate their effects in the airway. For example, drugs such as 5-lipoxygenase inhibitors prevent the synthesis of cysteinyl leukotrienes and leukotriene receptor antagonists interfere with the binding of cysteinyl leukotrienes to their cellular receptor. Leukotriene receptor antagonists are used across the world and demonstrate a safe adverse-effect profile, although 5-lipoxygenase inhibitors are less widely used mainly due to multiple daily dosing regimes and concerns relating to hepatotoxicity. Over the past decade, leukotriene receptor antagonists have emerged as useful oral nonsteroidal antiinflammatory adjuncts both as monotherapy and in combination with other classes of drugs across the full spectrum of asthma severities. Moreover, they may have a particularly beneficial role in exercise-induced


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asthma, aspirin-sensitive asthma, and individuals with concomitant allergic rhinitis. As more data emerges, it is becoming increasingly noticeable that monitoring the effects of leukotriene receptor antagonists in terms of lung function alone may result in clinicians missing potentially beneficial effects on inflammatory biomarkers, airway hyperresponsiveness, and subsequent exacerbations. Recent studies have also suggested that some individuals with asthma may be predisposed to exhibit a preferential response to antileukotrienes according to genotype, which may well tailor the decision to initiate such treatment in the future.

Introduction A variety of proinflammatory cells, mediators and cytokines orchestrate the development of airway hyperresponsiveness, which results in the episodic airflow obstruction characteristic of asthma. As a consequence, suppression of inflammation using inhaled corticosteroids forms the gold standard treatment and are advocated in all but the mildest of disease (British guideline on the management of asthma 2003; GINA Workshop Report 2004). Once bound to cytoplasmic receptors, inhaled corticosteroids increase and decrease the gene transcription of antiinflammatory and proinflammatory mediators respectively. They also exert a direct inhibitory effect on a number of cells such as eosinophils, T lymphocytes, and epithelial cells implicated in the asthmatic inflammatory process. As a result, they attenuate airway hyperresponsiveness over several weeks, although the maximal effect may not be achieved until after several months of regular use (Barnes 1990; Barnes et al. 1998). It is important to note that inhaled corticosteroids do not attenuate all the effects of inflammatory cells and mediators involved in the pathogenesis of asthma (Schleimer et al. 1989; Cox 1995; Gyllfors et al. 2006). Moreover, their effects on the synthesis and release of cysteinyl leukotrienes, which are potent bronchoconstrictor and proinflammatory mediators, are limited (Booth et al. 1995; Wenzel et al. 1997; Pavord et al. 1999). After being established on inhaled corticosteroids, additional second-line preventative therapy is advocated in cur-

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Antileukotriene Agents

Phospholipid bilayer Phospholipase A2 Cyclooxygenase

Arachidonic acid

5-Lipoxygenase/ FLAP

Prostaglandins Thromboxanes

5-HPETE Leukotriene A4 hydrolase Leukotriene B4

5-Lipoxygenase/ FLAP Leukotriene A4 Leukotriene C4 synthase Leukotriene C4 g-Glutamyl transpeptidase

Fig. 30.1 Synthesis pathway of cysteinyl leukotrienes and their effects in the airway. FLAP, 5-lipoxygenase-activating protein; HPETE, 5-hydroxyperoxyeicosatetraenoic acid.

rent guidelines for patients with persistent symptoms (British guideline on the management of asthma 2003; GINA Workshop Report 2004). The use of concomitant second-line therapy has become increasingly important as the dose–response effect (in terms of lung function) for inhaled corticosteroids such as fluticasone propionate becomes relatively flat at daily doses greater than 500 μg (Holt et al. 2001). Moreover, there is a greater propensity to develop systemic adverse effects with increasing doses (Lipworth 1999), while patients may express a preference for treatment that avoids an excessive corticosteroid burden. This all suggests that alternative nonsteroidal antiinflammatory therapy may play an increasingly prominent role in the successful management of asthma. Over the past few decades, a substantive amount of data has emerged indicating that cysteinyl leukotrienes, a family of bioactive fatty acids, play an important part in mediating crucial aspects of the asthmatic inflammatory cascade (Drazen 1998). In this chapter we provide a summary of the pharmacologic and clinical effects of drugs which block the actions of cysteinyl leukotrienes in the airway (antileukotrienes). We also highlight the role of antileukotriene drugs across a variety of asthma phenotypes and severities, and of their effects when used as both monotherapy and concomitant second-line treatment.

Pathophysiology Cysteinyl leukotriene biosynthesis The cysteinyl leukotrienes (LTC4, LTD4 and LTE4) are lipid mediators produced from an arachidonic acid precursor fol-

Cysteinyl leukotriene receptor

Leukotriene D4 Dipeptidase Leukotriene E4

lowing a series of enzymatic steps. Arachidonic acid is firstly released from the phospholipid bilayer by phospholipase A2 and may be metabolized by either the cyclooxygenase (COX) or 5-lipoxygenase pathway (Fig. 30.1) (Lam et al. 1990; Horwitz et al. 1998). Once the unstable precursor LTA4 has been produced, it may be converted in neutrophils or monocytes to the noncysteinyl LTB4 by LTA4 hydrolase. In mast cells, eosinophils, macrophages, and basophils, LTA4 may alternatively be converted into LTC4 by LTC4 synthase and subsequently into LTD4 and LTE4. The cysteinyl leukotrienes then exert their effects following activation of specific receptors located on cell membranes of airway smooth muscle and macrophages. LTB4 activates the BLT receptor, while LTC4, LTD4 and LTE4 activate cysteinyl leukotriene receptor subtypes 1 and 2.

Effects of cysteinyl leukotrienes The cysteinyl leukotrienes exhibit a variety of effects associated with perpetuating the asthmatic inflammatory process (Table 30.1). LTC4, LTD4 and LTE4 are also potent bronchoconstrictor stimuli and have been shown to increase the

Table 30.1 Effects of cysteinyl leukotrienes in asthma leading to airway hyperresponsiveness and airflow obstruction. Mucus hypersecretion Inhibition of mucociliary clearance Hypertrophy and proliferation of smooth muscle Increased pulmonary vascular permeability Recruitment of inflammatory cells Release of acetylcholine from nerve fibers

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extent of airway hyperresponsiveness following exposure to histamine and methacholine (O’Hickey et al. 1991). Cysteinyl leukotrienes are also found in sputum, urine, nasal secretions, plasma, and bronchoalveolar lavage fluid following bronchial challenge tests and in individuals with spontaneous exacerbations of asthma (Lam et al. 1988; Wenzel et al. 1990; Wenzel et al. 1995). LTB4 is a neutrophil chemoattractant and is involved in the production of interleukin (IL)-5 (Yamaoka & Kolb 1993), although not thought to be closely implicated in the pathophysiology of asthma. The cysteinyl leukotrienes therefore appear to be potentially important mediators intrinsically associated with the asthmatic inflammatory response, and this knowledge has prompted the development and subsequent introduction of drugs which attenuate their effects in the airway.

Antileukotriene drugs Two main strategies have been developed to block the effects of cysteinyl leukotrienes in the airway. One method is to use drugs that prevent their synthesis (using a 5-lipoxygenase inhibitor) and the other involves interfering with the binding of cysteinyl leukotrienes to their cellular receptor using a leukotriene receptor antagonist (LTRA).

Leukotriene receptor antagonists LTRAs such as montelukast (the most commonly used), zafirlukast, and pranlukast selectively antagonize the cysteinyl leukotriene 1 receptor. These drugs have been shown to reduce airway hyperresponsiveness following exposure to both direct and indirect bronchoconstrictor stimuli. For example, in a metaanalysis of 12 trials (N = 353 subjects with asthma) (Currie & Lipworth 2002), significant effects on attenuating airway hyperresponsiveness was observed amounting to a 0.85 doubling dose/dilution shift (95% CI 0.69–1.02) from baseline. LTRAs have also been shown to reduce a variety of surrogate inflammatory biomarkers such as airway and blood eosinophils, eosinophil cationic protein, and exhaled nitric oxide (Yoshida et al. 2000; Wilson et al. 2001a; Minoguchi et al. 2002; Bjermer et al. 2003; Currie et al. 2003a; Ilowite et al. 2004). Moreover, LTRAs are effective following single oral doses (Dempsey et al. 2000), and unlike long acting β2 agonists (Grove & Lipworth 1995), tolerance to their bronchoprotective effects has not been demonstrated (Wilson et al. 2001a; Sims et al. 2003). There are also animal data which suggest that LTRAs may significantly reduce parameters of airway remodeling (Henderson et al. 2002).

it is generally no longer widely prescribed mainly due to concerns regarding hepatotoxicity. Far fewer data have been published regarding the putative effect of 5-lipoxygenase inhibitors and effects on surrogate inflammatory biomarkers and airway hyperresponsiveness. However, there is evidence in both animals and asthmatics that zileuton does have inhibitory effects on eosinophils (Munoz et al. 1997; Dahlen et al. 1998). Single doses of zileuton have been shown to significantly attenuate airway hyperresponsiveness to direct bronchoconstrictor stimuli (Dekhuijzen et al. 1997). For example, in a randomized, double-blind, placebo study in mild asthmatics receiving inhaled corticosteroids, a single dose of zileuton 400 mg increased the provocative dose of histamine causing a 20% fall in forced expiratory volume in 1 s (FEV1) by 2.1 doubling doses compared with placebo (P < 0.03). Chronic dosing with zileuton has also been shown to attenuate airway hyperresponsiveness to a significant degree (Fischer et al. 1995).

Prescribing antileukotrienes In the UK, Europe and the USA, montelukast is licensed for once-daily oral administration in adults and is also available as a cherry flavored pink tablet or as granules for use in children over the age of 6 months. Zafirlukast is licensed for use in individuals over 12 years of age (Fig. 30.2). In some countries such as Japan, another LTRA, pranlukast, is available for use. In the USA, zileuton is licensed for use in those over 12 years of age (Table 30.2).

CH3

Zafirlukast

N

O N H

O

CH3 O N H O

O

S O CH3

COONa

Montelukast

OH S

5-Lipoxygenase inhibitors These drugs are divided into those which bind and deactivate 5-lipoxygenase-activating protein or directly inhibit 5lipoxygenase. Zileuton is the only 5-lipoxygenase inhibitor available for use in some countries such as the USA. However,

696

N CI

Fig. 30.2 The chemical structures of zafirlukast and montelukast.

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Table 30.2 Adult prescribing information and pharmacokinetics relating to antileukotrienes.

Use in pregnancy Protein binding Half-life Time to peak levels Bioavailability

Montelukast Singulair Inhibitor of leukotriene receptor 10 mg daily Yes No dose adjustment No dose adjustment in mild-tomoderate dysfunction Limited data > 99% 2.7–5.5 hours 3–4 hours 64%

Special instructions Interaction with warfarin

Can be taken with food Not known to interact

Generic name Brand name Mode of action Adult dose Pediatric licence Prescribing in renal impairment Prescribing in hepatic impairment

Advantages of oral treatment over inhaled treatment Patients with asthma notoriously find difficulty in using inhalers. For example, children and the elderly frequently have problems coordinating some hand-held devices, while spacers (which are designed to help overcome this problem) tend to be cumbersome, less portable, and pose difficulties in optimal use and care. This in turn may lead to suboptimal adherence to inhaled treatment regimens. Patients started on inhaled drugs for asthma require initial education and subsequent reinforcement in terms of correct use of a particular device. As a consequence, it is conceivable that oral asthma medication (such as with antileukotrienes) is advantageous in the “real world” and may confer some benefit over and above inhaled antiinflammatory therapy. Indeed, previous data have shown that patient compliance and acceptance of treatment are significantly superior for oral compared with inhaled regimens (Kelloway et al. 1994; Bukstein et al. 2003).

Adverse effects LTRAs are generally well tolerated (Storms et al. 2001), even in children (Price 2001). However, adverse effects such as hypersensitivity reactions, arthralgia, pulmonary eosinophilia, gastrointestinal disturbances, sleep disorders, respiratory tract infections, hallucinations, seizures, and raised transaminases have been reported (www.bnf.org). Due to lack of data, their use in pregnancy is not advised. Concerns have been raised regarding the development of Churg–Strauss syndrome and administration of LTRAs (Hosker & Tuggey 2001). Many, although not all, of the documented cases of Churg–Strauss syndrome have been in patients in whom concomitant LTRA treatment has permitted a reduction in dose of inhaled corticosteroid. This in turn suggests that latent Churg–Strauss syndrome may have been unmasked by a reduction in anti-

Zafirlukast Accolate Inhibitor of leukotriene receptor 20 mg twice daily No No dose adjustment Reduced clearance

Zileuton Zyflo 5-lipoxygenase inhibitor 600 mg four times daily No No dose adjustment Contraindicated

Limited data > 99% 10 hours 3 hours Unknown, but reduced by 40% with food Avoid with food Increases prothrombin time

Limited data 93% 2.5 hours 1.7 hours Unknown Can be taken with food Increases prothrombin time

inflammatory therapy delivered to the lungs (Lipworth & Wilson 2001). Liver function derangement has been reported with zileuton (elevated transaminase levels) and may be a greater problem in females over 65 years of age and in those with preexisting elevated transaminases. As a consequence, liver function tests should be monitored and the drug discontinued if signs or symptoms of liver dysfunction were to develop. Other reported adverse effects include headache, dyspepsia, nausea, abdominal pain, leukopenia, asthenia, insomnia, rashes, and myalgia.

Current position of antileukotrienes Mild persistent asthmatics should preferably be treated with a low to medium dose (400–800 μg/day of beclomethasone dipropionate or equivalent) of inhaled corticosteroid, although guidelines suggest that an alternative option, especially in individuals with prominent exercise-related symptoms, is monotherapy with an antileukotriene. When symptoms persist, despite good compliance with inhaled corticosteroids, patients should preferentially be started on a long-acting β2 agonist. An antileukotriene should then be considered if symptoms persist or the therapeutic trial of long-acting β2 agonist is unsuccessful (British guideline on the management of asthma 2003; GINA Workshop Report 2004).

Antileukotrienes as monotherapy in asthma Patients frequently express a preference for oral rather than inhaled treatments, which can translate into greater acceptance and better compliance with oral formulations of antiasthma drugs (Kelloway et al. 1994). It therefore appears logical to consider whether antileukotrienes might be a satisfactory

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nonsteroidal antiinflammatory alternative to inhaled corticosteroids as first-line controller therapy in mild persistent asthmatics. In a double-blind, multicenter, placebo-controlled trial, 146 mild-to-moderate asthmatics not using inhaled corticosteroids were randomized to receive zafirlukast 20 mg twice daily or placebo for 13 weeks (Suissa et al. 1997). The zafirlukast group (mean FEV1 74% predicted) had 89% more days without symptoms (P = 0.03), 89% more days without reliever use (P = 0.001), and 98% more days without episodes of asthma (P = 0.003) compared to the placebo group (mean FEV1 84% predicted). Individuals randomized to receive zafirlukast also experienced 55% fewer healthcare contacts (P = 0.007) and 55% fewer days of absence from work or school (P = 0.04). Thus, the clinical and putative economic benefits associated with zafirlukast in this study indicate a possible role for LTRAs as monotherapy in patients with mild to moderate asthma. Another study collected data from five randomized double-blind trials where the effects of zafirlukast 20 mg twice daily was compared to placebo in corticosteroid-naive asthmatics in terms of effects on exacerbations (Barnes & Miller 2000). Overall, the risk of an asthma exacerbation requiring withdrawal from zafirlukast-treated groups was approximately half that of placebo (odds ratio 0.45, 95% CI 0.26– 0.76; P = 0.003). Similar beneficial effects with zafirlukast versus placebo were also observed for exacerbations requiring additional medication (odds ratio 0.47, 95% CI 0.30–0.74; P = 0.001) and oral corticosteroid use (odds ratio 0.53, 95% CI 0.32– 0.86; P = 0.010). Studies with montelukast have also shown that benefits occur when LTRAs are used as monotherapy (Laviolette et al. 1999; Malmstrom et al. 1999). For example, 895 asthmatics (mean FEV1 65% predicted) were randomized to receive montelukast 10 mg/day, inhaled beclomethasone 400 μg/day or placebo (Malmstrom et al. 1999). Over the 12-week treatment period, the average improvement from baseline in FEV1 was 13.1% with beclomethasone, 7.4% with montelukast, and 0.7% with placebo (P < 0.001 for each active treatment vs. placebo and P < 0.01 for beclomethasone vs. montelukast). The average reduction from baseline in daytime symptom score was 0.62 for beclomethasone, 0.41 for montelukast, and 0.17 for placebo (P < 0.001 for each active treatment vs. placebo and P < 0.01 for beclomethasone vs. montelukast). Both active treatments significantly improved peak expiratory flow (PEF) and quality of life, reduced nocturnal awakenings, and decreased the number of days with asthma exacerbations (P < 0.001 for each active treatment vs. placebo and P < 0.01 for beclomethasone vs. montelukast for each end point). Treatment with montelukast and inhaled beclomethasone also resulted in similar significant reductions in blood eosinophils (P < 0.05 vs. placebo). Thus, beclomethasone demonstrated superior clinical benefits versus montelukast, although montelukast conferred significant superiority over placebo. In the same study, montelukast had a faster onset

698

*

8 * Fold difference from placebo at 4 weeks

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4 *

*

2

1

0.5 Methacholine

AMP

Fig. 30.3 Effects on airway hyperresponsiveness with methacholine and adenosine monophosphate (AMP). Geometric fold differences (95% CI) from placebo for inhaled corticosteroid (open bars) and leukotriene receptor antagonist (blue bars) are shown. *, P < 0.05 vs. placebo. (From Dempsey et al. 2002, with permission.)

of action and a greater initial effect than beclomethasone. In another study comparing once-daily hydrofluoroalkane triamcinolone 450 μg/day versus montelukast 10 mg/day as monotherapy in mild persistent asthma, significant and similar improvements were observed on the primary outcome of airway hyperresponsiveness to methacholine and adenosine monophosphate after 4 weeks (Fig. 30.3) (Dempsey et al. 2002). However, triamcinolone was superior to montelukast in attenuating other surrogate inflammatory biomarkers such as exhaled nitric oxide, blood eosinophils, and eosinophil cationic protein. Other head-to-head comparisons of inhaled corticosteroids with LTRAs have tended to favor the former across a variety of end points. For example, a metaanalysis of 13 trials demonstrated that inhaled corticosteroids equivalent to beclomethasone 400 μg/day were more effective than LTRAs in adults with mild or moderate asthma (Ducharme 2003). Individuals treated with LTRAs were 60% more likely to experience an exacerbation requiring oral corticosteroids (relative risk 1.6, 95% CI 1.2–2.2), while a 130-mL greater improvement (95% CI 80–170 mL) in FEV1 and a 19 L/min greater increase (95% CI 14–24 L) in morning PEF was found when inhaled corticosteroids were compared to LTRAs. However, in one large study over 6 weeks evaluating mild asthmatics (mean FEV1 67% predicted, N = 782), montelukast was as effective as 400 μg/day of inhaled beclomethasone in preventing asthma exacerbations (Israel et al. 2002), despite the latter being significantly superior in improving the FEV1. This in turn leads to the notion that monitoring lung function alone may be a poor indicator of overall asthma control (Teeter & Bleecker 1998), and doing so may miss potentially beneficial effects. A disconnection between effects of antiinflammatory

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treatment on lung function and exacerbations has also been observed in more severe asthmatics (Pauwels et al. 1997). For example, a fourfold increase from 200 to 800 μg/day of budesonide conferred no further improvement in lung function but did result in a further significant reduction in exacerbations (P < 0.001) (Pauwels et al. 1997). Thus, when optimizing antiinflammatory treatment in asthma, lung function is relatively distant from the underlying inflammatory process, and despite little change further beneficial clinical effects may actually occur. Thus, while end points such as lung function are of undoubted value, clinicians must not lose sight of the basic pathophysiologic hallmarks of the asthma syndrome (i.e., airway hyperresponsiveness and inflammation) along with the impact of asthma pharmacotherapy on exacerbations. Indeed, since LTRAs principally exhibit antiinflammatory actions and attenuate airway hyperresponsiveness, and are only weak bronchodilators, monitoring lung function alone may miss beneficial effects occurring in both “real-life” and in randomized trials (Currie et al. 2003a; Currie & Lee 2005). The current literature presently suggests that when used as first-line therapy, LTRAs confer significant improvements in parameters of asthma control, although they tend to be clinically inferior to low to moderate doses of inhaled corticosteroids. Asthmatics with mild disease, especially those with exercise-induced symptoms (Leff et al. 1998), who are disinclined to use regular inhaled corticosteroids, LTRAs appear to be suitable asthma controller therapy. As several inhaled corticosteroids are now licensed for once daily use, including easy to use breath-actuated devices (e.g., budesonide dry powder, mometasone dry powder, ciclesonide pressurized metereddose inhalers), there would appear to be little rationale for choosing an antileukotriene as first-line antiinflammatory therapy in persistent asthmatics, unless patients are unable or unwilling to use inhaler devices. However, one potential advantage of using montelukast in concomitant atopic asthma and rhinitis is its efficacy in allergic rhinitis which would not occur with orally inhaled corticosteroids alone.

Antileukotrienes as add-on therapy to inhaled corticosteroids in asthma Despite optimum drug delivery and good compliance with inhaled corticosteroids, many patients experience symptoms and exacerbations. Dose–response studies using inhaled corticosteroids have generally been unable to demonstrate any significant difference between individual doses of inhaled corticosteroids (Holt et al. 2001; Masoli et al. 2005). For example, a metaanalysis evaluated eight studies (2324 asthmatics) where the effects of at least two doses of fluticasone were measured (Holt et al. 2001). Most therapeutic gain (in terms of airway caliber, symptoms, and effects on exacerbations) was achieved at daily doses of fluticasone between 100 and 250 μg. Indeed, it is generally accepted that at daily doses


greater than 800 μg of beclomethasone or equivalent in adults, the dose–response curve for desired effects becomes flat, while that for systemic adverse effects becomes steep (Lipworth & Wilson 1998). Since inhaled corticosteroids have only limited impact on the synthesis or release of cysteinyl leukotrienes (O’Shaughnessy et al. 1993; Dworski et al. 1994; Booth et al. 1995; Wenzel et al. 1997; Pavord et al. 1999), concomitant inhaled corticosteroid plus antileukotriene therapy may facilitate more complete attenuation of the inflammatory pathway. Moreover, even a high dose of inhaled fluticasone propionate has been shown to confer no significant effect on airway hyperresponsiveness to LTD4 or urine cysteinyl leukotriene concentrations (Gyllfors et al. 2006). Numerous studies, including a Cochrane metaanalysis, have suggested that LTRAs are useful as add-on therapy to inhaled corticosteroids across a range of outcome parameters (Laviolette et al. 1999; Lofdahl et al. 1999; Virchow et al. 2000; Ducharme 2002; Vaquerizo et al. 2003). For example, in a 16-week, multicenter, double-blind study, the efficacy of adding montelukast to a constant dose of inhaled budesonide (400 –1600 μg/day) was evaluated (Vaquerizo et al. 2003). A total of 639 symptomatic asthmatics (mean FEV1 81% predicted) were randomized to receive add-on montelukast 10 mg/day (N = 326) or placebo (N = 313). The frequency of asthma exacerbation days was 35% lower (P = 0.03) and asthma-free days was 56% higher (P = 0.001) in the montelukast versus placebo group. Individuals receiving montelukast also benefited from significant reductions in nighttime symptoms and reliever use (P < 0.05), and a significant improvement in PEF (P < 0.05) compared to placebo. In the same study, improvements in asthma control occurred despite no significant improvement in FEV1. This may well be because the mean FEV1 at baseline was within the lower limits of normal (81%) thus allowing little further room for improvement. Similar to the findings by Israel et al. (2002), this provides further evidence that FEV1 may not be the most sensitive parameter by which to detect improvements in asthma control. In other words, significant reductions in exacerbation frequency can occur (due to reduction in inflammation and airway hyperresponsiveness) without altering airway caliber to any great extent. Similar effects have also been observed with zafirlukast as add-on therapy to inhaled corticosteroids. In a double-blind parallel group study, 368 symptomatic asthmatics receiving high-dose inhaled corticosteroids (1000–4000 μg/day) were randomized to receive add-on zafirlukast 80 mg twice daily over 6 weeks (Virchow et al. 2000). Compared with placebo, zafirlukast produced a significant improvement over baseline in the primary end point of morning PEF (18.7 vs. 1.5 L/min, P < 0.001), in addition to evening PEF (P < 0.01), FEV1 (P < 0.05), daytime symptom scores (P < 0.001), and reliever use (P < 0.001). Furthermore, zafirlukast significantly reduced the risk of an exacerbation of asthma (odds ratio 0.61, 95% CI 0.38–0.99) and the risk of patients requiring a further

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increase in asthma controller therapy (odds ratio 0.4, 95% CI 0.2–0.8). In another study, the inhaled corticosteroid-sparing effects of montelukast was evaluated in a double-blind, randomized, placebo-controlled, parallel group design (Lofdahl et al. 1999). Following a single blind placebo run-in period, during which at least two decreases in inhaled corticosteroid dose occurred, patients (N = 226) were randomized to receive montelukast 10 mg/day or matching placebo for up to 12 weeks. Every 2 weeks, the inhaled corticosteroid dose was tapered, maintained, or increased based on a standardized clinical score. Compared with placebo, montelukast allowed a significant (47% vs. 30%, P = 0.046) reduction in the inhaled corticosteroid dose. Although LTRAs confer additive effects when used as second-line therapy, few studies have directly examined the effects of LTRAs plus inhaled corticosteroid versus a higher dose of the latter. However, in a double-blind, randomized, parallel group, multicenter 16-week study, the effects of adding montelukast to budesonide, compared to doubling the budesonide dose in adult asthmatics was evaluated (Price et al. 2003). Following a 4-week run-in period, patients inadequately controlled on 800 μg/day of inhaled budesonide were randomized to receive add-on montelukast 10 mg (N = 448) or double the dose of budesonide (N = 441) for 12 weeks. Both groups showed progressive improvement in several measures of asthma control compared with baseline. For example, the mean PEF improved to a similar extent in the last 10 weeks of treatment compared with baseline in both the LTRA and higher inhaled corticosteroid dose group (34 vs. 30 L/min respectively). Moreover, patients in the former group experienced a quicker improvement in PEF. Both randomized treatments showed similar improvements in terms of reliever requirement, daytime symptom score, nocturnal awakenings, exacerbations, asthma-free days, blood eosinophils, and asthma-specific quality of life. The implication from this study is that adding montelukast conferred a steroid-sparing effect equivalent to 800 μg of budesonide, although no intermediate dose of budesonide (such as 1200 μg) was evaluated. In another study, adding twice-daily zafirlukast 20 mg to extra-fine hydrofluoroalkane beclomethasone dipropionate 100 μg/day produced the same effect as increasing the dose of beclomethasone alone to 400 μg/day in terms of attenuation of methacholine hyperresponsiveness and reducing exhaled breath nitric oxide (Fig. 30.4) (Dempsey et al. 2002).

Antileukotriene or long-acting b2 agonist as add-on therapy to inhaled corticosteroids in persistent asthmatics? Current internationally recognized guidelines indicate that symptomatic asthmatics using a low to medium inhaled

700

60 Per cent fall NO from baseline

PART 3

+ ‡

+ ‡

BDP 400 mg

BDP 100 mg + Zafirlukast

40

20

0 BDP 100 mg

BDP 100 mg + Theophylline

Fig. 30.4 Effects of beclomethasone (BDP) 100 mg/day, beclomethasone 400 mg/day, beclomethasone 100 mg/day plus zafirlukast, and beclomethasone 100 mg/day plus theophylline on exhaled nitric oxide (NO). Values are shown as percentage fall from pretreatment baseline. *, P < 0.05 vs. baseline; +, P < 0.05 vs. beclomethasone 100 mg/day alone. (From Dempsey et al. 2002, with permission.)

corticosteroid dose (400–800 μg/day of beclomethasone or equivalent) alone should preferentially be commenced on a long-acting β2 agonist prior to an LTRA (British guideline on the management of asthma 2003; GINA Workshop Report 2004). However, two recent large trials have performed headto-head comparisons of add-on long-acting β2 agonist versus LTRA as therapeutic adjuncts to inhaled corticosteroids, using exacerbation frequency (rather than lung function and symptoms) as the primary end point (Bjermer et al. 2003; Ilowite et al. 2004). Indeed, studies comparing the effects of either long-acting β2 agonist or LTRAs on measures of airway caliber, reliever use, and symptoms will tend to favor the former since they relax airway smooth muscle more completely and thereby exhibit superior bronchodilator effects. Moreover, since many trials evaluating the effects of asthma treatment require reversibility of at least 12% to short-acting β2 agonist to be demonstrated as a prerequisite to entry, long-acting β2 agonists will frequently appear superior to antiinflammatory therapy. In a study involving 1490 persistent asthmatics, the effects of montelukast 10 mg/day versus salmeterol was evaluated in patients whose symptoms were inadequately controlled with fluticasone 100 μg twice-daily alone (Bjermer et al. 2003). Following 52 weeks of treatment, 20.1% of patients receiving add-on montelukast had an asthma exacerbation compared with 19.1% in the group receiving add-on salmeterol, with no significant difference (95% CI −3.1 to 5%) between randomized treatments (Fig. 30.5). As expected, treatment with add-on salmeterol resulted in a significantly greater improvement in lung function, while treatment with montelukast resulted in a significantly greater reduction in peripheral blood eosinophils. A similar nonsignificant difference in exacerbation frequency was observed in another 52-week study evaluating effects of add-on montelukast 10 mg/day

9781405157209_4_030.qxd 4/1/08 17:33 Page 701

Per cent of patients with an asthma exacerbation

CHAPTER 30

25

Montelukast plus fluticasone Salmeterol plus fluticasone

20

15

10

5

0 0

12

24 36 Weeks since randomization

48

Fig. 30.5 Effects of fluticasone plus salmeterol or montelukast in terms of cumulative percentage of patients with asthma exacerbations (P = 0.599 by log rank test). (From Bjermer et al. 2003, with permission.) (See CD-ROM for color version.)

versus salmeterol in 1473 randomized asthmatics using inhaled corticosteroids (Ilowite et al. 2004); 80% of patients receiving montelukast and 83.3% of patients receiving salmeterol remained free of exacerbations during the treatment period (95% CI 0.96–1.49 for the difference between randomized


treatments). In the same study, montelukast significantly reduced blood eosinophils compared with salmeterol, whereas salmeterol significantly increased FEV1, asthma-specific quality of life and morning PEF and decreased nocturnal awakenings compared with montelukast. It is pertinent to point out that most studies (Nelson et al. 2000; Fish et al. 2001; Nelson et al. 2001; Wilson et al. 2001a; Bjermer et al. 2003; Currie et al. 2003a; Ringdal et al. 2003; Ilowite et al. 2004; Storms et al. 2004) making head-tohead comparisons have demonstrated that the addition of an LTRA is generally as effective at reducing exacerbations as adding a long-acting β2 agonist to an inhaled corticosteroid (Table 30.3) (Currie et al. 2005a). However, add-on long-acting β2 agonist tends to be superior to add-on LTRA in improving lung function, while the latter treatment confers superior antiinflammatory activity and attenuates airway hyperresponsiveness to a greater extent (Currie et al. 2005a). Moreover, it could be argued that some of these studies were biased in favor of long-acting β2 agonists, since individuals were required to exhibit a high degree of reversibility to shortacting β2 agonist (salbutamol) as an entry requirement. In other words, adding a long-acting β2 agonist would always be destined to be more effective in terms of lung function in study participants who had been selected a priori to respond favorably.

Table 30.3 Studies comparing effects of long-acting b2 agonists with leukotriene receptor antagonists as add-on therapy to inhaled corticosteroids. Duration (weeks)

Treatment

ICS (mg)

FEV1

PEF

Inflammatory biomarker

AHR

Exacerbations

QOL or symptoms

13–14

SM vs. ML

606

NA

*LABA+

NA

NA

↔

LABA+

1490

52

SM vs. ML

200

LABA+

LABA+

LRTA+

NA

*↔

↔

PG

1473

52

SM vs. ML

220

LABA+

LABA+

LRTA+

NA

*↔

LABA+

XO

22

8

SM vs. ML

466

↔

LABA+

LRTA+

*LRTA+

NA

↔

PG

447

15

SM vs. ML

200

LABA+

*LABA+

NA

NA

LABA+

↔

XO

20

6

SM vs. ML

↔

↔

LRTA+

*↔

NA

↔

PG

725

16

SM vs. ML

800 (median) 200

LABA+

*LABA+

NA

NA

LABA+

LABA+

PG

122

6

SM vs. ML

200

*LABA+

NA

NA

NA

NA

↔

PG

429

4–6

SM vs. ZL

NA

LABA+

*LABA+

NA

NA

↔

LABA+

Reference

Design

N

Fish et al. (2001) Bjermer et al. (2003) Ilowite et al. (2004) Currie et al. (2003a) Nelson et al. (2000) Wilson et al. (2001a) Ringdal et al. (2003) Storms et al. (2004) Nelson et al. (2001)

PG

948

PG

PEF, peak expiratory flow; FEV1, forced expiratory volume in 1 s; QOL, quality of life; N, number of randomized subjects; ICS, mean daily inhaled corticosteroid dose at study entry (unless specified); SM, salmeterol; ML, montelukast; PG, parallel group; XO, crossover; AHR, airway hyperresponsiveness; LABA, long-acting b2 agonist; LTRA, leukotriene receptor antagonist. LABA+, significant superiority vs. LTRA; LTRA+, significant superiority vs. LABA; ↔, no significant difference between randomized treatments; *, primary end point; NA, not documented or measured.

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PART 3

Adenosine monophosphate PC20 doubling dose difference vs placebo

2.5

2.0

1.5

*+

* *

1.0

NS 0.5

0.0 Montelukast 1 day

Salmeterol 2 wks

1 day

2 wks

Fig. 30.6 Means (SE) for the doubling dilution difference from placebo for the adenosine monophosphate threshold concentration after the first and last doses of montelukast and salmeterol. *, P < 0.05 difference from placebo; +, P < 0.05 between the first and last doses of each drug. (From Wilson et al. 2001a, with permission.)

A further issue encountered when prescribing long-acting β2 agonists (but not with antileukotrienes) is the development of tolerance to their bronchoprotective effects (Grove & Lipworth 1995). Regular use of long-acting β2 agonists results in β2-adrenoceptor downregulation, receptor internalization, and uncoupling of the G protein–adenyl cyclase unit with subsequent subsensitivity (or tachyphylaxis) of response to effects on airway smooth muscle and inflammatory cells. As

a result, an attenuated bronchoprotective response can be observed following exposure to different types of inhaled stimuli (Lipworth 1997). For instance, in a randomized, placebo-controlled, crossover study, 20 asthmatics not controlled on inhaled corticosteroids received either add-on montelukast 10 mg/day or salmeterol for 2 weeks each (Wilson et al. 2001a). For the primary end point of adenosine monophosphate threshold concentration (a marker of mast cell-mediated activity), compared to placebo there were significant differences with montelukast for first and last doses, but only after the first dose of salmeterol (Fig. 30.6). Similar results were also observed in another study where montelukast conferred sustained bronchoprotection against inhaled adenosine monophosphate over 2 weeks of treatment, whereas formoterol only conferred significant protection following first but not last dose (Sims et al. 2003). Whether these observations lead to future guidelines in asthma management acknowledging that addition of an LTRA is as effective in reducing exacerbations as adding a long-acting β2 agonist to inhaled corticosteroids remains to be seen. However, it appears reasonable to consider that in persistent asthmatics using inhaled corticosteroids who have impaired FEV1 (e.g., FEV1 < 80% predicted) and who exhibit a high degree of reversibility to short-acting β2 agonist, should proceed to have a therapeutic trial with a long acting β2-agonist as add-on therapy. However, perhaps those patients with preserved lung function (e.g., FEV1 > 80%), especially those with symptomatic allergic rhinitis or prominent exercise-induced symptoms, and who are therefore less likely to benefit from the bronchodilator effects of a longacting β2 agonist, should be started on an LTRA (Fig. 30.7).

Persistent asthmatic using 400–800 µg/day of inhaled corticosteroid (beclomethasone equivalent)

• • • •

Assess inhaler technique Check compliance Exclude avoidable trigger factors Exclude concomitant diseases

Persistent asthmatic with preserved FEV1, symptomatic allergic rhinitis or exerciseinduced symptoms

Persistent asthmatic with impaired FEV1 and with reversibility to short-acting b2 agonist > 12%

Add a LTRA

Add a LABA

Symptoms controlled?

Yes

No

No Add a LABA

702

Symptoms controlled?

Arrange further review

Add a LTRA

Fig. 30.7 Suggested algorithm for guiding clinicians as to whether a long-acting b2 agonist (LABA) or leukotriene receptor antagonist (LTRA) should be used as additional second-line therapy in persistent asthmatics using a low to moderate dose of inhaled corticosteroid. (From Currie et al. 2005a, with permission.)

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asthma severities, montelukast was observed to be an effective and well-tolerated treatment in everyday life in as many as 66% of individuals, including symptomatic individuals already using inhaled corticosteroids plus long-acting β2 agonists (Barnes et al. 2005). In a randomized placebo-controlled trial evaluating 72 moderate-to-severe asthmatics maintained on inhaled corticosteroids and mostly taking long-acting β2 agonists, the addition of montelukast 10 mg/day for 2 weeks conferred no significant improvement in terms of PEF and symptom scores (Robinson et al. 2001). A major limitation of the study was failure to evaluate any antiinflammatory biomarkers or airway hyperresponsiveness, while the relative short treatment duration would have precluded identification of any reduction in exacerbation frequency (Green & Pavord 2001). The effects of “triple therapy” with inhaled corticosteroid, long-acting β2 agonist plus LTRA was evaluated in 22 mild-to-moderate persistent asthmatics (mean FEV1 80% predicted) in a randomized, double-blind, placebo-controlled, crossover study (Currie et al. 2003a). After a 2-week run-in using twice-daily fluticasone propionate 250 μg with salmeterol, patients entered a randomized crossover period where they received additional montelukast 10 mg daily or identical placebo for 3 weeks each. Treatment with montelukast was significantly (P < 0.05) better than placebo in reducing airway hyperresponsiveness to adenosine monophosphate (Fig. 30.8a) and surrogate biomarkers of inflammation including exhaled nitric oxide (Fig. 30.8b) and blood eosinophils (Fig. 30.8c), despite conferring no further improvement in lung function. This in turn implies that serial monitoring of airway caliber does not necessarily reflect any potential benefit of nonsteroidal antiinflammatory treatment in patents already receiving inhaled corticosteroids and long-acting β2 agonists. Longer studies with greater numbers of patients

Antileukotrienes as add-on therapy to inhaled corticosteroids plus long-acting b2 agonists in persistent asthma Over and above inflammation of underlying endobronchial mucosa, intermittent bronchial smooth muscle dysfunction (generally responsive to β2 agonists) is a fundamental component in the overall asthma syndrome. This knowledge has led to increased use of inhalers using fixed combinations of corticosteroids and long-acting β2 agonists (Currie et al. 2005b), which facilitate patient compliance by reducing the numbers of inhalations and inhalers required. Current guidelines (British guideline on the management of asthma 2003; GINA Workshop Report 2004) advocate that in individuals with persistent symptoms despite regular use of fixed combination inhalers, the main options consist of a therapeutic trial of LTRA, oral theophylline, or higher dose of inhaled corticosteroid. In these symptomatic patients, the widespread use of theophylline is often limited due to unwanted drug interactions, adverse effects, and need to monitor plasma levels. Moreover, some patients are reluctant to use higher inhaled corticosteroid doses in view of greater risk of unwanted local and systemic sequeluae, while dose–response studies have suggested that little further therapeutic benefit may be gained from doing so (Holt et al. 2001; Masoli et al. 2005). It is important to point our that few studies have actually examined whether additional treatment with a LTRA in conjunction with an inhaled corticosteroid plus long-acting β2 agonist (“triple therapy”) does in fact confer further clinical benefit. However, in a large retrospective study (1351 patients) evaluating the effects of LTRAs across a range of

P < 0.05 1.5

P < 0.05

2

P < 0.05

P < 0.05

P < 0.05

0.5

FP/SM +ML

FP +ML

FP/SM +PL

FP +PL

0

–2

FP/SM +ML

FP +ML FP/SM +PL

FP +ML

FP/SM +PL

FP +PL

–100

–200

–4 (b)

FP/SM +ML

FP +PL Change in eosinophils (×106/L) from run-in

1.0

0 (a)

Change in exhaled NO (ppb) from run-in

Doubling dilution difference in AMP PC20 threshold from run-in

0

P < 0.05 P < 0.05

(c)

Fig. 30.8 Change from fluticasone/salmeterol (FP/SM) 250 mg b.i.d. run-in with add-on montelukast 10 mg (ML) or placebo (PL) for (a) AMP PC20 threshold doubling dilution shift, (b) exhaled nitric oxide, and (c) blood eosinophils. Montelukast or placebo was given with fluticasone (FP) alone or fluticasone plus salmeterol in combination (FP/SM). All changes are referred to baseline after run-in on FP/SM. (From Currie et al. 2003a, with permission.)

703

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Pharmacology

Antileukotrienes in acute asthma Leukotrienes can be found in the airway and urine following both spontaneous exacerbations of asthma and acute exposure to bronchoconstrictor stimuli in the laboratory (Hui et al. 1991; Wenzel et al. 1995). This in turn indicates that they may have a role in the pathogenesis of acute episodes of bronchoconstriction. Although antileukotrienes are not currently advocated in the management of acute asthma, there are data to suggest that they might be of some potential benefit. Prior treatment with montelukast has been shown in several studies to significantly shorten the time taken to recover (in terms of FEV1) following exposure to a bronchoconstrictor stimulus (Brannan et al. 2001; Currie et al. 2003a,b). For example, in a study evaluating mild atopic asthmatics, single doses of montelukast 10 mg given 14 hours earlier, either alone or in combination with an antihistamine, resulted in a significantly (P < 0.05) shortened recovery time back to baseline FEV1 compared with placebo following indirect bronchial challenge with adenosine monophosphate (Fig. 30.9) (Currie et al. 2003b). This amounted to approximately 30 min of a difference compared to placebo. These data suggest that cysteinyl leukotrienes are important mediators in prolonging the bronchoconstrictor response following an acute episode of airway narrowing. The “real-life” implication of this was observed in a study by Camargo et al. (2003). In a doubleblind, parallel-group controlled trial, 210 patients with acute asthma were randomized to receive either intravenous montelukast (7 or 14 mg) or placebo along with standard treatment. Montelukast improved FEV1 over the first 20 min after intravenous administration (mean percentage change from baseline, 14.8% vs. 3.6% for pooled montelukast and placebo groups respectively; P = 0.007). Moreover, patients given active drug had a significantly quicker improvement in FEV1 over the entire 2-hour period and received less inhaled β2 agonist compared with placebo. In another study, the effects of zafirlukast was evaluated in patients with acute asthma over a 28-day follow-up period (Silverman et al. 2004). In a double-blind multicenter study, 641 acute asthmatics presenting to the emergency room were randomized to receive single doses of either 160 mg zafirlukast, 20 mg zafirlukast, or placebo as an adjunct to standard care. Patients who were discharged after 4 hours continued randomized treatment over 28 days and received either zafirlukast 20 mg twice daily (276 patients) or placebo (270 patients) in addition to prednisolone and their previous asthma medications. At the end of the emergency room treatment period, 16 of 162 patients (10%) treated initially

704

100 Per cent subjects with FEV1 > 5% from baseline

are required to establish whether these preliminary findings extrapolate into long-term clinical advantages in asthma control such as reductions in exacerbation frequency and airway remodeling.

Placebo ML ML/DL

80

60

40

20

0

2

4

7

10

15

20

25

30

40

50

100

Placebo ML ML/DL

80

60

40

20

0 (b)

60

Time (min)

(a)

Per cent subjects with FEV1 > 5% from baseline

PART 3

2

4

7

10

15

20

25

30

40

50

60

Time (min)

Fig. 30.9 Percentage of patients during recovery with a fall in FEV1 > 5% from baseline following (a) adenosine monophosphate challenge and (b) mannitol challenge. ML, montelukast alone; ML/DL, montelukast plus desloratadine in combination. (From Currie et al. 2003b, with permission.)

with a single dose of zafirlukast 160 mg, 26 of 158 patients (17%) treated with a single dose of zafirlukast 20 mg, and 48 of 321 patients (15%) treated with placebo required extended care (P = 0.052 for the difference between zafirlukast 160 mg and placebo). Moreover, at the end of the 28-day treatment period, 65 of 276 patients (24%) treated with twice-daily zafirlukast 20 mg and 78 of 270 patients (29%) treated with placebo relapsed (P = 0.047 for the difference). These findings were supported by a significant improvement in lung function and reduction in breathlessness in the emergency room with zafirlukast 160 mg and with similar improvements during the 28-day treatment period for patients receiving twice-daily zafirlukast. Some benefit with LTRAs was also observed in a randomized, double-blind, placebo-controlled

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study evaluating 51 preschool children with a mild to moderate exacerbation of asthma (Harmanci et al. 2006). At presentation, subjects received a single dose of montelukast 4 mg or placebo in addition to inhaled salbutamol and were followed up over a 4-hour period. Active treatment was associated with a significantly lowered respiratory rate at all time points, while 38.5% in the placebo group versus 20.8% in the montelukast group required oral steroid (P = 0.22). Whether LTRAs prove to be useful adjuncts in the management of acute asthma (in conjunction with oral corticosteroids and nebulized bronchodilators) remains to be seen. Further studies are required to confirm whether administration of a LTRA along with conventional treatment confers any advantage over addition of intravenous magnesium or aminophylline in the setting of an acute episode of asthma. Moreover, whether treatment with an LTRA at the onset of an exacerbation of asthma confers clinically relevant and cost-effective advantages in the short and medium term needs prospective evaluation.

Antileukotrienes in allergic rhinitis Allergic rhinitis is a common inflammatory condition of the upper airway characterized by sneezing, nasal pruritus, rhinorrhea, and nasal obstruction (Haberal & Corey 2003). Current management strategies are centered around the use of allergen avoidance, immunotherapy, corticosteroids, and antihistamines. Since the upper and lower airways have a direct anatomic connection, share similar epithelial lining, and release similar inflammatory mediators (Busse 1996; Lipworth & White 2000), it has been suggested that asthma and allergic rhinitis represent a continuation of the same inflammatory disease process. Indeed, 20–40% of individuals with allergic rhinitis are thought to have concomitant asthma, while 30– 90% of asthmatics are thought to have allergic rhinitis (Simons 1999; Leynaert et al. 2000). Uncontrolled allergic rhinitis is known to precipitate and exacerbate asthma, with the inference that clinicians should positively search for typical nasal and ocular symptoms (Rachelefsky 1999). Moreover, successful treatment of allergic rhinitis can confer benefits in overall asthma control (Henriksen & Wenzel 1984; Reed et al. 1988; Ragab et al. 2006). Nasal allergen challenge has been shown to result in dose-related increased levels of cysteinyl leukotrienes in nasal lavage fluid (Naclerio et al. 1983; Creticos et al. 1984; Pipkorn et al. 1987), while other studies have demonstrated the importance of these mediators in individuals with allergic rhinitis following exposure to natural allergens (Skoner et al. 1990; Knani et al. 1992). Moreover, activated eosinophils and mast cells, both of which release cysteinyl leukotrienes, play a prominent role in the pathophysiology of allergic rhinitis (Knani et al. 1992). This knowledge has raised interest and prompted considerable research into the potential therapeutic role of antileu-


kotrienes in individuals with allergic rhinitis both with and without concomitant asthma. It is important to note that montelukast has gained approval from the US Food and Drug Administration for the management of allergic rhinitis alone, although LTRAs are not currently licensed for this use in the UK. They can however be prescribed for patients with uncontrolled asthma who may have active symptoms of allergic rhinitis. Montelukast as monotherapy has been shown to be effective in the treatment of allergic rhinitis. For example, a study of 1302 patients with allergic rhinitis demonstrated that montelukast 10 mg/day improved daytime and nighttime nasal symptoms plus quality-of-life parameters (Philip et al. 2002). Moreover, the adverse-effect profile with montelukast was comparable to placebo. In two other large studies (van Adelsberg et al. 2003a,b), montelukast 10 mg/day was significantly superior in reducing symptoms of allergic rhinitis compared to placebo, and was similar to treatment with loratadine. A study of 1862 symptomatic patients with allergic rhinitis showed superiority of montelukast over placebo in improving nasal symptoms, and also demonstrated a superior response in individuals who were exposed to a higher pollen counts (Chervinsky et al. 2004). Similarly, zafirlukast has been shown to significantly reduce nasal symptoms, nasal resistance, and lavage eosinophil counts in patients with allergic rhinitis (Piatti et al. 2003). A recent systematic review and metaanalysis examined the efficacy of LTRAs in patients with allergic rhinitis incorporating 11 randomized controlled trials (Wilson et al. 2004). Treatment with LTRA reduced mean daily rhinitis symptom scores 5% (95% CI 3–7%) more than placebo. In the same study, antihistamines improved nasal symptom scores 2% (95% CI 0–4%) more than LTRAs, and nasal corticosteroids improved scores 12% (95% CI 5–18%) more than LTRAs. This all suggests that LTRAs appear to be superior to placebo, generally as effective as antihistamines, but less effective than topical corticosteroids in improving symptoms and quality of life in patients with allergic rhinitis. Similar findings were also obtained in an another metaanalysis evaluating the effects of LTRAs as monotherapy or when combined with other drugs in patients with allergic rhinitis (Rodrigo & Yanez 2006). Seventeen randomized controlled trials (N = 6231 subjects) were eligible for inclusion, with 16 of the studies using montelukast and one zafirlukast. LTRAs significantly reduced daytime and nighttime nasal symptoms and eye symptoms, and also significantly improved quality of life compared to placebo. Moreover, no significant differences were observed between LTRAs and antihistamines, although nasal corticosteroids were significantly superior to LTRAs. Data relating to the combined effects of LTRAs plus antihistamines in allergic rhinitis have shown conflicting results. One study of 460 patients with seasonal allergic rhinitis demonstrated that the combination of montelukast plus loratadine improved daytime nasal symptoms, while less benefit

705

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Pharmacology

was observed with either drug as monotherapy (Meltzer et al. 2000). In contrast, in another larger study of 907 patients with seasonal allergic rhinitis, both drugs alone improved daytime nasal symptoms with no further additional benefit from the combination (Nayak et al. 2002). A study of 62 patients with seasonal allergic rhinitis found that the combination of montelukast and loratadine was no more effective than montelukast alone on daytime or nighttime nasal symptoms (Pullerits et al. 2002), while a study of 60 patients with seasonal allergic rhinitis found that the combination of montelukast and cetirizine was more effective than cetirizine alone (Kurowski et al. 2004). When compared to intranasal corticosteroids, the combination of LTRA plus antihistamine has been shown to be similar in some studies (Wilson et al. 2001b,c) and less effective in others (Pullerits et al. 2002; Saengpanich et al. 2003; Di Lorenzo et al. 2004) in terms of effects on nasal symptoms in allergic rhinitis.

Antileukotrienes in concomitant asthma and allergic rhinitis In a multicenter trial, the efficacy of antileukotrienes in individuals (N = 831) with both symptomatic allergic rhinitis and active asthma was evaluated (Philip et al. 2004). Subjects were randomized to receive daily montelukast 10 mg (N = 415) or placebo (N = 416) over a 2-week, double-blind treatment period. Montelukast significantly (P = 0.001) reduced rhinitis symptoms scores compared to placebo, with improvements also being observed in nasal and eye symptoms. Moreover, active treatment provided greater benefit in overall asthma control and reduced the requirement for reliever use. A post hoc analysis from the COMPACT study (Price et al. 2003) examined whether those asthmatics with concomitant allergic rhinitis responded differently depending on whether they were randomized to receive add-on montelukast (N = 216) or double the dose of inhaled corticosteroid (N = 184) (Price et al. 2006). In terms of the primary outcome, there was a 9.2% increase in morning PEF from baseline in the montelukast group compared with a 6% increase in the higher inhaled corticosteroid group (P = 0.028 for the difference), although secondary end points were not significantly different between randomized groups. Thus, in the subgroup of asthmatic patients with allergic rhinitis, a combined treatment approach that included montelukast and budesonide provided significantly greater efficacy in improving lung function compared with doubling the dose of budesonide. This in turn supports the notion that a unified approach aimed at treating airway inflammation common to both diseases is beneficial for the large proportion of asthmatics who have concomitant allergic rhinitis. In a double-dummy crossover study, patients with both asthma and concomitant allergic rhinitis were randomized to

706

receive 14 days of 400 μg/day of orally inhaled budesonide plus 200 μg/day intranasal budesonide or once-daily 10 mg montelukast plus 10 mg oral cetirizine (Wilson et al. 2000). Both treatments were significantly (P < 0.05) better than placebo, but of similar efficacy in terms of improving lung function, symptoms and inflammatory biomarkers. In another study of patients with asthma and allergic rhinitis, montelukast was as effective as orally inhaled and intranasal budesonide on lower airway parameters, with both treatments conferring benefit on symptoms of allergic rhinitis (Wilson et al. 2001d). Whether in the future a therapeutic trial of LTRA becomes standard practice in individuals symptomatic of both upper and lower airways inflammation remains to be seen. Indeed, it could be argued that asthmatics using inhaled corticosteroids with concomitant allergic rhinitis requiring additional second-line therapy should preferentially be commenced on an LTRA (instead of a long-acting β2 agonist), which in turn may confer a positive effect on both the upper and lower airways (see Fig. 30.7).

Other uses of antileukotrienes Aspirin-induced asthma The prevalence of aspirin-sensitive asthma is uncertain although it may exist in up to 20% of all asthmatics (Jenkins et al. 2004). The characteristic features include profound bronchoconstriction following aspirin ingestion, rhinosinusitis, nasal polyps, and abdominal cramps. Aspirin and nonsteroidal antiinflammatory drugs selectively inhibit COX-1, which in turn shunts arachidonic acid down the 5-lipoxygenaseactivating protein pathway, causing overproduction of cysteinyl leukotrienes. As a consequence, elevated levels of cysteinyl leukotrienes can be found in bronchial and nasal aspirates, and in urine following aspirin challenge (Christie et al. 1991; Knapp et al. 1992). Moreover, the rate-limiting enzyme, LTC4 synthase, which converts LTA4 to LTC4, is overexpressed in eosinophils and mast cells in patients with aspirin-sensitive asthma (Cowburn et al. 1998). Since cysteinyl leukotrienes appear therefore to be important mediators in the pathogenesis of aspirin-sensitive asthma, it is logical to consider that antileukotrienes may prevent development of symptoms. In one study, 80 aspirin-sensitive asthmatics receiving moderate to high doses of inhaled corticosteroid were randomized to receive placebo or montelukast 10 mg/day for 4 weeks (Dahlen et al. 2002). Compared to placebo, significant improvements in FEV1 and morning PEF (P < 0.001) were observed along with reductions in symptoms and reliever use (P < 0.05). Other studies have specifically evaluated the airway response following exposure to aspirin (Christie et al. 1991; Dahlen et al. 1993; Israel et al. 1993; Lee et al. 2004). In one randomized study of 12 patients with aspirin-sensitive

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asthma, nasal lysine aspirin challenge was performed after single doses of montelukast 10 mg, montelukast 40 mg, or placebo taken 12 hours earlier (Lee et al. 2004). The former treatment partially protected against the local effects (in terms of peak nasal inspiratory flow) of nasal lysine aspirin challenge compared to placebo, with no further benefit being observed with the higher dose of montelukast. In another randomized crossover study, bronchial provocation with lysine aspirin was performed at baseline and 1 hour after placebo or single dose of another LTRA (MK-0679) (Dahlen et al. 1993). Following active treatment, a rightward shift in the dose–response relationship for all eight subjects was observed (median shift 4.4-fold), with three individuals failing to produce a 20% decrease in FEV1 despite inhalation of the highest dose of lysine aspirin. In aspirin-sensitive asthmatics, zileuton decreased baseline urinary LTE4 excretion (P < 0.02) and blunted the maximum increase in urinary LTE4 after ingestion of aspirin (P < 0.01) (Israel et al. 1993). In the same randomized crossover study in contrast to placebo, zileuton prevented any significant fall in FEV1 following ingestion of aspirin (P < 0.014). However, in a further small study evaluating aspirin-sensitive asthmatics, montelukast was only partially effective in preventing bronchoconstriction following exposure to aspirin (Stevenson et al. 2000). Similarly in another study, 3 weeks of montelukast 10 mg/day conferred an overall improvement in asthma control, although no differences in the clinical response between aspirin-sensitive and aspirin-tolerant individuals were observed (Mastalerz et al. 2002). Antileukotrienes therefore appear to be of some value in preventing the airway response following exposure to aspirin in predisposed asthmatics, although they do not appear to attenuate the effects completely. Further large-scale prospective studies are required to further delineate the place of chronic dosing with antileukotrienes, perhaps in conjunction with antagonists of other mediators such as histamine, in aspirin-sensitive asthmatics.

Exercise-induced asthma Many patients with asthma develop symptoms as a response to exercise. This is thought to be due to drying and cooling effects occurring in the airway, with the subsequent release of proinflammatory mediators such as cysteinyl leukotrienes and histamine (Anderson & Brannan 2002). Indeed, cysteinyl leukotrienes can be found in urine following exerciseinduced bronchoconstriction (Kikawa et al. 1992; Reiss et al. 1997). LTRAs and other inhibitors of the cysteinyl leukotriene pathway have been shown to protect against exerciseinduced bronchoconstriction in a number of studies in both adults and children (Meltzer et al. 1996; Bronsky et al. 1997; Reiss et al. 1997; Kemp et al. 1998; Leff et al. 1998; de Benedictis et al. 2006). For example, in 100 corticosteroid-naive asthmatics with a mean FEV1 of 83% predicted, the effects of montelukast 10 mg/day was evaluated over a 12-week period


(Leff et al. 1998). Compared to placebo, montelukast was significantly superior in protecting against exercise-induced bronchoconstriction, while patients also experienced better asthma control during active treatment. In the same study, tolerance to its effects were not observed, which is often a problem encountered with long-acting β2 agonists (Ramage et al. 1994). In a double-blind multicenter trial, asthmatics (N = 122) with a history of exercise-induced symptoms uncontrolled on low-dose fluticasone were randomized to receive montelukast 10 mg/day, salmeterol, or placebo for 4 weeks (Storms et al. 2004). The aim of this study was to compare the effects of chronic dosing on airway responses following exercise challenge and rescue short β2 agonist. The maximum FEV1 after short-acting β2 agonist improved in the montelukast (1.5%) and placebo (1.2%) groups at 4 weeks, but diminished in the salmeterol group by 3.9% (P < 0.001 for salmeterol vs. placebo and montelukast). Although the pre-exercise FEV1 was greatest with salmeterol (P = 0.10), individuals taking montelukast had significantly greater protection from an exercise-induced decrease in FEV1 than those taking salmeterol (P < 0.001). Both the magnitude and rate of rescue bronchodilation were greater with montelukast compared with salmeterol (P < 0.001). Moreover, 5 min after rescue short-acting β2 agonist, 92% of patients taking montelukast and 68% of those taking placebo had recovered to preexercise levels, while only 50% of those taking salmeterol had recovered. In other words, add-on therapy with montelukast facilitated a greater and more rapid degree of rescue bronchodilation with short-acting β2 agonist than addition of salmeterol, and provided superior protection against exerciseinduced bronchoconstriction. In another randomized doubleblind study, the effects of 8 weeks of montelukast 10 mg/day versus salmeterol was evaluated in individuals with exerciseinduced asthma (N = 191, mean FEV1 88% predicted) who were not using inhaled corticosteroids. Montelukast provided significantly greater protection against exercise-induced bronchoconstriction versus salmeterol after both 4 weeks (P = 0.015 for the difference) and 8 weeks (P = 0.002 for the difference) (Edelman et al. 2000). Similar beneficial effects on attenuation of exerciseinduced bronchoconstriction have also been observed with zafirlukast (Dessanges et al. 1999). In a randomized, doubleblind, three-way, crossover trial, 20 mg or 80 mg twice daily of zafirlukast or placebo was given for 14 days with a 7-day washout period between treatments. Exercise challenges were performed at 2 and 8 hours after the morning dose on day 14. Compared to placebo, both doses of zafirlukast significantly reduced exercise-induced bronchoconstriction as measured by the area under the FEV1 time curve after the 2-hour (P < 0.001) and 8-hour (P < 0.001) exercise challenges and maximum fall in FEV1 at the 2-hour challenge (P < 0.001). Mannitol is a bronchoconstrictor stimulus which can be used to asses the extent of airway hyperresponsiveness in

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asthmatics (Anderson et al. 1997). When given as a dry powder, inhaled mannitol increases the surface osmolarity of the bronchial mucosa resulting in the release of inflammatory and bronchoconstrictor mediators from a variety of cells. Bronchoprovocation with mannitol has been shown to demonstrate a high degree of sensitivity and specificity to eucapnic voluntary hyperpnea, and as a result suggested to be an alternative and more convenient means by which to identify exercise-induced bronchoconstriction (Holzer et al. 2003). Several studies evaluating the effects of prior dosing with montelukast (Brannan et al. 2001; Currie et al. 2003b) have demonstrated a significantly quicker rate of recovery following bronchoprovocation with mannitol, in turn suggesting that such drugs may be advantageous in individuals with exercise-induced symptoms. These data all indicate that drugs which antagonize the effects of cysteinyl leukotriene pathway should be considered in patients who require prevention of symptoms related to activity. Moreover, LTRAs may facilitate a simple and effective treatment option in patients with exerciseinduced symptoms, when either used alone or in combination with inhaled corticosteroids. The fact that they are effective even following single doses (Currie et al. 2003b) suggests that they may be a particularly attractive pharmacologic option in individuals with only mild, intermittent exercise-induced asthma who do not require daily inhaled corticosteroids.

Pharmacogenetic determinants of responsiveness to antileukotrienes An interesting property of antileukotrienes in “real life” is their apparent ability to demonstrate preferential therapeutic effects in subgroups of asthmatics. This in turn has prompted research into possible pharmacogenetic determinants that may govern the response to antileukotriene agents. LTC4 synthase is the terminal and rate-limiting enzyme involved in the production of cysteinyl leukotrienes. Polymorphisms of this critical enzyme, characterized by adenine (A) to cytosine (C) translocations at the −444 nucleotide, have been identified and prompted investigation into whether they might be related to asthma severity or phenotype, or in determining a preferential response to antileukotrienes. In aspirin-sensitive asthmatics, the variant C allele has been reported to be present in as many as 76% of cases, compared with around 40% in normal individuals and asthmatics without aspirin sensitivity (Sanak et al. 1997). However, the association between polymorphisms of LTC4 synthase and asthma phenotype and severity is controversial, and has generally revealed conflicting results. One large study of mild, moderate, and severe asthmatics failed to demonstrate a link between the variant C allele and disease severity or sensitivity to aspirin (Kedda et al. 2004). Similarly

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in another study, no significant association was observed between the presence of the C allele and asthma severity or presence of aspirin intolerance (Isidoro-Garcia et al. 2005). Conversely, a study in children did show that, compared to controls, those possessing the C allele had a lower mean percent predicted FEV1 (97.4% vs. 92.7%, P = 0.005) (Sayers et al. 2003). Only a few studies have examined the effects of polymorphisms of LTC4 synthase and clinical response to antileukotriene treatment. In a study by Sampson et al. (2000), the effects of zafirlukast 20 mg twice daily for 2 weeks in 23 asthmatics was evaluated according to genotype. In those with the variant LTC4 synthase genotype (AC and CC, N = 13), FEV1 and forced vital capacity increased by 9% and 15% respectively with zafirlukast compared to baseline levels, whereas individuals with the wild type (AA, N = 10) had a falls of 12% and 18% respectively. Although the differences were nonsignificant in this small sample (P = 0.1), these findings did raise the possibility that individuals with the variant C allele might represent those with a preferential response to antileukotrienes. Furthermore, Asano et al. (2002) evaluated the effects of pranlukast 225 mg twice daily for 4 weeks in 48 patients with moderate-to-severe persistent asthma according to genotype. Those with genotypes AC or CC experienced a greater improvement in FEV1 (P = 0.01) following LTRA treatment compared to those with the wild type. In a retrospective analysis (Currie et al. 2003c), polymorphisms of LTC4 synthase were not associated with clinical response to LTRAs in terms of surrogate inflammatory biomarkers and lung function. However, it may not be surprising to discover that a single allelic variation does not determine the response to LTRAs, since cysteinyl leukotrienes are synthesized via a cascade of enzymes. Indeed, in patients using montelukast over a 6-month period, a variety of single nucleotide polymorphisms in different genes were found to be implicated in determining preferential response to montelukast in terms of improving FEV1 and reducing exacerbations (Lima et al. 2006). Further large prospective studies specifically designed to evaluate whether interindividual variability in response to antileukotrienes in asthmatics is determined according to genotype are required.

Conclusion Due to greater understanding of the underlying inflammatory process implicated in the pathogenesis of asthma, antileukotrienes, particularly LTRAs, have emerged as useful oral nonsteroidal antiinflammatory agents that are effective in patients with symptomatic allergic airways disease (Currie et al. 2005c). Accumulating evidence indicates that there is a role for antileukotrienes across a broad spectrum of asthma severities, both as monotherapy and in conjunction with other pharmacologic adjuncts. Indeed, although corticosteroids

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are the most potent antiinflammatory drugs available, they fail to completely attenuate all aspects of the inflammatory disease process and many patients remain symptomatic despite their use. This in turn suggests that when used in combination with inhaled corticosteroids, antileukotrienes may facilitate more complete attenuation of the inflammatory cascade. Moreover, they also appear to confer some benefit in asthmatics with aspirin sensitivity, exercise-related symptoms, and concomitant allergic rhinitis. There is little doubt that inhaled corticosteroids remain the cornerstone of asthma management in all but the mildest of disease. However, antileukotrienes do allow an alternative therapeutic option in mild asthmatics perhaps unwilling or unable to use low to medium doses of inhaled corticosteroids. Indeed, this approach may well be best suited in those with predominant exercise-related symptoms. In the “real-world” setting, patient preference with oral treatment and a superior adverse-effect profile compared to inhaled corticosteroids may in turn lead to better compliance rates and superior long-term effect on airway inflammation. Current guidelines (British guideline on the management of asthma 2003; GINA Workshop Report 2004) suggest that antileukotrienes should be considered when the combination of a low to medium inhaled corticosteroid dose plus long-acting β2 agonist fails to adequately control symptoms. Alternatively, LTRAs may be tried in patients maintained on inhaled corticosteroids after a failed therapeutic trial of long-acting β2 agonist. However, randomized controlled trials suggest that when evaluating parameters such as exacerbation frequency, LTRAs may in fact have similar efficacy as long-acting β2 agonists. Indeed, it is important to note that many patients already using inhaled corticosteroids have relatively preserved lung function, with the inference that underlying inflammation and airway hyperresponsiveness are the driving forces behind episodic airflow obstruction and persistent symptoms. In these patients, add-on therapy with an LTRA would therefore appear to be a reasonable therapeutic option in view of its dual actions of attenuating airway hyperresponsiveness and suppressing inflammation. Indeed, the use of a long-acting β2 agonist, which as a class lacks intrinsic antiinflammatory properties (Gardiner et al. 1994; Roberts et al. 1999), would do little to deal with these underlying problems. This option may also be particularly attractive since concerns have been raised regarding the long-term safety of long-acting β2 agonists and their associated risks of increased asthma exacerbations and deaths (Nelson et al. 2006; Salpeter et al. 2006). However, in individuals with persistent symptoms and impaired lung function (e.g., FEV1 < 80% predicted), adding in a long-acting β2 agonist, while keeping the inhaled corticosteroid dose the same, would appear to be a logical step. Indeed, further increases in antiinflammatory therapy (either with an LTRA or increased inhaled corticosteroid dose) would be unlikely to improve lung function (Holt et al. 2001), and a combined inhaled cor-


ticosteroid plus long-acting β2 agonist inhaler would ensure maximal bronchodilation. While not universally licensed for use in patients with allergic rhinitis alone, LTRAs have demonstrated efficacy in asthmatics with concomitant upper airway inflammation. The treatment of “one airway” therefore appears a logical step by which to attenuate both lower and upper airway inflammation and symptoms. Moreover, this rationale would confer a more favorable adverse-effect profile when compared to patients using both inhaled and nasal corticosteroids. In conclusion, antileukotrienes are one of the first asthma drugs developed as a consequence of exploring the possibility of antagonizing the effects of a specific inflammatory mediator. They not only provide a further therapeutic tool with which to attenuate inflammation and airway hyperresponsiveness, but facilitate an orally active means to target the clinical burden of asthma in both primary and secondary care settings. Indeed, the challenge facing clinicians must surely be to decide where this class of drug sits most comfortably in stepwise treatment algorithms favored by guidelines.

Conflicts of interest G.P.C. has received funding from MSD (who make montelukast) and AstraZeneca (who make zafirlukast) for attending postgraduate international conferences, and for giving talks. B.J.L. has received funding from MSD for giving talks and grant support for clinical trials.

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31

Glucocorticosteroids Peter J. Barnes

Summary

Table 31.1 Effect of glucocorticoids on gene transcription.

Glucocorticoids are the most effective controllers of asthma and other allergic diseases. They suppress inflammation by switching off multiple activated inflammatory genes through reversing histone acetylation via the recruitment of histone deacetylase 2. Through suppression of airway inflammation inhaled glucocorticoids reduce airway hyperresponsiveness and control asthma symptoms. Inhaled glucocorticoids are now first-line therapy for all patients with persistent asthma, controlling asthma symptoms and preventing exacerbations. Inhaled long-acting β2 agonists added to inhaled glucocorticoids further improve asthma control and are commonly given as combination inhalers, which improve compliance and control asthma at lower doses of glucocorticoid. Inhaled glucocorticoids, which are absorbed from the lungs into the systemic circulation, have negligible systemic side effects at the doses most patients require. Systemic glucocorticoids are used in the treatment of acute exacerbations of asthma and as maintenance treatment in patients with severe asthma not controlled by maximum inhaled therapy. Oral steroids have numerous metabolic and endocrine side effects and the lowest dose needed to control the disease should be used. A few patients with asthma are resistant to the antiinflammatory effects of glucocorticoids and several molecular mechanisms for this have recently been described.

Increased transcription Lipocortin 1 b2-Adrenoceptor Secretory leukocyte inhibitory protein IkB-a (inhibitor of NF-kB) Antiinflammatory or inhibitory cytokines: IL-10, IL-12, IL-1 receptor antagonist Mitogen-activated protein kinase phosphatase 1 (inhibits MAP kinase pathways) Decreased transcription Inflammatory cytokines: IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, IL-13, IL-15, TNF-a, GM-CSF, SCF Chemokines: IL-8, RANTES, MIP-1a, eotaxin Inducible nitric oxide synthase (iNOS) Inducible cyclooxygenase (COX-2) Inducible phospholipase A2 (cPLA2) Endothelin 1 NK1 receptors Adhesion molecules (ICAM-1,VCAM-1) ICAM, intercellular adhesion molecule; SCF, stem cell factor; VCAM, vascular cell adhesion molecule. See text for definition of other abbreviations.

Introduction

Mechanisms of action

Glucocorticosteroids (also known as glucocorticoids, corticosteroids, steroids) are by far the most effective controllers used in the treatment of asthma and the only drugs that can effectively suppress the characteristic inflammation in asthmatic airways. They are also highly effective in the treatment of rhinitis and other allergic diseases. In this chapter, the mechanism of action and pharmacology of glucocorticoids and their use in the treatment of asthma will be discussed.

There have been major advances in understanding the molecular mechanisms whereby glucocorticoids suppress inflammation, based on recent developments in understanding the fundamental mechanisms of gene transcription (Rhen & Cidlowski 2005; Barnes 2006a). Glucocorticoids activate and suppress many genes relevant to understanding their action in asthma and other allergic diseases (Table 31.1).

Cellular effects Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

At a cellular level glucocorticoids reduce the numbers of inflammatory cells in the airways, including eosinophils, T lymphocytes, mast cells, and dendritic cells (Fig. 31.1). These effects of glucocorticoids are produced through inhibiting the

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Inflammatory cells

Structural cells

Eosinophil ↓ Numbers (apoptosis)

Epithelial cell ↓ Cytokines mediators

T-lymphocyte ↓ Cytokines Mast cell

Endothelial cell ↓ Leak

CORTICOSTEROIDS

↓ Numbers

Airway smooth muscle ↑ b2-Receptors ↓ Cytokines

Macrophage ↓ Cytokines

Mucus gland Dendritic cell

Mucus ↓ secretion

↓ Numbers

Fig. 31.1 Cellular effect of glucocorticoids. (See CD-ROM for color version.)

recruitment of inflammatory cells into the airway by suppressing the production of chemotactic mediators and adhesion molecules and by inhibiting the survival in the airways of inflammatory cells, such as eosinophils, T lymphocytes, and mast cells. Epithelial cells may be the major cellular target for inhaled glucocorticoids, which are the mainstay of modern asthma management. Inhaled glucocorticoids suppress many activated inflammatory genes in airway epithelial cells (Fig. 31.2). Epithelial integrity is restored by regular inhaled glucocorticoids. The suppression of mucosal inflammation is relatively rapid, with a significant reduction in eosinophils detectable within 6 hours and associated with reduced airway hyperresponsiveness (AHR) (Gibson et al. 2001; Ketchell et al. 2002). Reversal of AHR may take several

Epithelial cells

Enzymes iNOS COX-2 cPLA2

Peptides ET-1

Adhesion molecules ICAM-1

↓ INFLAMMATION

Fig. 31.2 Inhaled glucocorticoids may inhibit the transcription of several “inflammatory” genes in airway epithelial cells and thus reduce inflammation in the airway wall. GM-CSF, granulocyte–macrophage colony-stimulating factor; IL, interleukin; iNOS, inducible nitric oxide synthase; COX-2, inducible cyclooxygenase; cPLA2, cytoplasmic phospholipase A2; ET, endothelin; ICAM, intercellular adhesion molecule.

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Glucocorticoid receptors Glucocorticoids diffuse across the cell membrane and bind to glucocorticoid receptor (GR) in the cytoplasm (Rhen & Cidlowski 2005). There is only one form of GR that binds glucocorticoids termed GRα. GRβ is an alternatively spliced form of GR that interacts with DNA but not with glucocorticoids, so may act as a dominant negative inhibitor of glucocorticoid action by interfering with the binding of GR to DNA (Lewis-Tuffin & Cidlowski 2006). Whether GRβ is involved in steroid resistance in asthma is controversial. Activated GRs rapidly translocate to the nucleus where they produce their molecular effects. A pair of GRs (GR dimer) bind to glucocorticoid response elements (GREs) in the promoter region of steroid-responsive genes and this interaction switches on (and sometimes switches off) gene transcription (Fig. 31.3). Examples of genes that are activated by glucocorticoids include genes encoding β2-adrenergic receptors and the antiinflammatory proteins secretory leukoprotease inhibitor and mitogenactivated protein kinase phosphatase-1 (MKP-1) which inhibits mitogen-activated protein (MAP) kinase pathways. These effects may contribute to the antiinflammatory actions Glucocorticoid

Glucocorticoid receptor trans-activation

GRE Antiinflammatory Annexin-1 SLPI MKP-1 IkB-a

Inhaled glucocorticoids

Cytokines IL-1b IL-6 GM-CSF RANTES Eotaxin MIP-1a

months to reach a plateau, probably reflecting recovery of structural changes in the airway (Juniper et al. 1990a).

cis-repression

Negative GRE Side effects POMC CRF-1 Osteocalcin Keratin

trans-repression NF-kB CBP Inflammatory Cytokines Chemokines Adhesion molecules Inflammatory enzymes Inflammatory receptors Inflammatory proteins

Fig. 31.3 Glucocorticoids may regulate gene expression in several ways. Glucocorticoids enter the cell to bind to glucocorticoid receptors (GR) in the cytoplasm that translocate to the nucleus. GR homodimers bind to glucocorticoid-response elements (GRE) in the promoter region of steroid-sensitive genes, which may encode antiinflammatory proteins. Less commonly, GR homodimers interact with negative GREs to suppress genes, particularly those linked to side effects of corticosteroids. Nuclear GR also interact with coactivator molecules, such as CREB-binding protein (CBP), which is activated by proinflammatory transcription factors such as nuclear factor (NF)-kB, thus switching off the inflammatory genes activated by these transcription factors. SLPI, secretory leukoprotease inhibitor; MKP-1, mitogen-activated kinase phosphatase-1; IkB-a, inhibitor of NF-kB; GILZ, glucocorticoid-induced leucine zipper protein; POMC, proopiomelanocortin; CRF, corticotrophin-releasing factor. (See CD-ROM for color version.)

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of glucocorticoids (Clark 2003; Barnes 2006b). GR interaction with negative GREs may suppress gene transcription and it is thought that this may be important in mediating many the side effects of glucocorticoids. For example, glucocorticoids inhibit the expression of osteocalcin involved in bone synthesis (Dostert & Heinzel 2004).

Switching off inflammation The major action of glucocorticoids is to switch off multiple activated inflammatory genes that encode for cytokines, chemokines, adhesion molecules, inflammatory enzymes, and receptors (Barnes & Adcock 2003). These genes are switched on in the airways by proinflammatory transcription factors, such as nuclear factor (NF)-κB and activator protein (AP)-1, both of which are activated in asthmatic airways and switch on inflammatory genes by interacting with coactivator molecules, such as CREB-binding protein, that have intrinsic histone acetyltransferase activity, resulting in acetylation of core histones, which opens up the chromatin structure so that gene transcription is facilitated (Barnes et al. 2005). In artificial overexpression systems, activated GR may directly interact with NF-κB and AP-1 to inhibit their activity, but this does not appear to occur in asthmatic patients treated with inhaled glucocorticoids (Hart et al. 2000). Glucocorticoidactivated GR also interact with coactivator molecules and this inhibits the interaction of NF-κB with coactivators, thus reducing histone acetylation (Ito et al. 2000; Barnes 2006b). Reduction of histone acetylation also occurs through the recruitment of histone deacetylase 2 (HDAC2) to the activated inflammatory gene complex by activated GR, thereby resulting in effective suppression of all activated inflammatory genes within the nucleus (Fig. 31.4). This accounts for why glucocorticoids are so effective in the control of asthmatic inflammation, but also why they are safe, since other activated genes are not affected. There may be additional mechanisms that are also important in the antiinflammatory actions of glucocorticoids. Glucocorticoids have potent inhibitory effects on MAP kinase signaling pathways through the induction of MKP-1 and this may inhibit the expression of multiple inflammatory genes (Clark 2003; Barnes 2006a). Some inflammatory genes, for example granulocyte–macrophage colony-stimulating factor (GM-CSF), have an unstable mRNA that is rapidly degraded by certain RNAses but which is stabilized when cells are stimulated by inflammatory mediators. Glucocorticoids reverse this effect, resulting in rapid degradation of mRNA and reduced inflammatory protein secretion (Bergmann et al. 2004). This may be through the inhibition of proteins that stabilize mRNAs of inflammatory proteins, such as tristretraprolin (Brook et al. 2006).

Interaction with b2-adrenergic receptors

Inhaled β2 agonists and glucocorticoids are frequently used together in the control of asthma and it is now recognized

Glucocorticosteroids

Inflammatory stimuli e.g. IL-1b, TNF-a

Glucocorticoid (low dose)

IKK-2 NF-kB

p65 p50

GR

AF

pC kB Inflammatory genes, cytokines, chemokines, adhesion molecules, inflammatory receptors, enzymes, proteins

CBP p65 HAT p50 Acetylation –

↑ Gene transcription

GR

HDAC2 Deacetylation

Gene repression

Fig. 31.4 Glucocorticoid suppression of activated inflammatory genes. Inflammatory genes are activated by inflammatory stimuli, such as interleukin (IL)-1b or tumor necrosis factor (TNF)-a, resulting in activation of IKK2 (inhibitor of IkB kinase-2) which activates the transcription factor nuclear factor (NF)-kB. A dimer of p50 and p65 NF-kB proteins translocates to the nucleus and binds to specific kB recognition sites and also to coactivators, such as CREB-binding protein (CBP) or p300/CBP-activating factor (pCAF), which have intrinsic histone acetyltransferase (HAT) activity. This results in acetylation of core histone H4, resulting in increased expression of genes encoding multiple inflammatory proteins. Glucocorticoid receptors (GR) after activation by glucocorticoids translocate to the nucleus and bind to coactivators to inhibit HAT activity directly and recruiting histone deacetylase 2 (HDAC2), which reverses histone acetylation leading to suppression of these activated inflammatory genes. (See CD-ROM for color version.)

that there are important molecular interactions between these two classes of drug (Barnes 2002). As discussed above, glucocorticoids increase the gene transcription of β2-adrenergic receptors, resulting in increased expression of cell-surface receptors. This has been demonstrated in human lung in vitro (Mak et al. 1995a) and nasal mucosa in vivo after topical application of a glucocorticoid (Baraniuk et al. 1997). In this way glucocorticoids protect against the downregulation of β2 receptors after long-term administration (Mak et al. 1995b). This may be important for the nonbronchodilator effects of β2 agonists, such as mast cell stabilization. Glucocorticoids may also enhance the coupling of β2 receptors to G proteins, this enhancing β2-agonist effects and reversing the uncoupling of β2 receptors that may occur in response to inflammatory mediators, such as interleukin (IL)-1β through a stimulatory effect on a G protein-coupled receptor kinase (Mak et al. 2002). There is also evidence that β2 agonists may affect GR and thus enhance the antiinflammatory effects of glucocorticoids. β2 Agonists increase the translocation of GR from cytoplasm to the nucleus after activation by glucocorticoids (Roth et al.

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Pharmacology picin, phenobarbital or phenytoin, which induce cytochrome 450 enzymes, lower the plasma half-life of prednisolone (Gambertoglio et al. 1980). The plasma half-life is 2–3 hours, although its biological half-life is approximately 24 hours, so that it is suitable for daily dosing. There is no evidence that previous exposure to steroids changes their subsequent metabolism. Prednisolone is approximately 92% protein bound, the majority to a specific protein transcortin and the remainder to albumin; it is the unbound fraction that is biologically active. Some patients, usually with severe asthma, apparently fail to respond to glucocorticoids. “Steroid-resistant” asthma is not due to impaired absorption or metabolism of steroids, but is due to reduced antiinflammatory actions of glucocorticoids. Measurement of plasma concentrations of prednisolone are useful in monitoring compliance with inhaled glucocorticoids and in assessing whether a poor therapeutic response to glucocorticoids is due to poor absorption or increased metabolism.

2002). This effect has now been demonstrated in sputum macrophages of asthmatic patients after an inhaled glucocorticoid and inhaled long-acting β2 agonist (Usmani et al. 2005). This suggests that β2 agonists and glucocorticoid enhance each other’s beneficial effects in asthma therapy.

Chemical structures The adrenal cortex secretes cortisol (hydrocortisone) and, by modification of its structure, it was possible to develop derivatives, such as prednisolone and dexamethasone, with enhanced corticosteroid effects but with reduced mineralocorticoid activity. These derivatives with potent glucocorticoid actions were effective in asthma when given systemically but had no antiasthmatic activity when given by inhalation. Further substitution in the 17α ester position resulted in steroids with high topical activity, such as beclomethasone dipropionate (BDP), triamcinolone, flunisolide, budesonide, and fluticasone propionate (FP), which are potent in the skin (dermal blanching test) and were later found to have significant antiasthma effects when given by inhalation (Fig. 31.5).

Inhaled steroids The pharmacokinetics of inhaled glucocorticoids is important in relation to systemic effects (Barnes et al. 1998; Derendorf et al. 1998; Lipworth 1999). The fraction of steroid inhaled into the lungs acts locally on the airway mucosa, but may be absorbed from the airway and alveolar surface. This fraction therefore reaches the systemic circulation (Fig. 31.6). The fraction of inhaled steroid deposited in the oropharynx is swallowed and absorbed from the gut. The absorbed fraction may be metabolized in the liver before reaching the systemic circulation (first-pass metabolism). Budesonide and FP have a greater first-pass metabolism than BDP and are therefore less likely to produce systemic effects at high inhaled

Pharmacokinetics Prednisolone is readily and consistently absorbed after oral administration with little interindividual variation. Prednisone is converted in the liver to the active prednisolone. Enteric coatings to reduce the incidence of dyspepsia delay absorption but not the total amount of drug absorbed. Prednisolone is metabolized in the liver and drugs such as rifam-

CH2

HO

CH3

OH

CH2OCOC2H5 C=O

C=O 17

CH2

OH

C=O OCOC2H5 CH3

HO

HO

OH O O

C

H CH2CH2CH3

CH3 Cl O

O

O Hydrocortisone

Beclomethasone dipropionate

SCH2F

CH2 C=O

C=O HO

OCOC2H5 CH3

HO

Budesonide

OH O O

CH2 C

C=O

CH3

HO

CH3

O F Fluticasone propionate

Fig. 31.5 Chemical structures of inhaled glucocorticoids.

718

O O

F

F O

OH

O F Flunisolide

Triamcinolone acetonide

C

CH3 CH3

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MDI ~ 10–20% inhaled Mouth and pharynx ~ 80–90% swallowed (↓ spacer/mouth-wash)

Lungs

Systemic circulation

Absorption from GI tract Liver

GI tract

Inactivation in liver “first pass”

Systemic side effects

Fig. 31.6 Pharmacokinetics of inhaled glucocorticoids. GI, gastrointestinal. (See CD-ROM for color version.)

doses. The use of a large-volume spacer chamber reduces oropharyngeal deposition and therefore reduces systemic absorption of glucocorticoids, although this effect is minimal in glucocorticoids with a high first-pass metabolism (Brown et al. 1993). Mouth rinsing and discarding the rinse has a similar effect and this procedure should be used with highdose dry powder steroid inhalers, since spacer chambers cannot be used with these devices. The ideal inhaled corticosteroid with optimal therapeutic index should have high lung bioavailability, negligible oral bioavailability, low systemic absorption, high systemic clearance, and high protein binding (Derendorf 2005). A recently introduced glucocorticoid, ciclesonide, is an inactive prodrug that is activated by esterases in the lung to the active metabolite des-ciclesonide (Reynolds & Scott 2004). This may reduce oropharyngeal side effects as esterases appear to be less active in this site than in the lower airways. Ciclesonide is also claimed to be effective as a once-daily therapy.

Systemic steroids Hydrocortisone is given intravenously in acute severe asthma. While the value of glucocorticoids in acute severe asthma has been questioned, others have found that they speed the resolution of attacks. There is no apparent advantage in giving very high doses of intravenous steroids (such as methylprednisolone 1 g) as this only increases the risk of side effects, such as hyperglycemia and increased susceptibility to infections. Intravenous steroids are indicated in acute asthma if lung function is < 30% predicted and there is no significant improvement with a nebulized β2 agonist. Intravenous therapy is usually given until a satisfactory response is obtained and then oral prednisolone may be substituted. Oral prednisolone (40–60 mg) has a similar effect to intravenous hydrocortisone and is easier to administer (Harrison et al. 1986; Storr et al. 1987). High doses of inhaled glucocorticoids may also substitute for a course of oral steroids in controlling acute exacerbations of asthma. High-dose FP (2000 μg daily) was as effective as a course of oral prednisolone in controlling


acute exacerbations of asthma in a family practice setting and in children in an emergency room setting, although this route of delivery is more expensive (Levy et al. 1996; Manjra et al. 2000). Although doubling the dose of inhaled glucocorticoids has been recommended for mild exacerbations of asthma, this does not appear to be useful (FitzGerald et al. 2004; Harrison et al. 2004), but a fourfold increase in dose does appear effective (Foresi et al. 2000). Inhaled steroids have no proven effect in the management of severe acute asthma in a hospital setting (Edmonds et al. 2003), but trials with nebulized steroids, which can deliver large doses, are underway. Maintenance treatment with oral steroids are reserved for patients who cannot be controlled on maximum doses of other therapy, the dose being titrated to the lowest which provides acceptable control of symptoms. For any patient taking regular oral steroids, objective evidence of steroid responsiveness should be obtained before maintenance therapy is instituted. Short courses of oral steroids (30–40 mg prednisolone daily for 1–2 weeks) are indicated for exacerbations of asthma, and the dose may be tailed off over 1 week once the exacerbation is resolved (although the tail-off period is not strictly necessary, patients often find it reassuring).

Inhaled glucocorticoids There is no doubt that the early use of inhaled glucocorticoids has revolutionized the management of asthma, with marked reductions in asthma morbidity and improvement in health status. Inhaled steroids are now recommended as first-line therapy for all patients with persistent asthma (British Thoracic Society 2003; Global Initiative for Asthma 2006). Inhaled glucocorticoids are very effective in controlling asthma symptoms in asthmatic patients of all ages and severity (Kamada et al. 1996; Barnes 1999). Inhaled glucocorticoids improve the quality of life of patients with asthma and allow many patients to lead normal lives, improve lung function, reduce the frequency of exacerbations, and may prevent irreversible airway changes. They were first introduced to reduce the requirement for oral glucocorticoids in patients with severe asthma and many studies have confirmed that the majority of patients can be weaned off oral glucocorticoids (Barnes et al. 1998).

Studies in adults As experience has been gained with inhaled glucocorticoids, they have been introduced in patients with milder asthma, with the recognition that inflammation is present even in patients with mild asthma. Inhaled antiinflammatory drugs have now become first-line therapy in any patient who needs to use a β2-agonist inhaler more than two to three times a week and this is reflected in national and international guidelines for the management of chronic asthma. In patients with newly diagnosed asthma an inhaled corticosteroid (budesonide

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600 μg twice daily) reduced symptoms and β2-agonist inhaler usage and improved lung function. These effects persisted over the 2 years of the study, whereas in a parallel group treated with inhaled β2 agonists alone there was no significant change in symptoms or lung function (Haahtela et al. 1991). In another study patients with mild asthma treated with a low dose of inhaled corticosteroid (budesonide 400 μg daily) showed less symptoms and a progressive improvement in lung function over several months and many patients became completely asymptomatic (Juniper et al. 1990b). There was also a significant reduction in the number of exacerbations; in patients with mild asthma a low dose of glucocorticoids (budesonide 400 μg daily) significantly reduced exacerbation by around 40% over a 3-year period (Pauwels et al. 2003). Although the effects of inhaled glucocorticoids on AHR may take several months to reach a plateau, the reduction in asthma symptoms occurs much more rapidly (Vathenen et al. 1991) and reduced inflammation is seen within hours (Gibson et al. 2001; Ketchell et al. 2002). High-dose inhaled glucocorticoids may be used for the control of more severe asthma. This markedly reduces the need for maintenance oral glucocorticoids (Mash et al. 2000). With the use of add-on therapies, particularly long-acting β2 agonists, most patients can now be controlled on much lower doses of inhaled glucocorticoids so that high doses are needed in only a few patients with severe disease. Inhaled glucocorticoids are the treatment of choice in nocturnal asthma, which is a manifestation of inflamed airways, reducing nocturnal awakening and the diurnal variation in airway function. Inhaled glucocorticoids effectively control asthmatic inflammation but must be taken regularly. When inhaled glucocorticoids are discontinued there is usually a gradual increase in symptoms and airway responsiveness back to pretreatment values (Vathenen et al. 1991), although in patients with mild asthma who have been treated with inhaled glucocorticoids for a long time symptoms may not recur in some patients (Juniper et al. 1991). Reduction in the dose of inhaled glucocorticoids is associated with an increase in symptoms and this is preceded by an increase in exhaled nitric oxide (NO) and sputum eosinophils (Jatakanon et al. 2000; Leuppi et al. 2001).

Studies in children Inhaled glucocorticoids are equally effective in children. In an extensive study of children aged 7–17 years there was a significant improvement in symptoms, peak flow variability, and lung function compared to a regular inhaled β2 agonist which was maintained over the 22 months of the study (van Essen-Zandvliet et al. 1992), but asthma deteriorated when the inhaled glucocorticoids were withdrawn (Waalkens et al. 1993). There was a high proportion of drop-outs (45%) in the group treated with inhaled β2 agonist alone. Inhaled glucocorticoids are more effective than a long-acting β2 agonist in

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controlling asthma in children (Simons 1997). Inhaled glucocorticoids are also effective in younger children. Nebulized budesonide reduces the need for oral glucocorticoids and also improved lung function in children under the age of 3 years (Ilangovan et al. 1993; Berger 2005). Inhaled glucocorticoids given via a large-volume spacer improve asthma symptoms and reduce the number of exacerbations in preschool children and in infants.

Dose–response studies Surprisingly, the dose–response curve for the clinical efficacy of inhaled glucocorticoids is relatively flat and while all studies have demonstrated a clinical benefit of inhaled glucocorticoids, it has been difficult to demonstrate differences between doses, with most benefit obtained at the lowest doses used (Kamada et al. 1996; Barnes et al. 1998; Busse et al. 1998; Adams et al. 2002; Adams & Jones 2006). This is in contrast to the steeper dose–response for systemic effects, implying that while there is little clinical benefit from increasing doses of inhaled glucocorticoids, the risk of adverse effects is increased. However, the dose–response effect of inhaled glucocorticoids may depend on the parameters measured and, while it is difficult to discern a dose–response when traditional lung function parameters are measured, there may be a dose–response effect in prevention of asthma exacerbations. Thus, there is a significantly greater effect of budesonide 800 μg daily compared to 200 μg daily in preventing severe and mild asthma exacerbations (Pauwels et al. 1997). Normally, a fourfold or greater difference in dose has been required to detect a statistically significant (but often small) difference in effect on commonly measured outcomes such as symptoms, peak expiratory flow (PEF), use of rescue β2 agonist, and lung function, and even such large differences in dose are not always associated with significant differences in response. These findings suggest that pulmonary function tests or symptoms may have a rather low sensitivity in the assessment of the effects of inhaled glucocorticoids. This is obviously important for the interpretation of clinical comparisons between different inhaled glucocorticoids or inhalers. It is also important to consider the type of patient included in clinical studies. Patients with relatively mild asthma may have relatively little room for improvement with inhaled glucocorticoids, so that maximal improvement is obtained with relatively low doses. Patients with more severe asthma or with unstable asthma may have more room for improvement and may therefore show a greater response to increasing doses, but it is often difficult to include such patients in controlled clinical trials. More studies are needed to assess whether other outcome measures such as AHR or more direct measurements of inflammation, such as sputum eosinophils or exhaled NO, may be more sensitive than traditional outcome measures such as symptoms or lung function tests (Jatakanon et al. 1998, 1999; Lim et al. 1999; Kharitonov et al. 2002). Higher

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doses of inhaled glucocorticoids are needed to control AHR than to improve symptoms and lung function, and this may have a better long-term outcome in terms of reduction in structural changes of the airways (Sont et al. 1999). Measurement of sputum eosinophils to adjust the dose of inhaled glucocorticoids may reduce the overall dose requirement for inhaled glucocorticoids and exacerbations (Green et al. 2002; Jayaram et al. 2006). Monitoring of exhaled NO also reduces the requirement for glucocorticoids but is not yet practical in clinical practice (Smith et al. 2005).

Prevention of irreversible airway changes Some patients with asthma develop an element of irreversible airflow obstruction, but the pathophysiologic basis of these changes is not yet understood. It is likely that they are the result of chronic airway inflammation and that they may be prevented by treatment with inhaled glucocorticoids. There is some evidence that the annual decline in lung function may be slowed by the introduction of inhaled glucocorticoids (Dompeling et al. 1992) and this is supported by a 5-year study of low-dose budesonide in patients with mild asthma (Lange et al. 2006; O’Byrne et al. 2006). Increasing evidence also suggests that delay in starting inhaled glucocorticoids may result in less overall improvement in lung function in both adults and children (Agertoft & Pedersen 1994; Haahtela et al. 1994; Selroos et al. 1995). These studies suggest that introduction of inhaled glucocorticoids at the time of diagnosis is likely to have the greatest impact (Agertoft & Pedersen 1994; Selroos et al. 1995). So far there is no evidence that early use of inhaled glucocorticoids is curative and even when inhaled glucocorticoids are introduced at the time of diagnosis, symptoms and lung function revert to pretreatment levels when glucocorticoids are withdrawn (Haahtela et al. 1994).

Reduction in mortality Inhaled glucocorticoids may reduce the mortality from asthma but prospective studies are almost impossible to conduct. In a retrospective review of the risk of mortality and prescribed antiasthma medication, there was significant protection provided by regular inhaled corticosteroid therapy (Suissa et al. 2000). By contrast, asthma mortality appears to increase with increasing usage of short-acting β2 agonists, reflecting the fact that increased rescue therapy is a marker of poor asthma control (Ernst et al. 1993). The increase in use of rescue therapy should result in an increase in the maintenance dose of inhaled glucocorticoids. The long-acting inhaled β2 agonist salmeterol is associated with a small increase in asthma mortality, but the excess deaths appear to be related to underuse of inhaled glucocorticoids (Nelson et al. 2006).

Comparison between inhaled glucocorticoids Several inhaled glucocorticoids are currently prescribable in asthma, although their availability varies between countries.


There have been relatively few studies comparing efficacy of the different inhaled glucocorticoids, and it is important to take into account the delivery system and the type of patient under investigation when such comparisons are made. Because of the relatively flat dose–response curve for the clinical parameters normally used in comparing doses of inhaled glucocorticoids, it may be difficult to see differences in efficacy of inhaled glucocorticoids. Most comparisons have concentrated in differences in systemic effects at equally efficacious doses, although it has often proved difficult to establish dose equivalence. There are few studies comparing different doses of inhaled glucocorticoids in asthmatic patients. Budesonide has been compared with BDP and in adults and children it appears to have comparable antiasthma effects at equal doses, whereas FP appears to be approximately twice as potent as BDP and budesonide (Adams & Jones 2006). Studies have consistently shown that FP and budesonide have less systemic effects than BDP, triamcinolone, and flunisolide (Lipworth 1999). The new inhaled glucocorticoids mometasone and ciclesonide are claimed to have less systemic effects (Nathan et al. 2001; Reynolds & Scott 2004).

Clinical use in asthma Inhaled glucocorticoids are now recommended as first-line therapy for all patients with persistent symptoms. Inhaled glucocorticoids should be started in any patient who needs to use a β2-agonist inhaler for symptom control more than three times weekly. It is conventional to start with a low dose of inhaled corticosteroid and to increase the dose until asthma control is achieved. However, this may take time and a preferable approach is to start with a dose of glucocorticoids in the middle of the dose range (400 μg twice daily) to establish control of asthma more rapidly (Barnes 1996). Once control is achieved (defined as normal or best possible lung function and infrequent need to use an inhaled β2 agonist), the dose of inhaled corticosteroid should be reduced in a stepwise manner to the lowest dose needed for optimal control. It may take as long as 3 months to reach a plateau in response and any changes in dose should be made at intervals of 3 months or more. When daily doses of ≥ 800 μg daily are needed, a large-volume spacer device should be used with a metered-dose inhaler (MDI) and mouth-washing with a dry powder inhaler in order to reduce local and systemic side effects. Inhaled glucocorticoids are usually given as a twice-daily dose in order to increase compliance. When asthma is unstable four times daily dosage is preferable (Malo et al. 1989). For patients who require ≤ 400 μg daily, oncedaily dosing appears to be as effective as twice-daily dosing, at least for budesonide (Jones et al. 1994). The dose of inhaled corticosteroid should be increased to 2000 μg daily if necessary, but higher doses may result in systemic effects. It may be preferable to add a low dose of oral corticosteroid, since higher doses of inhaled glucocorticoids are expensive and have a high incidence of local side effects.

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Nebulized budesonide has been advocated in order to give an increased dose of inhaled corticosteroid and to reduce the requirement for oral glucocorticoids (Otulana et al. 1992), but this treatment is expensive and may achieve its effects largely via systemic absorption. The dose of inhaled corticosteroid should be the minimal dose that controls asthma and once control is achieved the dose should be slowly reduced (Hawkins et al. 2003).

Add-on therapy Previously it was recommended that the dose of inhaled glucocorticoids be increased if asthma was not controlled, on the assumption that there was residual inflammation of the airways. However, the dose–response effect of inhaled glucocorticoids is relatively flat, so that there is little improvement in lung function after increasing the dose of inhaled glucocorticoids. An alternative strategy is to add some other class of controller drug and this is more effective than increasing the dose of inhaled glucocorticoids for most patients (Kankaanranta et al. 2004). In patients in general practice who are not controlled on BDP 200 μg twice daily, addition of salmeterol 50 μg twice daily was more effective than increasing the dose of inhaled corticosteroid to 500 μg twice daily, in terms of lung function improvement, use of rescue β2-agonist use, and symptom control (Greening et al. 1994). This has been confirmed in several other studies (Shrewsbury et al. 2000). Similar results have been found with another long-acting inhaled β2agonist, formoterol, which in addition reduced the frequency of mild and severe asthma exacerbations in patients with mild, moderate, and severe persistent asthma (Pauwels et al. 1997; O’Byrne et al. 2001). These studies showing the great efficacy of combined glucocorticoids and long-acting β2 agonist compared with increased doses of long-acting β2 agonist have led to the development of fixed combinations of glucocorticoids and long-acting β2 agonists, such as FP/ salmeterol and budesonide/formoterol, which may be more convenient for patients (Chapman et al. 1999; Shapiro et al. 2000). These fixed combination inhalers also ensure that patients do not discontinue their inhaled glucocorticoids when a long-acting bronchodilator is used. For patients with mild persistent asthma, combination inhalers are no more effective than the inhaled glucocorticoids alone in controlled trials (Ni et al. 2005), but may have an advantage in the real world where adherence to regular inhaled glucocorticoids is very low. Recently studies have demonstrated that when formoterol combined with budesonide is used as a reliever therapy this gives better control of asthma compared with the normally used short-acting β2 agonist as rescue therapy with either the same dose of combination inhaler or a high dose of inhaled glucocorticoids as maintenance treatment (O’Byrne et al. 2005; Rabe et al. 2006a). This advantage is particularly striking in terms of reducing the number of severe exacerbations. When formoterol was used as reliever therapy, exacerbations

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were reduced to a greater extent than with the short-acting β2 agonist terbutaline, but the combination was even more effective (Rabe et al. 2006b). This suggests that the “as-required” use of inhaled glucocorticoids contributes to the marked reduction in acute exacerbations. The mechanisms by which as-required glucocorticoids improve asthma control and reduce exacerbations are not completely understood, but exacerbations of asthma evolve over several days when patients take increasing amounts of rescue medication (Tattersfield et al. 1999). During this time there is increasing inflammation of the airways, as may be measured by exhaled NO and sputum eosinophils (Jatakanon et al. 2000). Taking the inhaled corticosteroid at the same time as the formoterol to relieve symptoms may suppress this evolving inflammation, particularly since glucocorticoids appear to have a relatively rapid onset of effect in suppressing airway inflammation (Barnes 2006c). Addition of low doses of theophylline (plasma concentration < 10 mg/L) is more effective than doubling the dose of inhaled budesonide, either in mild or severe asthma (Evans et al. 1997; Ukena et al. 1997; Lim et al. 2000). However this is less effective than using a long-acting inhaled β2 agonist as add-on therapy (Wilson et al. 2000). Antileukotrienes have also been used as an add-on therapy (Laviolette et al. 1999; Price et al. 2003), although this is less effective than addition of long-acting β2 agonists (Nelson et al. 2000; Ducharme et al. 2004).

Side effects The efficacy of inhaled glucocorticoids is now established in short- and long-term studies in adults and children, but there are still concerns about side effects, particularly in children and when high inhaled doses are used. Several side effects have been recognized (Table 31.2).

Local side effects Side effects due to the local deposition of the inhaled corticosteroid in the oropharynx may occur with inhaled glucocorticoids, but the frequency of complaints depends on Table 31.2 Side effects of inhaled glucocorticoids. Local side effects Dysphonia Oropharyngeal candidiasis Cough Systemic side effects Adrenal suppression Growth suppression Bruising Osteoporosis Cataracts Glaucoma Metabolic abnormalities (glucose, insulin, triglycerides) Psychiatric disturbances

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the dose and frequency of administration and on the delivery system used. The commonest complaint is of hoarseness of the voice (dysphonia) and may occur in over 50% of patients using an MDI. Dysphonia is not appreciably reduced by using spacers, but may be less with dry powder devices. Dysphonia may be due to myopathy of laryngeal muscles and is reversible when treatment is withdrawn (Williamson et al. 1995). For most patients it is not troublesome but may be disabling in singers and lecturers. Oropharyngeal candidiasis (thrush) may be a problem in some patients, particularly in the elderly, with concomitant oral glucocorticoids and more than twice-daily administration (Toogood et al. 1980). Large-volume spacer devices protect against this local side effect by reducing the dose of inhaled corticosteroid that deposits in the oropharynx. There is no evidence that inhaled corticosteroid, even in high doses, increase the frequency of infections, including tuberculosis, in the lower respiratory tract. There is no evidence for atrophy of the airway epithelium and even after 10 years of treatment with inhaled glucocorticoids there is no evidence for any structural changes in the epithelium. Cough and throat irritation, sometimes accompanied by reflex bronchoconstriction, may occur when inhaled glucocorticoids are given via an MDI. These symptoms are likely to be due to surfactants in pressurized aerosols as they disappear after switching to a dry powder corticosteroid inhaler device.

The systemic effect of an inhaled corticosteroid is dependent on the amount of drug absorbed into the systemic circulation (Fig. 31.6). Approximately 90% of the inhaled dose from an MDI deposits in the oropharynx and is swallowed and subsequently absorbed from the gastrointestinal tract. Use of a large-volume spacer device markedly reduces oropharyngeal deposition and therefore the systemic effects of inhaled glucocorticoids, although this is less important when oral bioavailability is minimal, as with FP. For dry powder inhalers similar reductions in systemic effects may be achieved with mouth-washing and discarding the fluid. All patients using a daily dose of ≥ 800 μg of an inhaled corticosteroid should therefore use either a spacer or mouth-washing to reduce systemic absorption. Approximately 10% of an MDI enters the lung and this fraction (which presumably exerts the therapeutic effect) may be absorbed into the systemic circulation. As the fraction of inhaled corticosteroid deposited in the oropharynx is reduced, the proportion of the inhaled dose entering the lungs is increased. More efficient delivery to the lungs is therefore accompanied by increased systemic absorption, but this is offset by a reduction in the dose needed for optimal control of airway inflammation. For example, a multiple dry powder delivery system, the Turbohaler, delivers approximately twice as much corticosteroid to the lungs as other devices, and therefore has increased systemic effects. However this is compensated for by the fact that only half the dose is required.

Systemic side effects

Adrenal suppression Glucocorticoids may cause hypothalamic–pituitary–adrenal (HPA) axis suppression by reducing corticotrophin (ACTH) production, which reduces cortisol secretion by the adrenal gland. The degree of HPA suppression is dependent on dose, duration, frequency, and timing of corticosteroid administration. There is no evidence that cortisol responses to the stress of an asthma exacerbation or insulin-induced hypoglycemia are impaired, even with high doses of inhaled glucocorticoids. However, measurement of HPA axis function provides evidence for systemic effects of an inhaled corticosteroid. Basal adrenal cortisol secretion may be measured by a morning plasma cortisol, 24-hour urinary cortisol, or plasma cortisol profile over 24 hours. Other tests measure the HPA response following stimulation with tetracosactrin (which measures adrenal reserve) or stimulation with metyrapone and insulin (which measure the response to stress). There are many studies of HPA axis function in asthmatic patients with inhaled glucocorticoids, but the results are inconsistent as they have often been uncontrolled and patients have also been taking courses of oral glucocorticoids (which may affect the HPA axis for weeks) (Barnes et al. 1998). BDP, budesonide, and FP at high doses by conventional MDI (> 1600 μg daily) give a dose-related decrease in morning serum cortisol levels and 24-hour urinary cortisol, although values still lie well within the normal range. However, when a large-volume

The efficacy of inhaled glucocorticoids in the control of asthma is undisputed, but there are concerns about systemic effects of inhaled glucocorticoids, particularly as they are likely to be used over long periods and in children of all ages (Kamada et al. 1996; Lipworth 1999). The safety of inhaled glucocorticoids has been extensively investigated since their introduction 30 years ago (Barnes et al. 1998). One of the major problems is to decide whether a measurable systemic effect has any significant clinical consequence and this necessitates careful long-term follow-up studies. As biochemical markers of systemic corticosteroid effects become more sensitive, then systemic effects may be seen more often, but this does not mean that these effects are clinically relevant. There are several case reports of adverse systemic effects of inhaled glucocorticoids, and these may be idiosyncratic reactions, which may be due to abnormal pharmacokinetic handing of the inhaled corticosteroid. The systemic effect of an inhaled corticosteroid will depend on several factors, including the dose delivered to the patient, the site of delivery (gastrointestinal tract and lung), the delivery system used and individual differences in the patient’s response to the corticosteroid. Recent studies suggest that systemic effects of inhaled corticosteroid are less in patients with more severe asthma, presumably as less drug reaches the lung periphery (Brutsche et al. 2000; Harrison et al. 2001).

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spacer is used, doses of 2000 μg daily of BDP or budesonide have little effect on 24-hour urinary cortisol excretion. Stimulation tests of HPA axis function similarly show no consistent effects of doses of 1500 μg or less of inhaled corticosteroid. At high doses (> 1500 μg daily) budesonide and FP have less effect than BDP on HPA axis function. In children no suppression of urinary cortisol is seen with doses of BDP of 800 μg or less. In studies where plasma cortisol has been measured at frequent intervals there was a significant reduction in cortisol peaks with doses of inhaled BDP as low as 400 μg daily, although this does not appear to be dose related in the range 400–1000 μg. The clinical significance of these effects is not certain.

Bone metabolism Glucocorticoids lead to a reduction in bone mass by direct effects on bone formation and resorption and indirectly by suppression of the pituitary–gonadal and HPA axes, effects on intestinal calcium absorption, renal tubular calcium reabsorption, and secondary hyperparathyroidism (Efthimou & Barnes 1998). The effects of oral glucocorticoids on osteoporosis and increased risk of vertebral and rib fractures are well known, but there are no reports suggesting that longterm treatment with inhaled glucocorticoids is associated with an increased risk of fractures. Bone densitometry has been used to assess the effect of inhaled glucocorticoids on bone mass. Although there is evidence that bone density is less in patients taking high-dose inhaled glucocorticoids, interpretation is confounded by the fact that these patients are also taking intermittent courses of oral glucocorticoids. Changes in bone mass occur very slowly and several biochemical indices have been used to assess the short-term effects of inhaled glucocorticoids on bone metabolism. Bone formation has been measured by plasma concentrations of bone-specific alkaline phosphatase, serum osteocalcin, or procollagen peptides. Bone resorption may be assessed by urinary hydroxyproline after a 12-hour fast, urinary calcium excretion, and pyridinium cross-link excretion. It is important to consider the age, diet, time of day, and physical activity of the patient in interpreting any abnormalities. It is also necessary to choose appropriate control groups as asthma itself may have an effect on some of the measurements, such as osteocalcin. Inhaled glucocorticoids, even at doses up to 2000 μg daily, have no significant effect on calcium excretion, but acute and reversible dose-related suppression of serum osteocalcin has been reported with BDP and budesonide when given by conventional MDI in several studies. Budesonide consistently has less effect than BDP at equivalent doses and only BDP increases urinary hydroxyproline at high doses. However, with a large-volume spacer, even doses of 2000 μg daily of either BDP or budesonide are without effect on plasma osteocalcin concentrations. Urinary pyridinium and deoxypyridinoline cross-links, which are a more

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accurate and stable measurement of bone and collagen degradation, are not increased with inhaled glucocorticoids (BDP > 1000 μg daily), even with intermittent courses of oral glucocorticoids. It is important to monitor changes in markers of bone formation as well as bone degradation, as the net effect on bone turnover is important. There is no evidence that inhaled glucocorticoids increase the frequency of fractures. Long-term treatment with high-dose inhaled glucocorticoids has not been associated with any consistent change in bone density. Indeed, in elderly patients there may be an increase in bone density due to increased mobility.

Connective tissue effects Oral and topical glucocorticoids cause thinning of the skin, telangiectasiae, and easy bruising, probably as a result of loss of extracellular ground substance within the dermis, due to an inhibitory effect on dermal fibroblasts. There are reports of increased skin bruising and purpura in patients using high doses of inhaled BDP, but the amount of intermittent oral glucocorticoids in these patients is not known. Easy bruising in association with inhaled glucocorticoids is more frequent in elderly patients (Roy et al. 1996) and there are no reports of this problem in children. Long-term prospective studies with objective measurements of skin thickness are needed with different inhaled glucocorticoids. Cataracts Long-term treatment with oral glucocorticoids increase the risk of posterior subcapsular cataracts and there are several case reports describing cataracts in individual patients taking inhaled glucocorticoids (Barnes et al. 1998). In a recent crosssectional study in patients aged 5–25 years taking either inhaled BDP or budesonide no cataracts were found on slitlamp examination, even in patients taking 2000 μg daily for over 10 years (Simons et al. 1993). However, epidemiologic studies have identified an increased risk of cataracts in patients taking high-dose inhaled steroids over prolonged periods (Cumming et al. 1997). A slight increase in the risk of glaucoma in patients taking very high doses of inhaled glucocorticoids has also been identified (Garbe et al. 1997). Growth There has been particular concern that inhaled glucocorticoids may cause stunting of growth and several studies have addressed this issue. Asthma itself (as with other chronic diseases) may have an effect on the growth pattern and has been associated with delayed onset of puberty and deceleration of growth velocity that is more pronounced with more severe disease (Pedersen 2001). However, asthmatic children appear to grow for longer, so that their final height is normal. The effect of asthma on growth makes it difficult to assess the effects of inhaled glucocorticoids on growth in cross-sectional

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studies, particularly as courses of oral glucocorticoids is a confounding factor. Longitudinal studies have demonstrated that there is no significant effect of inhaled glucocorticoids on statural growth in doses of up to 800 μg daily and for up to 5 years of treatment (Barnes et al. 1998). A metaanalysis of 21 studies, including over 800 children, showed no effect of inhaled BDP on statural height, even with higher doses and long duration of therapy (Allen et al. 1994) and in a large study of asthmatics treated with inhaled glucocorticoids during childhood there was no difference in statural height compared to normal children (Silverstein et al. 1997). Another long-term follow-up study showed no effect of glucocorticoids on final height in children treated over several years (Agertoft & Pedersen 2000). Short-term growth measurements (knemometry) have demonstrated that even a low dose of an oral corticosteroid (prednisolone 2.5 mg) is sufficient to give complete suppression of lower leg growth. However, inhaled budesonide up to 400 μg is without effect, although some suppression is seen with 800 μg and with 400 μg BDP. The relationship between knemometry measurements and final height is uncertain since low doses of oral corticosteroid that have no effect on final height cause profound suppression.

Metabolic effects Several metabolic effects have been reported after inhaled glucocorticoids, but there is no evidence that these are clinically relevant at therapeutic doses. In adults fasting glucose and insulin are unchanged after doses of BDP up to 2000 μg daily and in children with inhaled budesonide up to 800 μg daily. In normal individuals high-dose inhaled BDP may slightly increase resistance to insulin. However, in patients with poorly controlled asthma high doses of BDP and budesonide paradoxically decrease insulin resistance and improve glucose tolerance, suggesting that the disease itself may lead to abnormalities in carbohydrate metabolism. Neither BDP 2000 μg daily in adults nor budesonide 800 μg daily in children have any effect on plasma cholesterol or triglycerides. Psychiatric effects There are various reports of psychiatric disturbance, including emotional lability, euphoria, depression, aggressiveness, and insomnia, after inhaled glucocorticoids. Only eight such patients have so far been reported, suggesting that this is very infrequent and a causal link with inhaled glucocorticoids has usually not been established. Pregnancy Based on extensive clinical experience inhaled glucocorticoids appear to be safe in pregnancy, although no controlled studies have been performed. There is no evidence for any adverse effects of inhaled glucocorticoids on the pregnancy, the delivery, or the fetus (Schatz 1999). It is important to


recognize that poorly controlled asthma may increase the incidence of perinatal mortality and retard intrauterine growth, so that more effective control of asthma with inhaled glucocorticoids may reduce these problems.

Systemic glucocorticoids Oral or intravenous glucocorticoids may be indicated in several situations. Prednisone is converted in the liver to the active prednisolone. In pregnant patients prednisone is preferable as it is not converted to prednisolone in the fetal liver, thus diminishing exposure of the fetus to glucocorticoids. Enteric-coated preparations of prednisolone are used to reduce side effects (particularly gastric side effects) and give delayed and reduced peak plasma concentrations, although the bioavailability and therapeutic efficacy of these preparations is similar to uncoated tablets. Prednisolone and prednisone are preferable to dexamethasone, betamethasone or triamcinolone, which have longer plasma half-lives and therefore an increased frequency of adverse effects.

Short courses Short courses of oral glucocorticoids (30–40 mg prednisolone daily for 1–2 weeks or until the peak flow values return to best attainable) are indicated for exacerbations of asthma, and the dose may be tailed off over 1 week once the exacerbation is resolved. The tail-off period is not strictly necessary, but some patients find it reassuring.

Maintenance glucocorticoids Maintenance oral glucocorticoids are only needed in a small proportion of asthmatic patients (approximately 1%) with the most severe asthma that cannot be controlled with maximal doses of inhaled glucocorticoids (2000 μg daily) and additional bronchodilators. The minimal dose of oral corticosteroid needed for control should be used and reductions in the dose should be made slowly in patients who have been on oral glucocorticoids for long periods (e.g., by 2.5 mg per month for doses down to 10 mg daily and thereafter by 1 mg per month). Oral glucocorticoids are usually given as a single morning dose, as this reduces the risk of adverse effects since it coincides with the peak diurnal concentrations. There is some evidence that administration in the afternoon may be optimal for some patients who have severe nocturnal asthma (Beam et al. 1992). Alternate-day administration may also reduce adverse effects, but control of asthma may not be as good on the day when the oral dose is omitted in some patients. Intramuscular triamcinolone acetonide (80 mg monthly) has been advocated in patients with severe asthma as an alternative to oral glucocorticoids (McLeod et al. 1985; Ogirala et al. 1991). This may be considered in patients in whom

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compliance is a particular problem, but the major concern is the high frequency of proximal myopathy associated with this fluorinated corticosteroid. Some patients who do not respond well to prednisolone are reported to respond to oral betamethasone, presumably because of pharmacokinetic handling problems with prednisolone.

Acute severe asthma Intravenous hydrocortisone is given in acute severe asthma, with a recommended dose of 200 mg i.v. While the value of glucocorticoids in acute severe asthma has been questioned, others have found that they speed the resolution of attacks (Engel & Heinig 1991). There is no apparent advantage in giving very high doses of intravenous glucocorticoids (such as methylprednisolone 1 g). Indeed, intravenous glucocorticoids have occasionally been associated with an acute severe myopathy (Decramer et al. 1995). There was no difference in recovery from acute severe asthma when intravenous hydrocortisone in doses of 50, 200 or 500 mg 6-hourly was used (Bowler et al. 1992) and another placebo-controlled study showed no beneficial effect of intravenous glucocorticoids (Morell et al. 1992). Intravenous glucocorticoids are indicated in acute asthma if lung function is < 30% predicted and there is no significant improvement with nebulized β2 agonist. Intravenous therapy is usually given until a satisfactory response is obtained and then oral prednisolone may be substituted. Oral prednisolone (40– 60 mg) has a similar effect to intravenous hydrocortisone and is easier to administer (Harrison et al. 1986; Engel & Heinig 1991). Oral prednisolone is the preferred treatment for acute severe asthma, providing there are no contraindications to oral therapy (British Thoracic Society 1997). There is some evidence that high doses of nebulized glucocorticoids may also be effective in acute exacerbations of asthma, with a more rapid onset of action (Devidayal et al. 1999).

Corticosteroid resistance Although glucocorticoids are highly effective in the control of asthma and other chronic inflammatory or immune diseases, a small proportion of patients with asthma fail to respond even to high doses of oral glucocorticoids (Adcock & Lane 2003; Leung & Bloom 2003; Barnes 2004). Resistance to the therapeutic effects of glucocorticoids is also recognized in other inflammatory and immune diseases, including rheumatoid arthritis and inflammatory bowel disease. Steroidresistant patients, although uncommon, present considerable management problems. Steroid-resistant asthma is defined as a failure to improve FEV1 by > 15% after treatment with oral prednisolone 30– 40 mg daily for 2 weeks, providing the oral steroid is taken (verified by plasma prednisolone level or reduction in early-morning cortisol level). These patients are not addisonian and they do not suffer from the abnorm-

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alities in sex hormones described in the very rare familial glucocorticoid resistance. Plasma cortisol and adrenal suppression in response to exogenous cortisol is normal in these patients, so they suffer from side effects of glucocorticoids. Complete glucocorticoid resistance in asthma is very rare, with a prevalence of less than 1 in 1000 asthmatic patients. Much more common is reduced responsiveness to glucocorticoids, so that large inhaled or oral doses are needed to control asthma adequately (steroid-dependent asthma). It is likely that there is a range of responsiveness to glucocorticoids and that steroid resistance is at one extreme of this range. It is important to establish that the patient has asthma rather than chronic obstructive pulmonary disease (COPD), “pseudoasthma” (a hysterical conversion syndrome involving vocal cord dysfunction), left ventricular failure, or cystic fibrosis that do not respond to glucocorticoids (Thomas et al. 1999). Asthmatic patients are characterized by variability in PEF and, in particular, diurnal variability of > 15% and episodic symptoms. Biopsy studies have demonstrated the typical eosinophilic inflammation of asthma in these patients.

Molecular mechanisms The molecular mechanisms of steroid resistance are still not understood, but there are likely to be several mechanisms (Adcock & Lane 2003; Leung & Bloom 2003). GRs appear to function normally, but there are defects in the interaction of GR with other transcription factors. Certain cytokines are able to reduce steroid responsiveness by enhancing the activity of transcription factors, such as AP-1, which then combine with and divert GR (Adcock et al. 1995). Other patients appear to have a defect in nuclear localization of GR as a result of phosphorylation of the receptor by p38 MAP kinase (Irusen et al. 2002; Matthews et al. 2004). In other patients there is a defect in corticosteroid-induced histone acetylation, with defective acetylation of the lysine-5 residue on histone-4, presumably resulting in reduced transcription of important antiinflammatory genes (Matthews et al. 2004). It has been proposed that increased expression of GRβ interferes with the antiinflammatory action of corticosteroids by competing with GRα for binding to GREs or coactivator molecules (Leung et al. 1997), but this is unlikely as the amount of GRβ expressed in inflammatory cells is insufficient to interfere by competition with the effects of corticosteroids on GRα (Pujols et al. 2002). As discussed above, HDAC2 plays a critical role in mediating the antiinflammatory effects of corticosteroids. In COPD, corticosteroid resistance is explained by a marked defect in activity and expression of HDAC2 (Barnes 2006d; Ito et al. 2005, 2006). This defect in HDAC2 appears to be due to increased oxidative stress. There is also a defect in HDAC2 in patients with severe asthma who have a poor response to glucocorticoids (Hew et al. 2006). Asthmatic patients who smoke are also glucocorticoid insensitive (Thomson & Spears 2005) and this is likely to be explained by impairment in

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Steroid-resistant asthma Smoking asthma Severe asthma

Normal asthma

Cigarette smoke Stimuli Alveolar macrophage

Corticosteroids

a beneficial effect in some patients with steroid-dependent asthma (Kidney et al. 1995), and identification of the molecular mechanisms of theophylline-induced HDAC activation may lead to a new approach to the treatment of glucocorticoid resistance in some patients.

Oxidative stress Peroxynitrite

References

GR NF-kB

NF-kB ↑ HDAC2

↓ HDAC2

Histone acetylation ↑ TNF-a ↑ IL-8 ↑ GM-CSF


↓ Histone acetylation

Histone acetylation TNF-a IL-8 GM-CSF

Fig. 31.7 Proposed mechanism of glucocorticoid resistance in severe asthma and smoking asthma. Stimulation of normal and asthmatic alveolar macrophages activates nuclear factor (NF)-kB and other transcription factors to switch on histone acetyltransferase leading to histone acetylation and subsequently to transcription of genes encoding inflammatory proteins, such as tumor necrosis factor (TNF)-a, interleukin (IL)-8, and granulocyte–macrophage colony-stimulating factor (GM-CSF). Corticosteroids reverse this by binding to glucocorticoid receptors (GR) and recruiting histone deacetylase 2 (HDAC2). This reverses the histone acetylation induced by NF-kB and switches off the activated inflammatory genes. In smoking asthmatic patients, cigarette smoke generates oxidative stress (acting via the formation of peroxynitrite) and in severe asthma intense inflammation generates oxidative stress to impair the activity of HDAC2. This amplifies the inflammatory response to NF-kB activation, but also reduces the antiinflammatory effect of corticosteroids, as HDAC2 is now unable to reverse histone acetylation.

HDAC2 in airway cells as a result of oxidative stress (Barnes et al. 2004) (Fig. 31.7).

Therapy Patients with glucocorticoid-resistant asthma may be difficult to manage, as they do not benefit from corticosteroids or require high doses of oral steroids that are associated with unacceptable side effects. Steroid-sparing therapies, including methotrexate, cyclosporin A, and oral gold have proved to be disappointing. Alternative antiinflammatory therapies, including phosphodiesterase-4 inhibitors, p38 MAP kinase inhibitors and NF-κB inhibitors, may prove useful in the future but there is a concern about potential side effects with these agents. Omalizumab is effective in a few allergic patients with steroid-dependent asthma. Anti-tumor necrosis factor therapies have been reported to be beneficial in patients with refractory asthma in small trials (Berry et al. 2006), but this has not been confirmed in larger controlled trials. Theophylline is able to reverse the glucocorticoid resistance in COPD by restoring HDAC activity (Cosio et al. 2004) and has

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32

Immunomodulating Drugs Iain A.M. MacPhee

Summary

Overview

The immune response causing tissue-damaging inflammation can be modulated at a number of points. Drugs with different modes of action are often used in synergistic combinations in an attempt to maximize efficacy with reduced drug toxicity. The most established of the immunosuppressive drugs act by inhibiting lymphocyte proliferation, the most widely used of which (azathioprine, mycophenolate and cyclophosphamide) are described here. These agents have effects on both T and B lymphocytes but are relatively nonspecific in their action, resulting in problems with toxicity. The calcineurin inhibitors ciclosporin A and tacrolimus block the production of interleukin (IL)-2 by T lymphocytes and their introduction resulted in significant improvements in the outcome for solid organ transplant recipients with more recent application to a number of autoimmune diseases. Nephrotoxicity is a major problem with calcineurin inhibitor use and this has led to a continued search for equally effective but less toxic agents. The mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus inhibit the response of lymphocytes to cytokines. They also inhibit wound healing and angiogenesis, with some potential benefits at the expense of side effects. The development of monoclonal antibodies has offered the potential for more specifically targeted drugs. Antibodies with specificity for a broad range of lymphocytes such as antithymocyte globulin and alemtuzumab are potently immunosuppressive but probably increase the risk of infectious complications. Antibodies with specificity for the IL-2 receptor, expressed on activated T lymphocytes, have been used to good effect in solid organ transplantation but have not been applied to other conditions. Rituximab, an antibody specific for the CD20 molecule expressed on B lymphocytes has been used successfully in the treatment of a number of antibody-mediated autoimmune diseases. These are all potent drugs with a narrow therapeutic index requiring a high degree of caution in prescribing.

T lymphocytes and their products are known to play a critical role in the pathogenesis of asthma and atopic allergic diseases (see Chapters 2 and 3) and “anti-T lymphocytes” agents have been evaluated in treatment of these conditions for many years. For example, the calcineurin inhibitors pimecrolimus and tacrolimus have an established place as topical immunosuppressants in atopic dermatitis (Alomar et al. 2004; Iskedjian et al. 2004; Breuer et al. 2005) with antiinflammatory potency similar to corticosteroids of moderate potency. Immunomodulatory drugs have been used for many years in the treatment of asthma. A metaanalysis of the effects of concomitant methotrexate therapy in oral glucocorticoid-dependent asthmatics suggested an oral glucocorticoid-sparing effect (Marin 1997). Two blinded placebo-controlled trials of cyclosporin A in severe oral glucocorticoid-dependent asthmatics showed improved lung function while reducing oral glucocorticoid requirements (Alexander et al. 1992; Lock et al. 1996). In a small controlled study a single injection of an anti-CD4 antibody improved lung function in steroid-dependent asthmatics (Kon et al. 1998). The currently available range of immunosuppressive drugs target a number of distinct steps in the generation of the immune response (Table 32.1). The synergistic effect of drugs acting at different points in the response is often used to good effect, allowing reduced drug doses with minimization of drug-specific side effects. Many of these drugs have a narrow therapeutic index with wide variation between individuals in the blood concentration achieved by a given dose. Therapeutic drug monitoring is often employed in an attempt to maximize efficacy and safety. In general, the immunosuppressive effect is proportional to the area under the concentration–time curve (AUC). Clearly, measurement of the AUC is impractical in clinical practice and limited sampling strategies, usually based on a single time point that correlates best with the AUC, are employed routinely. This chapter provides an overview of the immunosuppressive drugs used in current practice, with the exception of steroids (see Chapter 31).


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Immunomodulating Drugs

Table 32.1 Targets of the immunosuppressive drugs. Site of action

Drugs

Inhibition of IL-2 production by T lymphocytes Inhibition of T-lymphocyte response to IL-2 Inhibition of proliferation of T and B lymphocytes Depletion of T and B lymphocytes Depletion of T lymphocytes Depletion of B lymphocytes Inhibition of costimulatory signals

Ciclosporin A, tacrolimus Anti-CD25 antibodies, sirolimus, everolimus Azathioprine, mycophenolate, cyclophosphamide, methotrexate, leflunomide Polyclonal antilymphocyte antibodies, alemtuzumab Muromonab CD3 Rituximab Azathioprine, abatacept, belatacept

Generic complications of immunosuppression Inhibition of the host defense mechanisms to prevent tissuedamaging inflammation has the inevitable consequence of increased risk of infection and malignant disease, primarily due to defective control of intracellular pathogens. The commonest opportunistic infections in immunosuppressed patients are caused by latent viruses, in particular those in the herpesvirus family that reactivate. Individuals with a history of exposure to tuberculosis are at risk of recurrence following immunosuppression and prophylactic antituberculous therapy should be considered. Fungal infections are a rare but serious complication. Malignancy caused by oncogenic viruses is a potentially life-threatening complication, particularly skin cancer and lymphoma (Opelz & Dohler 2004).

The immunosuppressive drugs Antiproliferative agents Suppression of lymphocyte proliferation has proven to be a very effective way of modulating the immune response. The choice of antiproliferative agent for a given indication is often rooted in history rather than the drug having specific benefits for a given condition. The most widely used antiproliferative agents are discussed here.

Azathioprine/6-mercaptopurine Azathioprine is the best-established immunosuppressive drug and remains a useful and effective agent. Azathioprine was initially introduced in the early 1960s for solid organ transplantation and was subsequently adopted as therapy for a wide range of immunologically mediated diseases.

Structure Azathioprine is a purine analog (an imidazolyl derivative of 6-mercaptopurine; Fig. 32.1). Essentially, the features of

N H3C

N NO2 S

H N

N N

N

Fig. 32.1 Azathioprine: chemical structure.

azathioprine discussed below also apply to 6-mercaptopurine, an alternative orally active agent.

Pharmacokinetics Azathioprine is well absorbed orally and is rapidly metabolized nonenzymatically through reduction by glutathione and other sulfhydryl-containing compounds to 6-mercaptopurine (approximately 90%). Subsequently, 6-mercaptopurine is metabolized along three competing pathways (summarized in Fig. 32.2). The pathway leading to the active metabolite is initially catalyzed by hypoxanthine phosphoribosyltransferase via several steps to 6-thioguanine nucleotides. Competing pathways leading to the generation of inactive metabolites are catalyzed by xanthine oxidase and thiopurine-S-methyltransferase (TPMT). The plasma half-life of 6-mercaptopurine is short (38–114 min) but persistence of 6-thioguanine nucleotides in the tissues provides a basis for the once-daily dosing that is conventional (Chan et al. 1990). A typical initial daily dose would be 1–5 mg/kg body weight given either orally or intravenously (no adjustment required for parenteral administration), with dose adjusted according to clinical response and toxicity. Pharmacogenetics Variable activity of TPMT is the best-characterized example of a pharmacogenetic effect on an immunosuppressive drug (reviewed in Evans 2004). Approximately 90% of the

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Azathioprine Xanthine oxidase

Glutathione

6-Thiouracil

6-Methylmercaptopurine

6-Mercaptopurine

Hypoxanthine phosphoribosyltransferase

Thiopurine-S-methyltransferase

6-Thioinosine-monophosphate

6-Methylmercaptopurine ribonucleotides

Inosine monophosphate dehydrogenase 6-Thioxanthine-monophosphate Guanosine monophosphate pathways

6-Thioguanine nucleotides Active metabolite

population are homozygous for normal alleles with normal enzyme activity, 10% are heterozygotes with reduced enzyme activity, and 0.3% are homozygotes for nonfunctional alleles with functional enzyme deficiency (Yates et al. 1997). At least 11 variant alleles have been associated with reduced TPMT activity, three of which (TPMT*3C, *3A and *2) account for in excess of 95% of inherited TPMT deficiency. TPMT deficiency has been linked to myelotoxicity but not hepatotoxicity. Patients with TPMT deficiency require 5–10% standard dose of thiopurines, while heterozygotes can be treated with standard doses but are more likely to require dose reduction. The most widely applied assay for TPMT deficiency is measurement of enzyme activity in erythrocytes. As with all erythrocyte-based assays, it is important to avoid the assay within 30– 60 days of blood transfusion. The guidelines for the management of a number of autoimmune diseases now recommend screening for TPMT deficiency prior to treatment, although this may not be cost-effective in the presence of regular pharmacodynamic monitoring in the form of testing for myelosuppression.

Mechanism of action Azathioprine is a purine analog and inhibits the de novo and salvage pathways of purine synthesis resulting in the inhibition of DNA synthesis and the proliferation of both T and B lymphocytes. An additional effect, described recently, is inhibition of the transduction of the costimulatory signal delivered to T lymphocytes by ligation of CD28 (Tiede et al. 2003). Method of monitoring Plasma concentrations of azathioprine or 6-mercaptopurine are not predictive of efficacy or toxicity, although some recent publications suggest that measurement of 6-thioguanine

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Fig. 32.2 Simplified metabolic pathway for azathioprine. Enzymes-catalyzing steps on the pathway to the active drug are shown in black boxes and enzymes involved in the generation of inactive metabolites are shown in blue boxes. (See CD-ROM for color version.)

nucleotide concentrations in erythrocytes may be helpful in planning drug doses (Gearry & Barclay 2005). Generally, a pharmacodynamic approach is employed. The principle dose-limiting toxicities are myelotoxicity and hepatotoxicity. Routine monitoring of full blood count and liver blood tests are essential, weekly for at least the first 4 weeks after initiation (the summary of product characteristics recommends 8 weeks but this may be overcautious), followed by a reduced frequency but not less than every 3 months. Dose reduction should be considered when the white blood cell count falls below 4 × 109/L or platelets below 100 × 109/L or there is evidence of hepatic injury.

Established indications Use of azathioprine is well established for solid organ transplantation and a number of autoimmune disorders, including systemic lupus erythematosus (SLE), inflammatory bowel disease, rheumatoid arthritis, systemic vasculitis, dermatomyositis and polymyositis, autoimmune chronic active hepatitis, pemphigus vulgaris, autoimmune hemolytic anaemia, and chronic refractory idiopathic thrombocytopenic purpura. Safety profile/side effects (including drug interactions) The most important side effect of azathioprine is dose-related myelosuppression, as noted above, and macrocytosis is an expected observation. Hepatic problems including cholestasis and hepatic venoocclusive disease are much less common. Immunosuppression per se increases the risk of skin cancer but azathioprine may also have an added specific effect through the potentiation of oxidative damage to DNA (O’Donovan et al. 2005). The epidemiology for this issue is difficult to interpret as the cohort of patients with the longest duration of exposure to immunosuppression were mostly treated with azathioprine. The single most important drug interaction for

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Pharmacokinetics MPA is well absorbed orally with a peak concentration in blood at around 2 hours and a plasma half-life of around 17 hours (Weber et al. 1998). The principle difference in pharmacokinetics between MMF and enteric-coated MPA is the time of peak blood concentrations (Tmax), which occurs later and is more variable with enteric-coated MPA. The principal metabolite is the pharmacologically inactive phenolic

CH3 O O

O O N O OH Mycophenolate

25 20 15 10 5 0 4

8

12

16

20

24

Hours after oral dose

The active drug, mycophenolic acid (MPA), was initially identified in the 1940s as a product of Penicillium brevicompactum with antifungal activity. Mycophenolate is an alternative purine synthesis inhibitor to azathioprine. It is a more potent agent and has largely replaced azathioprine in solid organ transplantation, with increasing use for autoimmune indications (Tricontinental Mycophenolate Mofetil Renal Transplantation Study Group 1996). Two preparations are available that deliver MPA, the morpholinoethyl ester mycophenolate mofetil (MMF, Cellcept) and enteric-coated mycophenolate sodium (Myfortic) (Fig. 32.3). Comparative studies suggest that these formulations have equivalent efficacy and safety (Salvadori et al. 2004). With orally administered MMF, the ester group is cleaved by first-pass metabolism in the enterocyte and liver resulting in undetectable concentrations of the parent drug in blood. Mycophenolate was initially introduced in solid organ transplantation but data are now accumulating for efficacy in the treatment of a number of autoimmune diseases, in particular SLE (Chan et al. 2000; Ginzler et al. 2005).

O

30

0

Mycophenolate

CH3

35

MPA (mg/mL)

azathioprine is that with allopurinol. Allopurinol is an inhibitor of xanthine oxidase, a key enzyme in purine metabolism, and inhibits the metabolism of azathioprine leading to severe myelotoxicity with potentially fatal consequences. Some would advocate the use of reduced-dose azathioprine with allopurinol but the only genuinely safe approach is to use an alternative antiproliferative agent such as mycophenolate or to avoid allopurinol. Caution must be exercised in the coprescription of other drugs that inhibit TPMT, including the 5-aminosalicylic acid derivatives. Azathioprine should not be coadministered with mycophenolate.


CH3 Mofetil

Fig. 32.3 Chemical structure of mycophenolate. Mycophenolic acid is available either as the morpholinoethyl ester (mofetil) or as a sodium salt.

Fig. 32.4 Pharmacokinetics of mycophenolate mofetil (MMF): a typical 24-hour pharmacokinetic profile for a patient given a single oral dose of 1 g MMF. Coprescription of drugs that inhibit ABCC2 (MRP2), e.g., cyclosporin A, inhibit enterohepatic recirculation resulting in loss of the second peak of absorption, which typically comprises 30–50% of the AUC.

glucuronide (MPAG) generated by uridine glucuronyltransferase but a small proportion is metabolized to the active acyl glucuronide that may be responsible for some of the side effects (Oellerich et al. 2000). These glucuronides are primarily cleared by renal excretion but a proportion are secreted in bile by the drug efflux pump ABCC2 (MRP2). Subsequently, a proportion is deconjugated by bacterial flora in the gut with a second peak of absorption at around 6–8 hours due to enterohepatic recirculation. This second peak accounts for 30–50% of the total AUC (Fig. 32.4). When coprescribed with drugs that inhibit ABCC2 (e.g., ciclosporin A, but not tacrolimus or sirolimus) the AUC is significantly reduced (Hesselink et al. 2005). It must be noted that the registration studies where the recommended dose of mycophenolate was defined were conducted in patients coprescribed ciclosporin A (Tricontinental Mycophenolate Mofetil Renal Transplantation Study Group 1996). It is often stated that patients genetically of sub-Saharan African origin require higher doses of mycophenolate but this is not supported by the available pharmacokinetic data (Shaw et al. 2000). Taking mycophenolate along with food delays absorption but has no effect on total exposure. MPA is highly protein bound (97–99%) and the free fraction is increased by conditions that result in displacement of MPA from its protein binding sites or reduced plasma protein concentration (Weber et al. 1998; Kuypers et al. 2003). In renal impairment, accumulation of MPAG displaces MPA and uremic plasma and acidemia have been shown to have an independent effect (Nowak & Shaw 1995; Kaplan et al. 1999; MacPhee et al. 2000). An increased free fraction results in increased conjugation to MPAG with reduced plasma MPA concentrations but an increase in the degree of enterohepatic recirculation, which correlates with an increased incidence of diarrhoea. Conditions that increase the free fraction of MPA

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may result in poor tolerance due to gastrointestinal toxicity in the face of subtherapeutic MPA concentrations. The current recommended initial oral dose in renal transplant recipients is 1 g MMF twice daily, and 720 mg twice daily as the bioequivalent dose of enteric-coated MPA (Johnston et al. 2006). There is no dose adjustment for intravenous administration.

Pharmacogenetics Individuals with the g275T > A and g2152C > T single nucleotide polymorphisms (SNPs) in the UGT1A9 promoter region, mutations associated with reduced in vitro enzyme activity, have significantly reduced exposure to the active drug MPA, of a magnitude likely to be significant clinically (Kuypers et al. 2005). The ABCC2 g24C > T and g3972C > T polymorphisms have been found to prevent the reduced exposure to MPA found in renal transplant recipients with mildly altered liver dysfunction (Naesens et al. 2006). These observations do not yet provide a basis for the application of pharmacogenetics in planning mycophenolate dosing. Mechanism of action MPA is an inhibitor of inosine monophosphate dehydrogenase (particularly the type II isoform), a key rate-limiting enzyme for the de novo pathway of purine synthesis. Both T and B lymphocytes are particularly dependent on this pathway (Allison & Eugui 1993). There is some experimental evidence to suggest the inhibition of smooth muscle cell proliferation and pathologic scarring processes (Romero et al. 1999). Method of monitoring The role of therapeutic drug monitoring for MPA is an area of current controversy. The available data apply to MMF only. Enteric-coated MPA has an unpredictable Tmax and no abbreviated sampling strategies have been shown to correlate with AUC. Two methods are available for the assay of MPA. The high performance liquid chromatography (HPLC)-based approach measures only MPA while the enzyme-multiplied immunoassay technique (EMIT) measures both MPA and the acyl glucuronide (Shipkova et al. 1999). There is no single time point where measurement of plasma MPA correlates sufficiently well with the AUC to be of practical value. The best clinically applicable sampling strategies are based on three samples collected predose and 30 and 120 min after drug dosing, with the use of established algorithms to estimate the AUC (Pawinski et al. 2002). In renal transplantation the established therapeutic range for the estimated AUC is 45–60 mg/L per hour (van Gelder et al. 1999). There are no published data on the therapeutic range for other indications. Some experimental work has correlated the degree of suppression of inosine monophosphate dehydrogenase with efficacy but this has yet to translate to use in clinical practice (Glander et al. 2004).

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Full blood count should be monitored weekly during the first month of treatment, twice monthly during the second and third months, and then monthly throughout the first year. Dose reduction should be considered when the white blood cell count falls below 4 × 109/L or platelets below 100 × 109/L.

Established indications Established indications include solid organ transplantation and a number of autoimmune diseases including SLE. Safety profile/side effects (including drug interactions) The principal side effects of MPA are gastrointestinal toxicity, which is less severe when the drug is coprescribed with inhibitors of ABCC2, and myelosuppression. A common practice when faced with severe diarrhoea is to increase the dosing frequency to three or four times daily with the same total daily dose. However, there are no data to support equivalent efficacy with this approach. Drugs that inhibit enterohepatic recirculation including cholestyramine may reduce exposure to MPA. The absorption of MPA is reduced when coadministered with antacids.

Cyclophosphamide Cyclophosphamide is a nitrogen mustard and was originally derived from mustard gas used as a weapon during the First World War. It was noted to have myelosuppressive effects in the 1940s. It is the most potent and most toxic of the antiproliferative agents used for immunosuppression, being more widely used as a chemotherapeutic agent for malignant disease. It is reserved for the treatment of life- or organthreatening disease, primarily in systemic vasculitis and SLE. Protocols have evolved, for example in the treatment of systemic vasculitis, employing cyclophosphamide to induce remission, with switch to a less toxic agent such as azathioprine for maintenance treatment (Jayne et al. 2003).

Structure The chemical structure of cyclophosphamide is shown in Fig. 32.5. Pharmacokinetics The oral bioavailability of cyclophosphamide is approximately 75%, with a plasma half-life of 3.5–12 hours (Colvin 1999). Cyclophosphamide is a prodrug that is metabolized Cl

Cl

N O

P

NH

O Fig. 32.5 Cyclophosphamide: chemical structure.

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by cytochrome P450 (CYP), primarily CYP2C9, CYP3A4 and CYP3A5, to a number of active metabolites. The most important of these is 4-hydroxycyclophosphamide, a circulating metabolite that enters cells and is cleaved nonenzymatically to phosphoramide mustard, the principle alkylating substance, and acrolein (Ren et al. 1998). Clearance with urinary excretion follows further metabolism by cytochrome P450, with 30% excreted unmodified in urine. For the treatment of systemic vasculitis, typical daily doses of oral cyclophosphamide are 2 mg/kg (maximum 200 mg/day), with a 25% dose reduction for patients aged greater than 60 years and 50% reduction for those aged greater than 75 years (Savage et al. 2000). It has been demonstrated clearly in the treatment of lupus nephritis that the complication rate of pulsed intravenous cyclophosphamide is significantly lower than that for regular oral cyclophosphamide. This is particularly true for gonadal toxicity and malignant disease (Austin et al. 1986). Typical pulsed intravenous regimens comprise 500– 750 mg/m2 every 2– 4 weeks (Boumpas et al. 1992; Houssiau et al. 2002).


reduced by provoking high urine flow (> 100 mL/hour) for 24–48 hours after intravenous infusion and administration of the sulfhydryl-containing compound mesna. Mesna is given intravenously at a total dose of 60% that of cyclophosphamide at 0, 4 and 8 hours or is available orally. There is an increased risk of malignancy, particularly bladder cancer. Gonadotoxicity is a particular problem that is minimized, though not eliminated, by the use of pulsed intravenous therapy. Patients should be counselled on this and given the opportunity for sperm or egg storage prior to treatment. Hair loss is more common than with the other antiproliferative drugs.

Other antimetabolites Several other antiproliferative agents are used for immunomodulation. Methotrexate inhibits the enzyme dihydrofolate reductase with consequent inhibition of purine and pyrimidine synthesis. Leflunomide is a pyrimidine synthesis inhibitor. Chlorambucil has been used with some success in the treatment of membranous nephropathy (Ponticelli et al. 1995).

Calcineurin inhibitors Pharmacogenetics There are some published data to suggest that mutations in the genes encoding CYP3A5, CYP2B6, and CYP2C19 had a significant influence on efficacy failure and toxicity in patients treated with pulsed intravenous cyclophosphamide for lupus nephritis (Takada et al. 2004). Mechanism of action Phosphoramide mustard is a bifunctional alkylating agent that cross-links DNA, preventing cell division. Method of monitoring Regular monitoring of full blood count is required and cyclophosphamide should be withheld when the white blood cell count falls below 4 × 109/L or platelets below 100 × 109/L until the counts recover. With pulsed intravenous cyclophosphamide the nadir of blood cell counts occurs at around 10 days after dosing and should be monitored at this point, with reduction in subsequent doses in the event of excessive myelosuppression. Established indications Cyclophosphamide is indicated for a number of autoimmune conditions including, lupus nephritis and systemic vasculitis. Safety profile/side effects (including drug interactions) Parenteral administration is often followed by nausea or vomiting after 24 hours and very high doses can cause diarrhoea. As with the other antiproliferative agents, myelosuppression is a major problem, cell counts reaching a nadir 10–12 days after administration of a single dose, with return to baseline within 18–21 days. Hemorrhagic cystitis is thought to be caused by the metabolite acrolein and risk can be

There are two licensed drugs in this class, ciclosporin A and tacrolimus, both of which are fungal products. Ciclosporin was initially introduced as Sandimmun in the early 1980s (European Multicentre Trial Group 1983), with the introduction of a microemulsion formulation with more predictable absorption (Neoral) (Kovarik et al. 1994) in the early 1990s. Generic forms of ciclosporin A are now available but must used with caution as they have different pharmacokinetic properties and cannot be used interchangeably with Neoral (Kees et al. 2006). Tacrolimus was known for many years by its experimental name FK506 and is available as Prograf given twice daily and the recently introduced delayed release preparation (Pirsch et al. 1997). The primary use of these drugs has been in solid organ transplantation but use for autoimmune indications is increasing. Prior to the introduction of ciclosporin A, when azathioprine and prednisolone were the primary drugs used in immunosuppression for renal transplantation, 40% of transplants were lost to acute rejection during the first year. With the introduction of ciclosporin A, 1-year graft survival improved to greater than 80% and it is now unusual to lose a transplanted kidney through acute rejection.

Mode of action These drugs bind to intracellular binding proteins, cyclophilin A in the case of ciclosporin A or FK-binding protein 12 (FKBP12) in the case of tacrolimus, and inhibit the phosphatase calcineurin (Colgan et al. 2005; Fruman et al. 1992). Calcineurin is essential for activating the transcription factor NF-AT (nuclear factor of activated T lymphocytes) that is required for transcription of the IL-2 gene. The most important toxicity of the calcineurin inhibitors is nephrotoxicity. There are two different processes involved.

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(a)

(b) Fig. 32.6 Chronic calcineurin inhibitor toxicity. Sections are from a renal transplant biopsy performed in a tacrolimus-treated patient in order to investigate chronic renal transplant dysfunction. (a) Typical change of stripe fibrosis; (b) typical arterial changes with a hyaline deposit (arrow). (See CD-ROM for color version.)

The first is an acute reversible vasoconstriction of the renal vasculature, possibly mediated by endothelin or sympathetic nervous stimulation (Murray et al. 1985; Curtis et al. 1986; Kon et al. 1990). The vasoconstriction can, to some extent, be reversed by the administration of calcium channel blockers (Ruggenenti et al. 1993). Acute tubular toxicity is also manifest by tubular vacuolation. A more chronic effect is marked by hypertrophy of the arterial media and striped fibrosis (Fig. 32.6). Clear evidence on the long-term nephrotoxic effects comes from recipients of transplants other than kidneys, where the 5-year risk of developing chronic renal failure with glomerular filtration rate < 30 mL/min is 7– 21%, depending on the type of transplant (Ojo et al. 2003). Serial renal transplant biopsies in kidney/pancreas transplant recipients demonstrated changes of acute calcineurin inhibitor toxicity peaking at around 20% by month 6 after transplantation but, more ominously, over 75% of patients had evidence of chronic calcineurin inhibitor toxicity by 4 years after transplantation and almost all patients by 10 years (Nankivell et al. 2004). There is probably little difference between calcineurin inhibitors in the degree of nephrotoxicity (Solez et al. 1998). In a recent metaanalysis of the calcineurin inhibitors in renal transplantation, tacrolimus was shown to be more efficacious than ciclosporin A but at the cost of a higher rate of new-onset diabetes after transplantation (Webster et al. 2005). Both calcineurin inhibitors cause hypertension, but this is probably less common with tacrolimus. The key side effects of ciclosporin A and tacrolimus are compared in Table 32.2. Thrombotic microangiopathy is a rare but serious complication common to both calcineurin inhibitors and should be managed by drug withdrawal (Ponticelli & Banfi 2006).

Oral bioavailability Ciclosporin A and tacrolimus are both metabolized by the oxidative enzymes CYP3A4 and 3A5 and are substrates for

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Table 32.2 Calcineurin inhibitor side effects. Side effect

Ciclosporin A

Tacrolimus

Nephrotoxicity Hypertension Hyperkalemia Diabetes mellitus Gum hyperplasia Hypertrichosis Hair loss

++ +++ + + + + −

++ ++ ++ ++ − − +

the drug efflux pump P-glycoprotein (P-gp). Both drugs have oral bioavailability of 25–30%, in part due to an active barrier to drug absorption mediated by a combination of P-gp in the enterocyte, and first pass metabolism by CYP in the enterocyte and liver. Drugs that inhibit CYP and P-gp significantly enhance the absorption of the CNIs (Hebert et al. 1992; Tuteja et al. 2001) and vice versa for inhibitors (Gupta et al. 1989). Patients on treatment with CNIs should be advised against the consumption of grapefruit juice which can result in significant inhibition of CYP3A with increased blood drug concentrations and subsequent toxicity. Unfortunately, there is batch to batch variability in this effect preventing the use of regular grapefruit consumption as a strategy for reducing drug dose requirements (Lown et al. 1997).

Ciclosporin A Structure The chemical structure of ciclosporin A is shown in Fig. 32.7.

Pharmacokinetics The barriers to the oral absorption of ciclosporin A have been described above and the recommended intravenous dose is

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H HO

H3C

H3CO H H3C

H (CH3)2CHCH2

HO

CH3

H

O H3C

N

N O O

(CH3)2CHCH2 N H3C

N H

O N

N O

CH3

CH3

H N

H O

O N

HO

H

N

CH3

CH2CH3 O

N O

H

CH2CH(CH3)2

H

O

CH3

O

CH3 O

O

N

N CH3 CH(CH3)2

CH3

H

CH3 OH

H3C

CH3

H

O O

· H2O

CH3

O

H

H CH2CH(CH3)2 H

O CH(CH3)2

H3CO

Ciclosporin A

H H

OCH3

Tacrolimus

Fig. 32.7 Calcineurin inhibitors: chemical structure.

30% of the standard oral dose. The peak concentration in blood (Cmax) occurs at around 2 hours with a highly variable half-life, generally between 5 and 10 hours (Kees et al. 2006). The CYP3A metabolites are primarily excreted in bile.

Pharmacogenetics Unlike tacrolimus (see below) no clear genetic influences on ciclosporin A pharmacokinetics have been described. Specifically, SNPs in the genes encoding CYP3A5 and ABCB1 (MDR-1) have been shown to have no significant impact (MacPhee et al. 2005a; Fredericks et al. 2007). Method of monitoring Therapeutic drug monitoring is employed routinely for ciclosporin A. In view of uptake by erythrocytes, whole-blood concentrations are assayed. Initially this was by sampling at 12 hours after the previous dose (trough or C0 concentration). However, most of the heterogeneity in ciclosporin pharmacokinetics is in the absorption phase and blood concentration measured at 2 hours after the last dose (C2) correlates better with the AUC (Canadian Neoral Renal Transplantation Study Group 2001; Clase et al. 2002). C2 monitoring requires more accurate timing of the blood samples than C0 sampling, with a tolerance of only ± 10 min, which has generated logistical problems in the introduction of this sampling strategy. The therapeutic range for cyclosporin A in solid organ transplantation is 75–300 μg/L for C0 or 800– 1700 μg/L for C2 (Clase et al. 2002; Cole et al. 2003). Established indications Established indications for ciclosporin A are solid organ and bone marrow transplantation and a number of autoimmune diseases, including, psoriasis, atopic dermatitis, rheumatoid arthritis, and immunologically mediated nephrotic syndrome.

Safety profile/side effects (including drug interactions) Apart from the general comments on calcineurin inhibitor toxicity above, ciclosporin A has some adverse cosmetic effects with hypertrichosis and gum hypertrophy. Ciclosporin A increases plasma uric acid concentrations with increased incidence of gout.

Tacrolimus Tacrolimus is a macrolide with structure shown in Fig. 32.7. Occasionally there is cross-reaction with allergy to the chemically related antibiotic erythromycin.

Pharmacokinetics The barriers to the oral absorption of tacrolimus have been described above and the recommended intravenous dose is 20% of the standard oral dose. The peak blood concentration occurs at 1–2 hours after an oral dose, with a half-life of around 40 hours (Bekersky et al. 1999). When taken along with food, absorption is significantly inhibited and increased absorption is seen in fasting patients. The increased oral bioavailability of tacrolimus in patients with diarrhoea is intriguing and has been ascribed to reduced efficiency in the active barrier to drug absorption (Lemahieu et al. 2005). Tacrolimus is metabolized by CYP3A4 and CYP3A5 (Kamdem et al. 2005), with eight known metabolites labeled M-I to M-VIII. M-II has significant immunosuppressive activity and cross-reacts with tacrolimus in some immunoassays (Iwasaki et al. 1995). The metabolites are eliminated by biliary excretion with essentially all clearance in stool. Pharmacogenetics There are important differences between ethnic groups in the blood concentration achieved by a given dose of tacrolimus. Individuals genetically of sub-Saharan African origin require

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twofold higher doses of tacrolimus to achieve target blood concentrations compared to individuals from other ethnic groups (MacPhee et al. 2002). This difference was demonstrated to be due to reduced absorption rather than more rapid clearance (Mancinelli et al. 2001) and is possibly due to the high prevalence of CYP3A5 expression in black individuals. There are several SNPs in the CYP3A5 gene that result in greatly reduced levels of expression in homozygotes, the most prevalent of which is CYP3A5*3. CYP3A5*3 homozygotes require twofold lower doses of tacrolimus to achieve target blood concentrations than individuals with at least one CYP3A5*1 allele (CYP3A5 expressers). CYP3A5 expressers experienced a significant delay in achieving target blood concentrations after renal transplantation (MacPhee et al. 2005a,b). The ABCB1 (MDR-1) gene codes for P-gp. SNPs in this gene have been associated with high and low levels of expression of P-gp. Individuals with the wild type at these SNPs have lower dose-normalized blood concentrations of tacrolimus, although this is a minor effect (MacPhee et al. 2002). However, the ABCB1 genotype may influence pharmacodynamics by controlling distribution of the drug between cellular compartments. This has been hypothesized to be the mechanism underlying the augmentation of calcineurin inhibitor nephrotoxicity by sirolimus (Anglicheau et al. 2006).

Method of monitoring Therapeutic drug monitoring is employed routinely with tacrolimus, with 12-hour postdose trough whole blood concentrations being used as standard. Tacrolimus is taken up by erythrocytes and results should be interpreted with caution in the presence of anaemia. Some older publications report plasma concentrations which are significantly lower. Dogma suggests that sampling at this time-point correlates well with AUC but this is controversial (Wallemacq et al. 1998). However, while more precise limited sampling strategies including measurement at 2 and 4 hours after dosing have been suggested, none have been adopted widely in routine clinical practice (Wong et al. 2000). The therapeutic range for solid organ transplantation lies between 5 and 15 μg/L (Bottiger et al. 1999; Undre et al. 1999; Staatz et al. 2001; Maes et al. 2001). Established indications The primary indication for tacrolimus is solid organ transplantation. A topical formulation is also available for use in atopic eczema. Safety profile/side effects (including drug interactions) Apart from the general comments on calcineurin inhibitor toxicity above, the main concern with tacrolimus is that it is diabetogenic, more so than ciclosporin A. Neurotoxicity, manifest most commonly by tremor or paresthesia, is more common with tacrolimus than ciclosporin A.

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OH OMe

O

OH

N O

O

HO O

O

O

MeO

O

OMe

Fig. 32.8 Sirolimus: chemical structure.

Mammalian target of rapamycin inhibitors Sirolimus and everolimus History of discovery Sirolimus (previously known as rapamycin) is available as Rapamune, a product of the fungus Streptomyces hygroscopicus and was initially isolated in soil samples from Easter Island. Everolimus (Certican) is a derivative of sirolimus with a 2hydroxyethyl chain substitution at position 40, resulting in more efficient absorption following oral administration and a shorter half-life.

Structure Sirolimus is a macrolide antibiotic with some similar structural features to tacrolimus (Fig. 32.8) and binds to the same intracellular protein, FKBP12. Despite initial concerns that this would result in mutual antagonism of tacrolimus and sirolimus used together, this has been shown not to be a problem. The sirolimus–FKBP12 complex does not inhibit calcineurin. Pharmacokinetics Sirolimus is conventionally administered as a once-daily oral dose. Oral bioavailability is around 14%, with peak absorption (Tmax) at 1–2 hours and a very long half-life of around 60 hours allowing once-daily dosing (MacDonald et al. 2000). Everolimus has a half-life of 16–19 hours requiring twicedaily dosing (Kahan et al. 1999). Metabolism is primarily by CYP3A4 and CYP3A5 with biliary excretion of metabolites. Pharmacogenetics Given that sirolimus is metabolized by CYP3A4 and CYP3A5 and is a P-gp substrate, it might be predicted to be susceptible to the same pharmacogenetic influences as tacrolimus. However, this issue remains somewhat unclear with two studies reporting lower dose-normalized sirolimus blood concentrations in CYP3A5 expressers (Anglicheau et al. 2005; Le Meur et al. 2006) and one negative study (Mourad et al. 2005). A caveat is that the association was lost in the event of coadministration of a calcineurin inhibitor. No influence of

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the MDR-1 SNP was identified in these studies. The use of a pharmacogenetic strategy would be particularly attractive for sirolimus given the long half-life, with a resultant long response loop for therapeutic drug monitoring.

Mechanism of action The sirolimus–FKBP12 complex binds to mammalian target of rapamycin (mTOR), a serine/threonine kinase involved in activating the phosphatidylinositol 3-kinase/AKT pathway. This pathway plays a key role in controlling the cellular response to cytokines, primarily by preventing cell cycle progression from G1 to S phase. Sirolimus inhibits the downregulation of IκB-α, a key step in the protection of cells from apoptosis mediated by ligation of CD28 (Hay & Sonenberg 2004). The effects of fibroblast growth factors and vascular endothelial growth factor (VEGF) are also inhibited, resulting in beneficial effects in diseases mediated by endothelial proliferation such as cardiac allograft vasculopathy (Guba et al. 2002; Eisen et al. 2003) but at the expense of a delay in wound healing (Dean et al. 2004). The rate of restenosis of vascular stents due to endothelial proliferation has been reduced by the use of sirolimus-eluting stents (Eisenberg & Konnyu 2006). These factors may underlie the observed inhibition of the growth of malignant cells in vitro and suppression of the growth of experimental tumors in mice (Luan et al. 2002). There are epidemiologic data suggesting a lower rate of new malignancies in immunosuppressed patients treated with sirolimus (Kauffman et al. 2005), in particular cutaneous lesions (Stallone et al. 2005; Euvrard et al. 2006). Method of monitoring There is wide variability between individuals in the blood concentrations achieved by a given dose of sirolimus. Therapeutic drug monitoring has been applied from the outset, initially by methods based on HPLC with mass spectrophotometric detection on whole blood. An immunoassay has now been introduced which produces results that are on average 24% higher than measurements with HPLC due to crossreactivity of the antibody with some metabolites. In interpreting blood concentrations it is essential to know which assay methodology was used. The usual sample is whole blood collected immediately before the next dose. The target concentration required in renal transplantation is now believed to be lower than when the drug was first introduced, typically 10–15 μg/L in the early period and 5–10 μg/L for stable maintenance patients (Kahan et al. 2000). Established indications The main indication for sirolimus is solid organ transplantation but potential as an anticancer drug is under investigation. Safety profile/side effects (including drug interactions) On initial introduction, it was hoped that the mTOR inhibitors would offer an alternative to the calcineurin inhibitors


by being nonnephrotoxic, nonhypertensive, and nondiabetogenic. However, a number of toxicities make the mTOR inhibitors difficult drugs to use (Kahan 2000). Hyperlipidemia is a common complication and it remains uncertain whether this will translate into an increased risk of atherosclerotic complications. Myelosuppression, particularly anemia and thrombocytopenia, are seen, especially when used with one of the antiproliferative agents. A range of other complications are likely to be related to the effects on wound healing or VEGF, including oral ulcers, skin rashes, ankle odema, and proteinuria. Interstitial pneumonitis is a rare but serious complication that may resolve on drug withdrawal (Haydar et al. 2004). Initially, the mTOR inhibitors were thought to be nondiabetogenic, but evidence for toxicity to pancreatic B cells and inhibition of intracellular signalling downstream from the insulin receptor is accumulating (Di Paolo et al. 2006). Several important drug interactions have come to light through the use of sirolimus in multiple drug combinations. Dose-normalized blood concentrations of sirolimus were significantly higher in patients coprescribed ciclosporin A than in those treated with MMF (Cattaneo et al. 2004). Sirolimus appears to increase the nephrotoxic effect of the calcineurin inhibitors, possibly through inhibition of P-gp, allowing increased entry of the calcineurin inhibitors to renal tubular epithelial cells (Anglicheau et al. 2006).

Antibodies A major problem with all the drugs described above is their lack of fine specificity for the immune response, resulting in a narrow therapeutic index and a significant burden of toxicity. The use of antibodies, initially polyclonal and more recently monoclonal, has offered the prospect of more specifically targeted therapy.

Polyclonal antibodies A number of different polyclonal preparations raised by the immunization of rabbits or horses with human thymocytes or T lymphocytes are available. These agents are conventionally known as antithymocyte globulin (ATG) or antilymphocyte globulin (ALG). They contain a broad range of antibody specificities including T and B lymphocytes and a number of more broadly expressed cell-surface molecules including adhesion molecules (Bourdage & Hamlin 1995). These agents are potent immunosuppressants, causing severe lymphopenia, and as a consequence are associated with a high rate of infectious complications. Allergic reactions to the foreign protein are a common problem, with the added potential for neutralization of the therapeutic antibody.

Monoclonal antibodies The development of monoclonal antibodies has allowed the possibility of drugs with a “designed” specificity. The use of genetic engineering to molecularly graft the antibody-binding domain of the primary mouse antibody onto the constant

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Mouse monoclonal antibody Muromonab . . . . .

Chimaeric antibody . . . . . . ximab

Humanized antibody . . . . . zumab

Fig. 32.9 Monoclonal antibodies showing the species origin of the immunoglobulin domains in genetically engineered antibodies. The murine component is shown in blue and the human component in black. The DNA encoding the variable domains of a murine monoclonal antibody can be grafted onto the genes for a human IgG molecule to form a chimaeric antibody, or for the hypervariable regions of the antibody-binding pocket to form a humanized antibody. The taxonomy used in naming these antibodies is indicated. (See CD-ROM for color version.)

region of a human IgG molecule has helped to address the problems associated with the administration of antigenic foreign protein. The taxonomy conventionally used for these antibodies is outlined in Fig. 32.9. The variable domains of a murine monoclonal antibody can be grafted onto the genes for a human IgG molecule to form a chimaeric antibody, or for the hypervariable regions of the antibody-binding pocket to form a humanized antibody. While the development of anti-murine immunoglobulin antibodies is minimized with chimeric antibodies, there can still be a human antichimaeric antibody (HACA) response resulting in either an allergic or neutralizing response. Typically these agents have the halflife of human IgG, around 20 days.

Muromonab CD3 Muromonab CD3 (OKT3) was the first therapeutic monoclonal antibody to be used for immunosuppression (Cosimi et al. 1981). This is a murine monoclonal antibody to the CD3 molecule in the T lymphocyte receptor complex. One major drawback of OKT3 is that it activates T lymphocytes before killing them, causing a cytokine release syndrome. It is potently immunosuppressive and as a consequence is associated with a high complication rate, including significantly increased risk of lymphoma (Opelz & Dohler 2004). The primary use of OKT3 was in renal transplantation but it is now rarely used due to concerns about toxicity. Antibodies to the IL-2 receptor (CD25) Basiliximab (Simulect) and daclizumab (Zenapax) were the first widely used genetically engineered antibodies. They bind the α-chain of the IL-2 receptor, which is only expressed on activated T lymphocytes. Basiliximab is a chimaeric antibody licensed for use in a regimen comprising two intravenous infusions on day 0 and on day 4 following renal

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transplantation (Nashan et al. 1997). Daclizumab is a humanized antibody licensed for use as an intravenous infusion at the time of transplantation followed by four further infusions at 2-weekly intervals (Vincenti et al. 1998). Both agents were effective in reducing the rate of acute rejection. Almost uniquely in the evolution of immunosuppression for solid organ transplantation, the introduction of the CD25 antibodies resulted in reduced rates of acute rejection of renal transplants without an increase in the infectious complication rate (Adu et al. 2003). While these antibodies are very effective in reducing the rate of rejection when present from the start of the evolution of the immune response, there is no evidence for their efficacy in reversing acute rejection, which may limit their application in autoimmunity where the tissue-damaging immune response is already established.

Alemtuzumab (Campath-1H) This humanized monoclonal antibody is specific for CD52 a cell-surface molecule expressed on a number of circulating mononuclear cells, including T and B lymphocytes and natural killer cells. Having been developed originally for the treatment of lymphoma, it is acquiring widespread use in solid organ transplantation (Magliocca & Knechtle 2006) and has been used in the treatment of systemic vasculitis (Lockwood et al. 1993). Despite profound immunosuppression, safety up to 5 years in renal transplant recipients appears to be acceptable (Watson et al. 2005). Rituximab Rituximab is a chimaeric monoclonal antibody specific for CD20, a molecule expressed on B lymphocytes but not plasma cells. It was initially developed for the treatment of B-cell lymphoma but has been used successfully in a number of autoimmune diseases including rheumatoid arthritis (Edwards et al. 2004), systemic vasculitis, and SLE (Smith et al. 2006). It has demonstrated efficacy in the prevention of rejection in solid organ transplant recipients in the presence of preformed anti-donor antibody and may have a role in the treatment of humorally mediated acute rejection (Salama & Pusey 2006).

Fusion proteins T lymphocytes require at least two signals for activation, the first delivered by the specific receptor for antigen and a second delivered through a number of costimulatory pathways. The most important of these in early T-lymphocyte activation is the cross-linking of CD28 on the T lymphocyte through interaction with the ligands CD80 and CD86. CTLA4 (CD152) also binds to these ligands but with higher affinity than CD28. Abatacept is a genetically engineered protein that combines a CTLA4 molecule with a human immunoglobulin domain. Abatacept has been demonstrated to be effective in the treatment of rheumatoid arthriris (Kremer et al. 2003). In contrast, abatacept was ineffective in transplantation, leading

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to the development of belatacept a molecule derived from abatacept but with several amino acid substitutions resulting in increased avidity for CD80 and CD86 and evidence of efficacy in renal transplantation (Vincenti et al. 2005).

Immunosuppression and pregnancy All the immunosuppressive drugs are either contraindicated in pregnancy or come with advice to avoid. However, there are circumstances where immunosuppression cannot be withdrawn during pregnancy. There are reasonable data from the solid organ transplant literature supporting the safe use of azathioprine, ciclosporin A, and tacrolimus in pregnancy. Cyclophosphamide, sirolimus, and mycophenolate carry a high risk of teratogenicity and should be avoided. These drugs are all present in breast milk and conventional advice is to avoid breast-feeding. While blood concentrations in the baby are likely to be small from the given dose, the immature cytochrome P450 system may result in drug accumulation.

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Physiologic Aspects of Asthma Philip W. Ind and Neil B. Pride

Summary Physiologic measurement of variable narrowing of intrathoracic airways remains essential to the diagnosis of asthma. Methods of determination of airway caliber and their practical use in asthma management are covered with emphasis on the primacy of forced expiratory volume in 1 s (FEV1) but usefulness of peak expiratory flow (PEF) in clinical practice. Airways resistance (Raw), usually measured by body plethysmography, gives an estimate of the dimensions of the total airway tree but is only available in lung function laboratories. Two other simpler methods of measuring resistance, airflow interruption and the forced oscillation technique, are becoming more widely used. Forced oscillation has usually been used to determine resistance of the total respiratory system but can potentially enable estimation of the relative contributions of central and peripheral airways to increased resistance. Measures from the latter part of maximum expiratory flow–volume (MEFV) curve (e.g., MEF25) have been proposed as a sensitive test of small airway function. Increase in maximum expiratory flow and reduction in pulmonary resistance breathing helium (less dense than air, preferentially affecting central rather than laminar peripheral airflow) has not been widely adopted. Endobronchial catheter techniques provide the best physiologic method of defining contributions of large and small airway narrowing but are invasive and unsuitable for wider application. Three-dimensional imaging techniques have increasingly been used to study airways in asthma despite limitations of resolution. Computed tomography (CT) techniques demonstrate increased airway wall thickness. Air trapping, regional hyperinflation, particularly on expiration, and lung attenuation reflect changes in small airways. Closed airway volume has been measured using single photon emission computed tomography (SPECT) using Technegas. Positron emission tomography (PET) using 13N injection quantifies regional hypoventilation and the accompanying hypoperfusion. Magnetic resonance imaging (MRI) using 3He demonstrates ventilatory defects more marked in more severe asthma. All these techniques confirm previous


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Kr radioisotopic scan demonstrations of gross ventilatory inhomogeneity as predicted from the classic multiple inert gas elimination technique (MIGET). PET modeling of 13N infusion has defined regions of severe hypoventilation and heterogeneity “clusters of self-organized patchiness” below the physical limits of PET resolution. Deep inspiration (DI) typically produces transient airway widening after induced bronchoconstriction in normal subjects while it leads to subsequent airway narrowing in spontaneous asthma. Prohibition of DI produces marked hyperresponsiveness in normal subjects and augments bronchoconstriction in asthma. DI-induced bronchoprotection appears to be absent to inhaled allergen in asthma and allergic rhinitis. Inhaled steroid therapy improves airway responses to DI. The potential importance of airway wall thickening in exaggerating the influence of airway smooth muscle shortening contributing to airway hyperresponsiveness is highlighted. The interaction of airway smooth muscle and lung volume and the possibility of increased shortening due to reduced alveolar wall coupling to intrapulmonary airways secondary to inflammation may contribute to “remodeling.” The use of MEFV and maximum inspiratory flow volume (MIFV) curves for clinically diagnosing extrathoracic airflow obstruction is emphasized. Vocal cord dysfunction can pose major clinical challenges as it often coexists with asthma.

Introduction Asthma is conventionally regarded as a syndrome involving narrowing of airways of all sizes. The magnitude of the narrowing varies over time, either spontaneously or following treatment, but can become persistent. In addition to variable airway narrowing, airway hyperresponsiveness (AHR) and airway inflammation are the most important characteristics of asthma. This chapter focuses on practical measurements of simple lung function and their clinical relevance (see Box 33.1) but includes sections on the site of airway narrowing, the small airways, ventilation inhomogeneity, extrathoracic airways with brief discussion of the nose and larynx, and some specific current areas of interest. Space does not allow a comprehensive description of applied physiology in asthma.

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Box 33.1 Clinical role of lung function testing in asthma.

Box 33.2 Methods for detecting peripheral airways obstruction.

Diagnosis Demonstration of airflow obstruction and reversibility (FEV1) Assessment of PEF variability/reversibility at home (PEF) Bronchial responsiveness testing (PC20 FEV1) Exercise-induced fall in FEV1 Assessment of occupational asthma (PEF, PC20 FEV1, challenge)

Reduced MEF25 Increased residual volume Nitrogen washout detection of increased closing volume Reduced response to breathing helium/oxygen mixture Forced oscillation technique Endobronchial catheter measurement of peripheral airway resistance Tests showing uneven distribution of ventilation Increased alveolar–arterial PO2 difference Single breath N2 test Multiple inert gas elimination technique Frequency dependence of lung compliance or respiratory resistance Radioisotope gas gamma camera imaging SPECT Technegas studies HRCT imaging MRI imaging 13 N2 PET studies

Assessment Demonstration of maximal attainable lung function (spirometry, lung volumes) Assessment for medicolegal purposes (e.g., scuba diving) Demonstration of parenchymal function (gas transfer) Assessment of comorbid lung disease (lung volumes, gas transfer) Assessment of breathlessness (lung volumes, gas transfer, exercise testing) Effect of therapy adjustment (home PEF, FEV1, lung volumes) Home monitoring (PEF) Acute severe asthma (PEF) Clinical research (FEV1, FOT, etc.) Pharmacologic assessment FEV1, PEF, MEFV, PEFV curve, VC, SGaw, PC20 FEV1, lung volumes

at home by the patient. These spirometry tests include forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), or peak expiratory flow (PEF). Measurement of airways resistance in the tidal range gives a direct indication of airway dimensions but is only available in lung function laboratories.

Standard lung function tests Airway function and assessment of airflow obstruction (Box 33.1)

Tests of forced expiration

The primary abnormality in asthma is variable narrowing of the intrathoracic airways. In clinical practice this is most commonly assessed by tests of forced expiration because these tests are simple and can be performed at the bedside or even

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Fig. 33.1 (a) Normal forced expiratory spirogram: change in expired volume (L) against time (s). FEV1 3.2 L, FEV1/FVC ratio 3.2/3.7 (86%). (b) Maximum expiratory flow (L/s) vs. expired volume (L) (MEFV) curve derived from the same spirogram. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; PEF, peak expiratory flow; MEF50, maximum expiratory flow when 50% of FVC remains to be expired; MEF25, maximum expiratory flow when 25% of FVC remains to be expired. Note that MEF50 and MEF25 may also be termed “forced expiratory flow” and abbreviated as FEF50 and FEF25.

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residual volume (RV). The same forced expiration can also be plotted as rate of change in volume (expiratory flow) versus expired volume to obtain a maximum expiratory flow–volume (MEFV) curve (Fig. 33.1b).

FEV1 and FVC In healthy subjects FEV1, which is reproducible in an individual to 5% or better, and FVC both depend on height, age, and gender. At a given height and age, FEV1 is about 20% greater in men than women. Maximum FEV1 is reached in the early twenties and begins to decrease after age 25 years; the rate of decline may slightly accelerate with increasing age but in practice reference values assume an unchanged rate of decline. When vital capacity (VC) is reduced by restrictive disease (pulmonary fibrosis, heart failure, etc.), FEV1 is also reduced, so to diagnose airflow obstruction both FEV1 and the FEV1/FVC ratio have to be reduced (a crude boundary for FEV1/FVC ratio is < 70%). Because of the wide range of values of FEV1 according to height and age, a reduced FEV1/FVC ratio is often used to diagnose airflow obstruction. Once this is established, FEV1 is used to establish reversibility for the diagnosis of asthma. An increase of 12% over baseline and > 200 mL absolute increase are considered significant, while > 400 mL is clinically important. FEV1 remains the gold standard in clinical research. Instruments for measuring FEV1 at home are available but so far have not come into widespread use (Reddel et al. 1998). Because the complete forced expiratory maneuver often causes coughing as RV is approached, the FVC is often less than the VC obtained with a slow expiratory maneuver, so the best practice is to express FEV1 as a ratio of a separate slow VC maneuver. In routine clinical practice in a series of measurements (such as during measurement of airway responsiveness) the forced expiration is often discontinued once the 1-s volume has been obtained. However, it is always useful to know change in (F)VC as well as change in FEV1. As severity of asthma increases, RV increases due to air trapping with reciprocal decreases in FVC and VC. This volume reduction inevitably itself reduces FEV1 and has led to the suggestion that the two components to the spirometric change in asthma, the volume change reflected by the decrease in (F)VC and the obstructive change reflected by the fall in FEV1/(F)VC ratio, should be distinguished (Gibbons et al. 1996). The extent of the volume change as asthma severity increases is quite variable and a disproportionately large increase in air trapping may be an important determinant of symptoms and an unfavorable clinical course. The underlying reason for the usefulness of tests of forced expiration is that when strongly positive pleural pressures are generated, the rate of volume change (flow) at any lung volume reaches a maximum level which cannot be increased by further increases in expiratory effort and pleural pressures (expiratory flow limitation). This occurs because although progressively increasing pleural pressure increases the driving

Physiologic Aspects of Asthma

pressure for expiratory flow, it also increasingly compresses the larger intrathoracic airways, increasing their resistance and opposing the increased driving pressure. The result is that submaximal expiratory efforts are sufficient to generate maximum expiratory flow (MEF). Furthermore, when asthma is active, the required expiratory pressure to achieve MEF is less than when the subject is in remission. Although airway dimensions are crucial in determining the absolute level of MEF, other factors such as lung volume (including reduced VC due to air trapping) and lung recoil pressures (a measure of elasticity of the lungs) are important.

PEF The measurement of PEF is simpler and easier to apply to routine monitoring but is less robust, less reproducible, and more effort dependent than FEV1. Because peak flow meters/gauges measure the highest expiratory flow developed during a forced expiration, the PEF value depends on rapidly developing a positive expiratory pressure early in the expiratory maneuver and close to TLC (in healthy subjects PEF is usually achieved within the first 100 ms after the start of the maneuver when only about 10–15% of the VC has been expired). When measured off a spirometric trace it is usually expressed in liters per second BTPS, whereas it is usually expressed as liters per minute from a PEF meter or gauge. True expiratory flow limitation is present even at volumes very close to TLC, but the precise lung volume at which PEF is measured depends on how rapidly a positive expiratory pressure can be generated at the start of forced expiration. This makes the value obtained more susceptible to technical errors than measuring the FEV1. Normal values of PEF depend on height, age, and gender and are reduced by restrictive lung diseases that reduce lung volumes and VC. Hence PEF should not be used to establish the presence of airflow obstruction (this requires full spirometry). Once obstruction is known to be the cause of a reduction in PEF, it is particularly useful for monitoring progress because of the number of measurements that can be recorded over time, at home, as part of a therapeutic trial or personal management plan. PEF gauges are plastic, portable, and inexpensive and most patients can produce reproducible measurements (see Box 33.3). It is important for patients to use the same meter when assessing trends over time. PEF can be expressed as percent of personal best as well as percent predicted. It is useful to record it first thing, on waking, before therapy, in the morning (PEFAM) when it is usually lower than later in the day and this value can then be compared with an evening reading (PEFPM); the difference between PEFPM and PEFAM constitutes diurnal variation, which has been found to correlate well with other direct measures of bronchial responsiveness. It is also useful to measure PEF at the time of increased symptoms, e.g., if the patient wakes at night and to assess reversibility after administration of a β2-agonist bronchodilator. The amplitude of variation in PEF has more formally been

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Box 33.3 Comparison of methods for monitoring airway obstruction. FEV1 Spirometer Relatively cheap Relatively simple Portable Accurate Less effort dependent Gives other information (FVC) Future telemedicine

PEF Meter or gauge Cheap Simple Hand-held Fairly accurate Effort dependent No other information Computerized with diary

Changes in PEF do not precisely equate with changes in FEV1; typically percent-predicted PEF is higher than percentpredicted FEV1. PEF will tend to underestimate airflow limitation as this worsens and gas trapping occurs. FEV1 and PEF are sometimes described as tests of large intrathoracic airways which are insensitive to change in more peripheral airways function. This view is based on the serial distribution of resistance in normal subjects where the peripheral airways (so-called “silent zone”) make little contribution to total resistance. In mild airflow obstruction this may continue to be the case, but in symptomatic asthma, obstruction of peripheral airways usually makes a larger contribution to the total resistance, which is reflected in reduction in FEV1 and PEF. Hence these tests reflect total resistance of the airways (and so are useful for following the severity of airway narrowing) but are not useful in determining where in the intrathoracic airways most narrowing is situated.

Box 33.4 Assessment of diurnal variation in PEF. Airways resistance Morning dip PEFPM – PEFAM Amplitude % mean (Maximum PEF – minimum PEF)/average daily PEF (over 1–2 weeks) × 100 Min%Max Minimum PEFAM pre bronchodilator/recent maximum PEF × 100 Diagnosis of asthma > 20% diurnal variation ≥ 3 days in a week for 2 weeks on PEF diary

suggested as maximum minus minimum PEF over the average daily PEF as a percentage over 1– 2 weeks (Aggarwal et al. 2002). An alternative method (see Box 33.4) is minimum morning PEF before bronchodilator over 1 week expressed as a percentage of recent maximum (Min%Max). Home PEF recording can be used for the diagnosis of asthma. Maximum daily variation in normal subjects is 5– 8%. A 60 L/min or ≥ 20% improvement post bronchodilator or diurnal variation of > 20% on ≥ 3 days in a week for 2 weeks suggests the diagnosis of asthma (Reddel et al. 1995; Aggarwal et al. 2002). Asthma outcomes have been shown to be improved with self-monitoring of symptoms and/or PEF. This may be particularly in “poor perceivers” (Killian et al. 2000) and in preventing exacerbations (Gibson & Powell 2004). Repeated PEF measurement throughout the day in the workplace (every 2 hours waking to sleeping for 4 weeks) is useful in identifying occupational asthma, with a reported sensitivity of 80% and a specificity of around 90% (Leroyer et al. 1998; Baldwin et al. 2002). The objective measurement of PEF has been repeatedly shown to aid clinical assessment of the severity of an acute exacerbation of asthma, improving outcome.

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The resistance of the airways (Raw) is the pressure generating flow (the difference between mouth and alveolar pressures) divided by the instantaneous flow. It is usually measured at the volumes used during tidal breathing, but can be measured at other lung volumes. Raw gives an estimate of the dimensions of the total airway tree but again gives no indication of the location of any airway narrowing. The main established method for measuring airflow resistance is body plethysmography. When there is airflow obstruction, expiratory resistance can be much greater than inspiratory resistance due to dynamic narrowing of the airways during expiration. If Raw is measured during tidal breathing, usually an average of inspiratory and expiratory resistance, which includes glottal changes, is measured. If the primary interest is in dimensions of the intrathoracic airways, it is preferable to measure Raw on inspiration during a shallow panting maneuver which widens the vocal cords and minimizes resistance of the extrathoracic airway. An advantage of the plethysmographic method is that usually absolute lung volume (VL), which is an important determinant of resistance, is also measured in the same maneuver. This strong dependence on lung size is included in the measurement of “specific” Raw (SRaw = Raw × VL). More commonly adjustment for VL is applied to the reciprocal of Raw, airway conductance (Gaw) yielding “specific” airway conductance (SGaw = Gaw/VL). SGaw, measured at functional residual capacity (FRC) in an individual, is remarkable in that its normal range of values does not change between childhood and old age and is similar in men and women. For example, values of Raw decrease as the lungs grow in childhood but SGaw, which corrects for the growth of the lung, stays constant. Women have higher Raw (and lower Gaw) than men due to the larger VL of men but again SGaw and SRaw are similar in men and women. The requirement for expensive and bulky instrumentation makes the method most suitable for elective full laboratory

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MEFV curve and other flow–volume tests The MEFV curve is a different method of displaying the forced expiratory maneuver used in spirometry in which flow is measured at the mouth with a pneumotachograph and plotted against expired volume (Fig. 33.1b). The MEFV curve shows impressive repeatability when performed properly, so that visual inspection provides excellent quality control of the forced expiratory maneuver, including how rapidly PEF was developed and whether expiration was continued until expiratory flow ceased. In asthma the MEFV curve is often curvilinear, with MEF being particularly reduced in the lower half of the FVC (Fig. 33.2). This qualitative evidence of airflow obstruction can be quantified by measuring MEF at specific points on the MEFV curve, usually when 50% or 25% of the FVC remains to be expired. In mild obstruction changes are characteristically in the last part of the expiration (in part due to the closure of some airways, see below) so MEF25 has been proposed as a sensitive test; its main weakness is that the normal range of values is very wide and not much reduced by adjusting for the factors influencing spirometry, such as age, height and gender. PEF can also be measured from the MEFV curve, but there is little point in doing this when a complete forced expiratory maneuver is obtained. In addition values may differ from those obtained from a peak flow gauge. Maximum inspiratory flow–volume (MIFV) curves can be obtained from a forced inspiratory maneuver started after a slow full expiration to RV and continuing to inspire until TLC is reached. In contrast to MEF, maximum inspiratory flow

100% Maximum expiratory flow (% of baseline)

assessments or the study of pharmacologic responses. Two other simpler methods of measuring resistance, airflow interruption and the forced oscillation technique (FOT), are less demanding experimentally and becoming more widely available. These methods are applied during tidal breathing and may be particularly useful in young children (Delacourt et al. 2001). The interruption technique simply estimates airflow resistance in either inspiration or expiration. The FOT technique is most commonly used to measure resistance of the total respiratory system, averaged over several breaths, but it can also be used to obtain a much fuller analysis of the mechanical properties of the lungs (Oostveen et al. 2003). This analysis depends on subdividing the primary measurement obtained, the instantaneous relation between the applied pressure and the resultant flow oscillation (impedance), into its in-phase (resistance) and out-of-phase (reactance) components. Furthermore, impedance can be measured over a wide range of frequencies (typically 4– 26 Hz) so that its frequency dependence can be determined. There are suggestions that by studying the frequency dependence of resistance and the values of reactance, the relative contributions of central and peripheral airways to increased resistance can be estimated. If these claims are confirmed, FOT may gain a significant role in assessment of airway function in asthma.


PEF

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100% Forced vital capacity (% of baseline) Fig. 33.2 Characteristic changes in maximum expiratory flow–volume (MEFV) curve as asthma becomes more severe. Curves were obtained in five patients admitted with severe asthma who were studied sequentially until normal airway function was obtained. The curves were normalized to the values obtained in full remission of asthma (top curve, 100% function). Note progressive convexity of curve to volume axis as airflow limitation worsens. Because of reduction in forced vital capacity and increase in residual volume, MEF50 and MEF25 are measured at successively larger volumes as airflow limitation worsens. With mild airflow limitation, the largest proportional changes are in MEF25 and MEF50. See Fig. 33.1 for definition of other abbreviations.

(MIF) depends on the driving pressure generated and so requires more trial efforts to achieve repeatable results. MIF is reduced in active asthma but to a lesser extent than MEF and has not found a useful place in routine assessment of asthma. Only in very severe asthma in children or the very elderly will MIF be reduced enough to compromise effective use of some of the dry powder inhalers. The main clinical relevance of the MIFV curve is that, when studied in conjunction with the MEFV curve, it helps distinguish extrathoracic obstruction, for instance in the larynx, from asthma. The shape of the MEFV curve may be distinctive (Fig. 33.3), with reduced MEF at high lung volume but a normal MEF as RV is approached (quite different from asthma) and MIF may be reduced throughout the inspiratory VC. Sometimes obstruction of the extrathoracic airways may only reduce MIF, the MEF curve remaining normal. Several other techniques using flow–volume curves have been developed. A full inspiration to TLC in healthy subjects leads to short-lived bronchodilatation which may increase flow during a subsequent complete forced expiratory maneuver; in contrast in many asthmatic subjects, a full inspiration causes transient bronchoconstriction. To study these effects, maximum expiratory flow and volume during a forced expiration started from the end of a tidal inspiration (partial expiratory

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flow–volume PEFV curve) may be compared with that during a conventional MEFV curve started from TLC. MEFV curves breathing air have been compared with MEFV curves after breathing helium/oxygen mixtures to investigate the predominant site of airway narrowing. Flow– volume curves during tidal breathing have been studied at baseline and after a small negative expiratory pressure (NEP) has been applied at the mouth during expiration (Koulouris et al. 1997); usually NEP results in an increase in tidal expiratory flow but in severe airflow obstruction there may be no increase, indicating the presence of expiratory flow limitation during tidal breathing.

Measurement of lung volumes: assessment of hyperinflation and air trapping The VC and FVC are frequently reduced in symptomatic asthma. They are usually measured in an expiratory maneuver and provide no information on absolute lung volumes (TLC or RV) or on the presence of hyperinflation. Absolute lung volumes can be measured in asthma by body plethysmography or by a multiple breath equilibration technique in which the subject re-breathes a helium mixture until a steady concentration is reached. Both methods have potential problems particularly when obstruction is severe. Plethysmography may overestimate lung volume because during the panting maneuver used mouth pressure may not equilibrate with alveolar pressure (Stanescu et al. 1982). In contrast, the helium equilibration method tends to underestimate volume because equilibration is incomplete. With both techniques the volume directly measured is usually the expiratory end-tidal volume, i.e., FRC; the volumes at the extremes of the VC (TLC and RV) are obtained by an additional two-part VC maneuver (full inspiration to TLC, full expiration to RV) started from FRC. Sometimes the volume directly measured using body plethysmography is termed “thoracic gas volume.” In healthy subjects FRC is the volume where the inward (expiratory) recoil of the lungs is exactly balanced by the outward (inspiratory) recoil of the relaxed chest wall (relaxation

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Fig. 33.3 Maximum expiratory and inspiratory flow–volume curves in extrathoracic airway obstruction. (a) Fixed narrowing of the glottis. Reduced maximum expiratory flow (MEF) over the first 75% of the forced vital capacity (FVC) with normal decline in MEF over the last 25% of the FVC. Maximum inspiratory FEV1/FVC remains normal flow (MIF) reduced at all volumes. (b) Variable narrowing in this patient due to subglottic polyp which occludes the glottis during a forced inspiration. Maximum expiratory flow–volume curve virtually normal but MIF reduced at all lung volumes.

volume). At rest the later part of a tidal expiration is passive so that there is no activation of respiratory muscles and FRC corresponds to the relaxation volume. Typically at rest FRC is about 50% TLC in young healthy subjects, rising to 55– 60% TLC in elderly subjects. During exercise there is activation of expiratory muscles and end-tidal volume is lower than the resting FRC. To reach TLC requires full activation of inspiratory muscles; conversely RV requires full activation of expiratory muscles to reduce the volume of the chest wall. In healthy young adults the increasing stiffness of the chest wall limits deflation but after age 40 years airway closure, probably in small bronchioles, determines RV. RV rises more than FRC with increasing age, and because TLC does not change, the rise in RV accounts for the reduction in VC with age. The difference between FRC and TLC is termed “inspiratory capacity”; this falls with increasing age but to a smaller extent than VC. Fewer measurements are made of closing volume (CV), which requires a separate single breath technique, usually based on the change in nitrogen concentration during a full expiration after a full inspiration of 100% oxygen from RV. CV is recognized by the beginning of a steeper rise in expired nitrogen concentration, which corresponds to the beginning of airway closure on expiration. CV is usually expressed as milliliters above RV or as percent VC but the absolute volume at which closure begins, closing capacity (CC), can be obtained as the sum of CV + RV. In young healthy subjects closure begins well below FRC, but CV (and CC) rise with age and may exceed FRC by 70 years of age. In active asthma RV is characteristically elevated due to enhanced airway closure (air trapping). It is often assumed that closure occurs in the terminal bronchioles (and so is an indicator of small airways disease), but this is not necessarily the case in asthma where airway smooth muscle contraction and mucoinflammatory plugs may result in closure of more proximal airways. Even in asthma remission, when VC and RV (and FEV1) are normal, CV is often increased (McCarthy & Milic-Emili 1973). Imaging with scintigraphy, high-resolution

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CT (HRCT), or MRI shows that in healthy subjects airway closure commences in the most dependent parts of the lungs and progresses up the lungs as full expiration continues (see below). In asthma this pattern of closure is much more disorderly, presumably reflecting difference in the extent of pathologic changes between adjacent regions of lung. FRC also increases as asthma worsens but to a lesser extent than RV. Some of this increase may be due to an increase in relaxation volume but usually FRC is greater than relaxation volume and is maintained by glottal “braking” of expiration, which is terminated by the next inspiration before relaxation volume is reached. The increase in FRC widens airways but at the cost of increased work by the inspiratory muscles to overcome the greater elastic load offered by lungs and chest wall as lung volume is increased. When spontaneous or bronchodilator-induced improvement occurs there is usually some reduction in FRC which removes part of the improvement gained in airway dimensions, but most often both airway resistance and FRC are reduced. TLC in stable asthma is usually normal or may be slightly increased, especially in patients who had asthma during growth (Greaves & Colebatch 1985). Whether TLC increases in acute asthma has been controversial since first proposed more than 40 years ago (Woolcock & Read 1966; Woolcock et al. 1971). Some of the early increases reported using body plethysmography were artifactual (Stanescu et al. 1982). Subsequently the consensus for many years was that TLC did not increase acutely when airway narrowing was induced in asthma (Kirby et al. 1986). Recently this controversy has been revived by an analysis suggesting that an acute increase in TLC is an important adaptation to a large increase in RV (Brown et al. 2006; and see commentary by Irvin 2006). It remains unclear where an increase in TLC could be accommodated within the thoracic cavity. Perhaps the controversy will be resolved by the sequential use of an imaging technique to estimate volume at full inflation during an acute episode of asthma. Woolcock and Read (1966) also first suggested that spontaneous or treatment-induced improvement in symptoms could be due to a fall in FRC without accompanying improvement in spirometry or PEF. Probably most such examples occur when overall physiologic changes are small; with large responses, there is usually both an improvement in spirometry and a fall in FRC. Because measurement of FRC cannot be routinely assessed in acute asthma, the true prevalence of such isolated volume responses remains uncertain. Measurement of any of these absolute volumes is important because FEV1 and PEF only assess airflow obstruction, while hyperinflation (breathing at large operating volumes) is an important cause of discomfort in acute asthma. “Hyperinflation” is probably best used to refer to increased FRC because this indicates tidal breathing is occurring at larger than normal operating volumes. It is sometimes used to indicate a large RV, RV/TLC ratio, or even TLC, none of which are


directly related to tidal breathing difficulty. The most practical method for monitoring hyperinflation in acute severe asthma is to measure inspiratory capacity (difference between FRC and TLC). This can be done with any spirometer which allows measurement of inspiratory volumes. As discussed above, a reduction in (F)VC indicates a reciprocal rise in RV.

Assessment of alveolar function Conventionally, asthma is regarded as a disease of the airways without significant changes in the lung tissue or airspaces. Although pulmonary gas exchange is inefficient and arterial oxygen pressure is reduced in attacks of asthma, this is caused by uneven distribution of ventilation (and blood flow) not by any alveolar abnormality. Carbon monoxide transfer (CO diffusing capacity) is usually normal or near normal in fully reversible asthma (Keens et al. 1979; Collard et al. 1994), but there is often a small loss of lung recoil pressure, especially at large volumes (Kraft et al. 2006) while in persistent asthma these losses may be larger (see below). Histologically human alveolar ducts contain smooth muscle and in animals increase in the resistance of lung tissue accounts for a considerable proportion of the response to bronchoconstrictor aerosols (Ludwig et al. 1989). In human asthma it is difficult to measure lung tissue resistance directly but modeling suggests that any contribution from lung tissue is certainly much smaller and is not found in all subjects (Kaczka et al. 1999).

Chronic asthma with persistent airflow obstruction In middle-aged and older patients with asthma, optimized airway function is often below predicted values (Brown et al. 1984). This confuses the distinction from, and results in genuine overlap with, smoking-related chronic obstructive pulmonary disease (COPD) (Boulet et al. 1998; Fabbri et al. 2003), although the prognosis in chronic asthma is much better. Airways resistance is increased in both central and peripheral airways (Yanai et al. 1992) but little is known about the pathophysiologic basis of these changes. Airway wall thickening is the most obvious and consistent finding on HRCT (Boulet et al. 1995; Awadh et al. 1998; Ketai et al. 2001; Nimi et al. 2003). Conventionally chronic asthma, in the absence of smoking, is not associated with the development of emphysema, although HRCT scans do show limited changes in a minority of patients (Boulet et al. 1995; Gelb et al. 2002). Carbon monoxide transfer may be modestly reduced but not to the extent commonly found in COPD (Boulet et al. 1998; Gelb et al. 2002; Fabbri et al. 2003). More striking reductions in lung recoil pressure, which would decrease MEF significantly, have been reported by Gelb and colleagues in a few patients with little or no emphysema on HRCT and normal carbon monoxide transfer (Gelb & Zamel 2000; Gelb et al. 2002); these findings need confirmation by further studies. Two factors that may contribute to loss of lung recoil are stress relaxation of alveolar tissue due to chronic hyperinflation

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(Gelb et al. 2002) and alveolar inflammation (Kraft et al. 2001). Peribronchial inflammation may damage alveolar walls (Mauad et al. 2004) and lead to loss of their recoil, while expansion of the airway outer perimeter may reduce coupling between the small airways and alveolar attachments. Hence even in the absence of overt destructive emphysema it is possible that more subtle alveolar disease contributes to the persistent airway obstruction of chronic asthma.

Changes in clinical remission Because of the enormous expansion of the cross-sectional area of the peripheral airways (their great increase in number far outweighing their much smaller diameter compared to more central airways) considerable disease can be present in small airways without obvious effect on spirometric function or total airways resistance. Many studies have shown that patients in clinical remission frequently show persisting abnormalities of RV, CV, abnormal distribution of ventilation, and peripheral lung resistance, changes all compatible with persistent obstruction in peripheral airways.

Site of increased airway resistance: large versus small airways Airway narrowing in episodic asthma is due to varying combinations of airway smooth muscle (ASM) contraction, mucosal swelling, and luminal secretions. These factors all vary during and between attacks. Pathologic studies have shown that inflammation of the airway mucosa and increased ASM mass extend throughout the tracheobronchial tree, with inflammation into the alveoli (Carroll et al. 1993; Kraft et al. 1996, 1999) indicating the potential for narrowing to develop in airways of all sizes. Studies of regional ventilation (see below) have consistently shown that airway narrowing varies greatly between parallel airways, despite the likely presence of inflammatory changes in all airways. Presumably there can also be longitudinal nonuniformity. Given such heterogeneity and the apparently invariable involvement of small airways, identifying precisely which airways contribute most to airflow obstruction is very difficult and may not even be clinically useful. Despite considerable research efforts, a simple robust measure of the predominant site of airflow obstruction is still needed (see Box 33.2).

receptors, and induce reflex bronchoconstriction and mucus production. The assumption is made that the peripheral lung beyond the catheter tip is “typical” of all other regions of peripheral lung (it has not been practical to place catheters in multiple regions of the lung) in an individual. Two different wedged-catheter techniques have been developed that measure pressure at a peripheral site; in one, a 3-mm catheter is wedged in a right lower-lobe bronchus and measuring lateral pressure just proximally to the wedged site allows the peripheral airways resistance of the adjacent ventilated lung to be determined (Yanai et al. 1992). Total lung resistance in normal subjects with this technique appears about 50% higher than without the presence of a catheter. In asthma in remission, inspiratory (but not expiratory) peripheral airway resistance was slightly increased. In chronic asthma with persistent airflow obstruction in middle-aged patients, both central and peripheral resistances were increased. Allowing for the contribution of the extrathoracic airway to total lung resistance, peripheral resistance accounted for about onethird of intrathoracic resistance in normal subjects and about 50% in chronic persistent asthma (Yanai et al. 1992). Another less physiologic technique measures pressure– flow relations within the occluded lung beyond a bronchoscope wedged in a segmental bronchus. Gas is introduced via the bronchoscope and slowly inflates the occluded lung while the pressure at the end of the bronchoscope is measured (Wagner et al. 1990). In asthmatic subjects in remission, with normal FEV1 and total airway resistance, a considerable increase in this peripheral lung resistance was found. This resistance not only reflects the resistance of peripheral airways, but also of collateral channels and lung tissue. These results directly confirm earlier suggestions from pathologic and physiologic studies that there are residual changes in the peripheral airways even in asthma in remission. This technique has also been used to show a rise in peripheral lung resistance at night in nocturnal asthma (Kraft et al. 2001) and to measure increases in the mean level and frequency dependence of this resistance after methacholine (Kaminsky et al. 2004). Although these results imply an increase in resistance of collateral channels in asthma, gas exchange studies suggest that these channels continue to allow some collateral ventilation except in the most severe asthma attacks.

Airway response to breathing a helium–oxygen mixture

Endobronchial catheter studies

MEFV curves

Endobronchial pressure measurements define the airway distribution of airflow resistance directly, and provide the best physiologic evidence for the respective contributions of large and small airways, but their invasive nature has restricted their use to stable patients. There are also important physiologic limitations. Endobronchial catheters inevitably reduce the area available for gas flow, may stimulate mucosal

The density of 80% helium/20% oxygen is approximately one-third that of air. In some (but not all) asthmatic subjects, MEF does not show the normal increase when breathing helium/oxygen (Despas et al. 1972) suggesting that the major site of flow limitation is no longer in central airways, as in normal subjects, but has moved to more peripheral airways, where flow is presumed to be laminar and independent of

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density (Ingram & McFadden 1977). This change is usually attributed to increased frictional pressure losses in narrowed peripheral airways. Some asthmatic subjects consistently lose or consistently retain density dependence of maximum flow with repeated attacks. In general, loss of density dependence becomes more common as expiratory airflow limitation increases in severity (Fairshter & Wilson 1980; Partridge & Saunders 1981) and is particularly observed in asthmatic subjects who smoke (Antic & Macklem 1976). Reduction in density dependence of MEF should not be interpreted as indicating that only peripheral airways are narrowed, even if they are the site of flow limitation. Considerable variation in size of the helium response in the normal population and in disease has been noted; changes in helium response in an individual before and after an acute intervention may be more reliable.

Pulmonary resistance An alternative method is to study the reduction in pulmonary resistance (RL) (measured by the esophageal balloon technique) when tidal breathing is switched from breathing air to a helium/oxygen mixture (Lisboa et al. 1980). A large decrease in RL suggests a large component is in the central conducting airways while a small response indicates that much of the resistance breathing air is in the peripheral airways. This tidal breathing technique is probably more relevant to the use of helium/oxygen in acute severe asthma to reduce resistive work of breathing. There are practical problems in clinical use because Heliox density rises once helium concentration falls below 70% due to increased inspired oxygen or entrainment of room air. A recent Cochrane metaanalysis of 544 patients in 10 studies concluded that there was no clinical role of Heliox, although FEV1 improved more in the subgroup with the most severe asthma (Rodrigo et al. 2006).


explains the variable changes in airway function after DI (Ingram 1987, 1990). When airway hysteresis exceeds that of parenchyma, DI results in temporary airway widening when tidal breathing is resumed. When parenchymal hysteresis exceeds that of the airways, DI results in airway narrowing. Equal degrees of hysteresis result in no effect of DI on resting airway caliber, as found in most normal subjects. A major interest of this idea is that it might localize the site of disease within the lung. Contraction of smooth muscle in conducting airways would be expected to increase airway hysteresis without affecting parenchymal hysteresis. This is the typical pattern of induced bronchoconstriction. Increased tone or other causes of airway narrowing in the extreme periphery of the lung (respiratory bronchioles and alveolar ducts) would be expected to increase parenchymal hysteresis with only a small increase in airway hysteresis. This is the typical pattern in spontaneous asthma.

Other evidence of small airway involvement in asthma Histologic and morphometric data have long suggested that important structural changes occur in the airway walls and ASM of the peripheral as well as central airways (Ebina et al. 1990; Carroll et al. 1993; Hamid 1997). The bronchoscopic catheter studies directly, and the frequency dependence of dynamic resistance and elastance indirectly, provide strong physiologic evidence of peripheral airway involvement. Multibreath nitrogen (Verbanck et al. 1999) and single-breath SF6 and helium washout studies have provided evidence of airway closure at acinar level contributing to peripheral airway response (Gustafsson et al. 2003). Mathematical modeling approaches (Lutchen et al. 2001; Kaminsky et al. 2004) relate inflammation, airway thickening, and amplification of heterogeneous constriction to determine mechanical response.

Airway response to a deep inspiration Deep inspiration (DI) produces airway widening transiently when tidal breathing is resumed in normal subjects after narrowing is induced by inhaled histamine or methacholine. In contrast, DI can lead to subsequent airway narrowing in subjects with acute spontaneous asthma (Simonsson et al. 1967). These differences between normal and asthmatic subjects have practical effects exaggerating differences in airway responsiveness as assessed by conventional tests of forced expiration. Partial expiratory flow–volume curves surmount some of these difficulties, with measurements usually made at volumes between 30 and 40% VC, thus balancing the conflicting demands of avoiding undue lung inflation and artifactual flow at the start of expiration, while still ensuring an adequate flow signal. Initially, changes after DI were attributed to a change in bronchial muscle activity (direct or reflex) produced by stretch (see further discussion in section Airway smooth muscle). An alternative hypothesis is that the relation between parenchymal and airway hysteresis

Imaging of the airways and ventilation (Box 33.5)

Visualization of the airways Direct visualization of airways, either at bronchoscopy or by external imaging (plain radiographs, bronchography, CT), is confined to the larger conducting airways. Narrowing of the large airways has occasionally been reported when acute bronchoconstriction has developed during bronchoscopy (or, in the past, at bronchography) or on segmental airway challenge. Airway lumens as small as 0.5 mm in diameter have been reportedly measured using CT but large errors are likely (De Jong et al. 2005). HRCT can interrogate small airways indirectly by demonstrating air trapping and regional hypoventilation (exaggerated by expiration), assumed to be due to enhanced closure of peripheral airways (reflecting increase in CV and RV on standard lung function testing).

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Computed tomography Airway size HRCT and multidetector row CT (see De Jong et al. 2005 for methodology) allow determination of airway luminal area, which has been reported to be both decreased (Lynch et al. 1993) but normalized after salbutamol in some patients (Beigelman-Aubry et al. 2002) but not in others with more severe asthma (Park et al. 1997; Nimi et al. 2000). However, all studies have demonstrated increased airway wall thickness in asthma of all severities in children as well as adults. Degree of wall thickening is related to duration and severity and level of airway obstruction (De Jong et al. 2005). Many authors have measured bronchoconstriction of larger airways following methacholine. However, the relationship between airway wall thickness and AHR is complex. Thicker airways may be less compliant and therefore less responsive: increase in wall thickness in larger airways or decrease in luminal diameter of medium airways (5–10 mm diameter) and dynamic hyperinflation with critical narrowing of large airways are important determinants of responsiveness (Brown et al. 2006).

Gas trapping Air trapping, and regional hyperinflation, particularly on expiration, allow indirect assessment of small airways qualitatively (Park et al. 1997) and semiquantitatively (Laurent et al. 2000). The distribution of Hounsfield units represents a frequency distribution of lung attenuation (LA) values and dynamic analysis of the distribution of LA yields reproducible quantitative data. Reduction of gas trapping by hydrofluoroalkane small-particle beclomethasone dipropionate (BDP) more than conventional chlorofluorohydrocarbon (CFC)-BDP (Goldin et al. 1999) and other inhaled corticosteroids (Aubier et al. 2001) and montelukast has been shown (Zeidler et al. 2006).

Single photon emission computed tomography SPECT measurements of closed airway volume (inaccessible to a Technegas bolus) correlated with CV and CC and did not differ in mild asthmatics with normal and abnormal spirometry from normal subjects (King et al. 1998), although the distribution differed in asthma. Regional expiratory flow limitation was demonstrated using Technegas with hotspots thought to represent exaggerated impaction of Technegas on severely narrowed airways (Pellegrino et al. 2001).

Positron emission tomography Early studies (Rhodes et al. 1989a,b) used inhaled 19Ne and intravenous infusion of 13N2 to measure regional ventilation– perfusion ratios in asthma. There was a greater variation in regional ventilation and ventilation–perfusion ratio than in normal subjects. Modeling of a 13N2 bolus injection followed by 30–60 s apnea enabled quantitation of “fast” (normal ventilation) and “slow” (hypoventilation) compartments (Musch & Venegas 2005) occurring below the limited resolution of PET

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(approx 1 cm). Regional hypoventilation and heterogeneity in asthma was quantified as injected tracer is retained locally after resumption of breathing (Venegas et al. 2005a). The relative contributions of large and small airways have been analyzed using an image functional modeling approach mapping three-dimensional ventilation images onto an anatomically explicit computational model using forced oscillometry at 0.15–8 Hz to determine dynamic lung resistance and elastance pre and post methacholine (Tgavalekos et al. 2005). Closure of airways < 2.39 mm and mainly < 0.44 mm in diameter best mimicked the experimental data. Patterns of constriction involving larger airways could mimic the results only in the presence of small airway closure.

MRI studies Gaseous MRI contrast agents such as 129Xe and 3He have led to new imaging of ventilation without the use of ionizing radiation (Middleton et al. 1995; de Lange et al. 1999). Single breaths of hyperpolarized 3He, which provides a large MRI signal of ventilated airspaces because of its low water and lipid solubility, have demonstrated wedge-shaped ventilatory defects, mostly < 3 cm in size, more consistently and more extensively in more severe asthma correlating with percentpredicted FEV1 (Altes et al. 2001; Samee et al. 2003). Ventilation defects per slice were significantly greater in moderate and severe persistent asthma than in mild intermittent or mild persistent asthma and correlated best with FEV1/FVC (r = −0.51, P < 0.002), MEF25–75 (r = −0.5, P = 0.001) and FEV1 (r = −0.40, P = 0.002) (de Lange et al. 2006). Many defects persisted when MRI was repeated. Induced bronchoconstriction resulted in the appearance of new defects; these are detected more sensitively by 3He MRI than change in spirometric indices including MEF25–75 (de Lange et al. 2007). Furthermore, defects often recurred in the same site when methacholine challenge was repeated. Dynamic imaging using ultra-fast pulse sequences to image the respiratory cycle has the potential to produce quantitative regional measurements of airflow (Salerno et al. 2001). Other MRI techniques including measurement of ventilation volumes, diffusion imaging (measurement of apparent diffusion coefficient), and regional oxygen tension mapping have not been applied specifically to asthma (van Beek et al. 2004).

Ventilation inhomogeneity Inhomogeneity of ventilation is well recognized clinically in asthma; breath sounds may be absent to auscultation regionally and segmental atelectasis may sometimes be visualized radiologically. The multiple inert gas elimination technique (MIGET) demonstrated a clear bimodal distribution of ventilation–perfusion ratios (Wagner et al. 1978) and different airways were shown to respond differently to inhaled histamine using the wedged bronchoscope technique (Wagner et al. 1998). Heterogeneity of conducting airways (Scond) using multiple breath nitrogen washout was recently suggested as

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Box 33.5 Imaging of airways and ventilation. Imaging of airways Chest radiography Bronchography CT fine section and reconstruction, multidetector row Imaging of ventilation Scintigraphy: 81Kr, 133Xe, 127Xe, 99mTc aerosols SPECT: xenon, Technegas MRI: hyperpolarized 3He, 129Xe PET: inhaled or intravenous 13N, inhaled 19Ne

a major determinant of responsiveness uncoupled from airway inflammation (using inhaled nitric oxide as a surrogate by treatment with BDP (uncontrolled) (Downie et al. 2007). A variety of three-dimensional imaging techniques demonstrate ventilatory inhomogeneity dramatically. Patchy defects in 81mKr ventilation scans have long been recognized (Fig. 33.4), particularly on induced bronchoconstriction and in severe asthma (Sovijarvi et al. 1982; Vernon et al. 1986; Orphanidou et al. 1986). HRCT has been used to demonstrate heterogeneous airway narrowing particularly of larger airways to inhaled methacholine in dogs, normals and asthmatics (Brown & Mitzner 2003; King et al. 2004; Brown 2007). Spotty deposition of Technegas was greater in the lower zones (and less in the upper zones) in asthma (King et al. 1998). Defects were larger, more patchy, and often peripheral and wedge-shaped (Fig. 33.5). Complete absence of Technegas in an entire lobe or part of it, mainly in dependent lung regions, occurring at high doses of methacholine were thought to suggest full airway closure (Pellegrino et al. 2001). Patchy ventilatory defects were also noted in early PET studies using infused 13N2 (Rhodes et al. 1989b; Rhodes & Hughes 1995). Quantification of regional hypoventilation and heterogeneity in asthma was possible using PET modeling of a 13N2 bolus injection (Venegas et al. 2005a). Methacholineinduced bronchoconstriction is associated with regions with severe hypoventilation or “clusters of self-organized patchiness” (Venegas et al. 2005b). Bronchoconstriction is characterized by patchy distribution of regional ventilation with bimodal V/Q distributions in line with classic physiologic studies in asthma using MIGET (Wagner et al. 1978; RodriguezRoisin et al. 1991). In mild asthma, ventilation defects (varying from subsegmental to lobar) induced by methacholine were associated with a systematic reduction in regional perfusion of the order of 30% (Harris et al. 2006). This would operate to reduce regional ventilation–perfusion mismatch, though it is not clear to what extent local regional increase in gas volume leads directly to reduced blood flow and to what extent active pulmonary vasoconstrictor mechanisms are responsible.


Airway smooth muscle and change in lung volume Both alveoli and airways are elastic structures, changing size when exposed to alterations in distending forces, as occurs every few seconds with tidal breathing, and to a greater extent with an occasional sigh or DI. However, whereas alveoli have rather fixed and predictable elastic properties, contraction of ASM changes airway dimensions and compliance drastically in a few seconds, putting ASM at the heart of the asthmatic process. The physiology of ASM is extensively discussed in Chapter 42. This brief section outlines the important interaction of ASM with lung volume and other determinants of its function in vivo.

Normal airways The area of a normal intrathoracic airway increases by 45–50% when a DI is made from FRC to TLC. This increase occurs due to increased distending forces by the lungs themselves as lung volume increases or, in the case of extrapulmonary intrathoracic airways, directly from the inspiratory reduction in pleural and mediastinal pressure. Normal airways have a baseline “tone” due to activation of ASM by vagal stimulation. This slightly reduces their dimensions from the relaxed state and accounts for the small response in airway function found with bronchodilators. Variations in this vagal “tone” determine the circadian rhythm in airway function found in normal subjects, which has the same timing but smaller amplitude compared with the circadian rhythm in asthma.

Asthma AHR to bronchoconstrictor challenge (see Chapter 38) is a central abnormality in asthma. For many years it has been known that the bulk of ASM is increased in asthma (Carroll et al. 1993; Woodruff et al. 2004) but there has been uncertainty whether it has increased contractility. Historically, AHR has been explained in terms of enhanced “twitchiness” and contraction of ASM. This could be due to external influences such as altered adrenergic receptors, altered neural control, inflammatory or other mediators, or the proximity of mast cells. Alternatively, it could be intrinsic to ASM itself: its increased mass or enhanced contractility. In recent years it has been emphasized that enhanced bronchoconstriction could occur without postulating any abnormality of ASM response because (i) a normal amount of shortening of ASM can give a disproportionate reduction in luminal caliber in the presence of relatively subtle wall thickening; or (ii) greater ASM shortening is produced for a given stimulus because the restraining force applied by surrounding alveolar walls attached to the external perimeter of intrapulmonary airways is reduced. Airway wall–parenchyma coupling might be reduced by peri-airway inflammation and damage to

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Mild asthma

Visit 1 FEV1 104%

Visit 2 FEV1 101%

Visit 1 FEV1 66%

Visit 2 FEV1 68%

Male Age 48 Grade 0

(a)

Male Age 42 Grade 1

(b) Severe asthma

Visit 1 FEV1 48%

Posterior ventilation

Visit 2 FEV1 52%

Male Age 66 Grade 2

(c) Visit 1 FEV1 41%

Visit 2 FEV1 43%

Male Age 53 Grade 3

(d)

alveolar walls and/or expansion of the airway perimeter. Although airway wall thickening (remodeling) might be induced by inflammation, if it does not completely regress with suppression of inflammation, AHR could be sustained solely by airway wall thickening.

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Fig. 33.4 Krypton-81m ventilation scans demonstrating heterogeneity, with defects visible in more severe asthma graded 0, 1, 2, and 3 in four patients studied on two occasions, when stable, 2 weeks apart. (a) Posterior scans in a 48-year-old mild asthmatic: FEV1 104% and 101% predicted on the two occasions, both graded 0 (normal). (b) Posterior scans in a 42-year-old asthmatic: FEV1 66% and 68% predicted on the two visits, both graded 1 (minor defects). (c) Posterior scans in a 66-year-old asthmatic: FEV1 48% and 52% predicted on the two visits, both graded 2. (d) Posterior scans in a 53-year-old asthmatic: FEV1 41% and 43% predicted on the two visits, graded 3 (multiple large defects). Note how ventilation has improved in the right lower lobe and deteriorated in the left between the two visits.

Effects of DI and its prohibition Further interest in the effects of DI and stretch on ASM was stimulated by the observation that prohibition of DI could produce striking hyperresponsiveness to methacholine in normal subjects (Skloot et al. 1995). This suggested that

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Fig. 33.5 Use of three-dimensional imaging to show heterogeneity of airway closure (at residual volume) and inhomogeneity of ventilation. (Top) Single coronal slices from three normal subjects aged 27, 32 and 43 years. Unventilated regions are relatively uniform and contiguous. (Bottom) Slices show typical large peripheral triangular defects in asthma suggesting airway closure. Superimposed emission and transmission images; ventilated lung is white, unventilated lung gray, and soft tissue black. (From King et al. 1998.) (See CD-ROM for color version.)

failure to periodically inflate the lung to large volumes results in loss of a bronchoprotective effect, which was subsequently shown to be considerably stronger than the bronchodilator effect of DI in normal subjects (Schichilone et al. 2000). However, this bronchoprotective effect was lacking in asthmatics and even in patients with rhinitis alone (Kapsali et al. 2000; Schichilone et al. 2001). However, using a different protocol administering a PC15 dose of methacholine every 5 min and measuring FEV1 at the beginning and then after five challenges, King et al. (2001) found that inhibition of DI during challenges had an augmenting effect on bronchoconstriction in asthma, suggesting that DI may limit induced bronchoconstriction in asthma as well. Pyrgos et al. (2003) showed no effect of DI-induced bronchoprotection against inhaled allergen in subjects with allergic rhinitis, who nevertheless demonstrated a protective effect of DI against methacholine-induced bronchoconstriction. Inhaled steroids (Corsico et al. 2000) and oral in addition to inhaled steroids (Slats et al. 2006) have been shown to improve airway responses to deep inspiration, while Schichilone et al. (2005) suggested that inhaled steroid treatment can normal-

ize the protective effect of DI in mild but not more severe hyperresponsiveness. Recently, inflammation in ASM and the submucosa on bronchial biopsies has been shown to be associated with an impaired response to DI measured by the FOT in asthma (Slats et al. 2007). In the last decade these clinical observations have stimulated many investigations into the effect of stretch and its absence on ASM both ex vivo and in intact animals (see extensive review by An et al. 2007). These experiments suggest that in the absence of repeated stretch ASM can stiffen and increase its bronchoconstrictor potential. Even quite small stretches, such as occur in normal tidal breathing, may be required to maintain ASM in its optimal compliant state. It is suspected that stretching of ASM during breathing might be reduced in asthma. Understanding of the dynamics of ASM has greatly advanced, but dissecting out which of several proposed abnormalities of ASM are important in vivo in asthma remains a formidable task, which is not assisted by great uncertainty about normal physiologic function (if any) of ASM in health (Mitzner 2004). Development of ablation of ASM by bronchial thermoplasty as a treatment for asthma

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may provide insights into its physiologic role (Solway & Irvin 2007).

Involvement of the extrathoracic airway

Bronchial thermoplasty

Nose

Radiofrequency ablation by controlled heating of the airway wall using a catheter system at bronchoscopy (bronchial thermoplasty) produced wide ablation of smooth muscle affecting the airway wall and immediate peribronchial tissue in airways over 3 mm diameter in dogs, lasting at least 3 years (Danek et al. 2004). Thermoplasty of segmental airways down to 3 mm diameter, in both lower lobes and both upper lobes, in three sessions of 30 min, under either general or local anesthesia, was shown to be feasible and safe in mild stable asthma (Cox et al. 2006). Modest increases in FEV1 were accompanied by geometric mean PC20 methacholine improvements of 2.4, 3.0 and 2.3 doublings at 12 weeks, 1 year, and 2 years. A recent randomized study (Cox et al. 2007) reported 12-month follow-up after thermoplasty in 55 asthma patients compared with a control group of 54 patients taking inhaled steroid and long-acting inhaled β2-agonist (LABA) therapy. Mild exacerbations were significantly reduced over 1 year in the thermoplasty group, with significant improvement in morning PEF, symptom scores, and quality of life with reduced rescue β2-agonist use. These effects were seen at 3 months, when all patients were taking LABA, and at 12 months in the treatment group when all patients had attempted to stop LABA (achieved in about half of all patients in both groups). Evaluations were carried out after 2 weeks off LABA. Bronchial thermoplasty improved asthma control in moderate to severe asthma. A placebo-controlled study is required to assess true clinical benefit and the contribution of more central airways to AHR.

Nasal obstruction due to rhinitis and/or polyps is the most obvious site of extrathoracic airway involvement. Rhinitis is common in the general population, affecting up to 30–40% at some age. However, in severe asthma several studies have suggested a prevalence approaching 100% (Bresciani et al. 2001). In normal subjects, during tidal breathing, the nose provides a resistance of about 2 cmH2O/L per s, about 50% of the total airflow resistance. Nasal resistance may be considerably higher in the presence of rhinitis, particularly when supine (Table 33.1) (Duggan et al. 2004). Presumably, if nasal resistance is greatly increased, tidal breathing is divided between the oral and nasal routes, with reduction of the protective and humidifying effects of the nose. Reflexes between the nose and intrapulmonary airways have been investigated extensively in experimental animals, but their importance in humans remains uncertain. The better-established interaction is between acute nasal stimulation and transient narrowing of intrapulmonary airways, but the reverse relationship, nasal congestion following the provocation of intrapulmonary airway narrowing, has been described (Yap & Pride 1994). Whether these interactions can lead to sustained airway narrowing or nasal congestion and whether they are exaggerated by chronic nasal or bronchial disease is unknown.

Pharynx, glottis and trachea Detailed studies of resistance during air and helium/ oxygen breathing suggest involvement of the glottis and/or

Table 33.1 Contributions of nasal resistance to total respiratory resistance during tidal breathing. (Data from Duggan et al. 2004.)

Total respiratory resistance (cmH2O/L per s) Mouth breathing (m)

Nose breathing (n)

Nasal airway resistance (n-m) (cmH2O/L per s)

Normal subjects (N = 10) Sitting 2.2 (0.1) Supine 3.0 (0.1)

4.2 (0.4) 5.7 (0.4)

2.1 2.7

Asthma alone (N = 7) Sitting 3.8 (0.5) Supine 4.6 (0.6)

6.7 (0.6) 8.2 (0.3)

2.9 3.7

7.6 (1.4) 11.2 (1.9)

3.9 5.8

Asthma with nasal symptoms (N = 10) Sitting 3.7 (0.2) Supine 5.4 (0.3)

Values are mean (SE). Resistance was measured by forced oscillation at 6 Hz at the airway opening.

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extrathoracic trachea is present in a significant proportion of asthmatic attacks (Lisboa et al. 1980). Expiratory narrowing of the glottis and supraglottic airway can be visualized during provoked bronchoconstriction in asthmatic subjects and may play a physiologic role in “braking” expiration and increasing FRC. The airway obstruction of asthma has to be distinguished from structural or functional obstruction of the extrathoracic airways which may cause similar symptoms (Bucca et al. 1995). Structural narrowing results in distinctive changes in maximum expiratory and inspiratory flow–volume curves (Miller & Hyatt 1973) (see Fig. 33.3). Functional obstruction may mimic acute asthma and because of its variability be more difficult to diagnose.

Vocal cord dysfunction Vocal cord dysfunction (VCD) is defined as paradoxical adduction of the vocal cords during inspiration or expiration or both. This causes airflow obstruction, obvious wheezing, and breathlessness. VCD frequently coexists with, and may be clinically indistinguishable from, asthma, with a spectrum from mild breathlessness to respiratory distress leading to intubation or tracheostomy (Newman et al. 1995). There may be accompanying hyperventilation, which could itself act as a bronchoconstrictor. There is often considerable shortterm variability which makes it difficult to obtain consistent measurements of lung function but this may assist the diagnosis. Typically, tidal flow–volume curves show that breathing is at very small lung volumes close to RV, while MEFV and MIFV curves when wheezing is present show reduction in MIF, with an MEF50/MIF50 ratio > 2.0. Inspiratory Raw may be greatly increased during panting in the body plethysmograph (Vlahakis et al. 2002). Bronchial challenge may be negative; if there is accompanying asthma, the level of responsiveness may be mild in relation to the prominence of symptoms. When VCD is the sole cause of wheeze, evidence of ventilatory inhomogeneity, so typical of even mild asthma, is absent. The wheeze characteristically disappears when the patient sleeps or is anesthetized but its pathogenesis, and whether psychopathologic factors are involved, is uncertain. An excessive response to such recognized stimuli as gastroesophageal reflux, postnasal drip, or inhaled irritants or autonomic imbalance (Ayres & Gabbott 2002) have all been proposed.

Bronchial blood flow Increase in bronchial blood flow in asthma has received relatively little attention because of the invasive methods required for measurement. Angiogenesis and vascular remodeling with increased vascularity (a hallmark of inflammation) have increasingly been recognized in asthma (Salvato 2001; Vignola et al. 2003). Recently a more rapid exponential rise in exhaled breath temperature (Δ°C/s) during a flow-


and pressure-controlled single exhalation in asthmatics compared to normal subjects (Paredi et al. 2002) was correlated with exhaled nitric oxide (NO). This was confirmed in a follow-up study (Paredi et al. 2005) with a faster rise of exhaled breath temperature (7.27 ± 0.6 in asthmatics vs. 4.23 ± 0.4 Δ°C/s in normal subjects). Furthermore, bronchial blood flow (Qaw) using the Fick principle and measuring the soluble gas acetylene with online mass spectrometry was elevated in mild persistent asthma (46.0 ± 51 vs. 31.6 ± 1.6 μL/mL per min in normal subjects). Qaw was weakly correlated with Δ°C/s (r = 0.52) and exhaled NO (r = 0.57). As measured, Qaw includes tracheal blood flow and dead space effects but is similar to other techniques that have previously been validated (Scuri et al. 1995). The effect of asthma drugs on Qaw has been studied: the inhaled steroid budesonide produced an approximate 60% reduction from 30–60 min in asthmatics while Qaw was increased by salbutamol about 67% in normal controls but not in asthmatics. Nonsignificant changes in Δ°C/s were seen after budesonide in asthmatics and normals, while in five normal subjects (but not in asthmatics) salbutamol significantly increased Δ°C/s. These findings are in line with older work in sheep and the established vasoconstrictor and vasodilator pharmacology of these agents. Furthermore, these recent results confirm others in asthma (Brieva et al. 2000; Horvath et al. 2007) suggesting that they may be of clinical significance. Increased bronchial blood flow in asthma probably contributes to increased heat transfer across the airway walls, accounting for the faster rise in breath temperature on exhalation. These may represent novel noninvasive markers of airway inflammation, potentially linked to the elevated levels of the potent endogenous vasodilator NO, found in asthma.

References Aggarwal, A.N., Gupta, D., Kumar, V. & Jindal, S.K. (2002) Assessment of diurnal variability of peak expiratory flow in stable asthmatics. J Asthma 39, 487–91. Altes, T.A., Powers, P.L., Knight-Scott, J. et al. (2001) Hyperpolarized 3 He MR lung ventilation imaging in patients with asthma: preliminary findings. J Magn Reson Imaging 13, 378–84. An, S.S., Bai, T.R., Bates, J.H.T. et al. (2007) Airway smooth muscle dynamics: a final common pathway of airway obstruction in asthma. Eur Respir J 29, 834–60. Antic, R. & Macklem, P.T. (1976) The influence of clinical factors on site of airway obstruction in asthma. Am Rev Respir Dis 114, 851–9. Aubier, M., Wettenger, R. & Gans, S.J. (2001) Efficacy of HFAbeclomethasone dipropionate extra-fine aerosol (800 μg/day) versus HFA-fluticasone propionate (1000 μg/day) in patients with asthma. Respir Med 95, 212–20. Awadh, N., Muller, N.L., Park, C.S., Abboud, R.T. & FitzGerald, J.M. (1998) Airway wall thickness in patients with near fatal asthma with or without fixed airflow obstruction. Thorax 53, 248–253.

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Aerosol Delivery Systems Thomas G. O’Riordan and Gerald C. Smaldone

Summary

Introduction

The inhaled route of administration is used for bronchodilator therapy for asthma as well as for delivery of antiinflammatory medications for allergic rhinitis and asthma. For most patients with asthma, pressurized metered-dose inhalers (pMDIs) and dry powder inhaler (DPI) devices are used for maintenance therapy but nebulizers are useful in young children and critically ill patients. Chlorofluorocarbons (CFCs) have been used as propellants in MDIs for 50 years but are now being replaced by hydrofluoralkane propellants in pMDIs because of concerns about the impact of CFCs on the ozone layer. Dry powder inhaler technology has been enhanced in recent years and modern DPIs produce aerosols of comparable quality and reproducibility as pMDIs. Aerosol quality control is important if the aerosolized medications are to be delivered in predictable quantities to the lower airways, as small changes in aerosol particle size or velocity can significantly alter lung deposition. In addition, breathing pattern and changes in airway anatomy due to disease can affect not only the quantity of medication delivered to the lung, but also its regional distribution within the lung (e.g., airway vs. alveolar deposition). Most aerosol delivery systems in current use cause significant extrapulmonary deposition of medication in the pharynx and can result in local irritation. Once extrapulmonary-deposited medication has been swallowed, it has the potential to be absorbed and cause systemic adverse events. Inhaled formulations of corticosteroids (ICS) have been developed to reduce this oral bioavailability and some preparations of ICS have negligible oral bioavailability. However it should be remembered that systemic exposure to ICS can also occur when medication that deposits in the lungs is absorbed. The delivery of aerosolized medications to children poses additional challenges. Masks used to facilitate the use of nebulizers or MDIs can also alter patterns of drug delivery. Nasal formulations of corticosteroids using aqueous suspensions or solutions and larger particles are effective at treating rhinitis.

Most medications used to treat asthma are delivered by the aerosol route. The rationale for therapeutic preference is that delivering a drug to the target organ will increase medication levels at the airway while reducing reduce systemic exposure. This assumption is on the whole valid but there are complicating factors that are the focus of this chapter. The human respiratory tract has evolved in a manner that makes it difficult for inhaled particles to enter the distal airspaces, thus protecting these delicate structures from the many noxious and infectious particles present in the atmosphere. The design of inhaled therapies must take these functional barriers to aerosol penetration into account. However, the principles of aerosol deposition and clearance apply not only to therapeutic aerosols but also to studies of how inhaled allergens, irritants, and infections also enter the lung.


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Definition and description of an aerosol An aerosol can be defined as a system of solid particles or liquid droplets that can remain dispersed in a gas, usually air (Agnew 1984). Because the aerodynamic behavior of an aerosolized particle is critically influenced by its mass, it is important to be able to precisely describe the size distribution of aerosolized particles. In clinical studies, the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (σg) are often used to characterize the dimensions of an aerosol. The MMAD represents the point in the distribution above which 50% of the mass resides, expressed as the diameter of a unit density (1 g/mL) sphere having the same terminal settling velocity as the aerosol particle in question, regardless of its shape and density. The σg is the ratio of the size at 84% (or 16%) to the MMAD and is an indicator of the variability in particle diameters (one geometric standard deviation, if the distribution is log-normal). If the particle size varies over a wide range (σg > 1.2), it is described as having a polydisperse particle distribution. Pharmaceutical aerosols are polydisperse. If all the

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particles in an aerosol are of similar size (σg < 1.2), the particle distribution is described as monodisperse. Monodisperse aerosols are usually only encountered in research studies where specialized generators are used to create such aerosols (Stahlhofen et al. 1980; Smaldone et al. 1983)

Aerodynamic behavior of aerosols Mechanisms of deposition in human subjects Therapeutic aerosols, with diameters of 0.5–10 μm, deposit in the lung by inertial impaction and gravitational sedimentation. Inertial impaction occurs when an inhaled particle is not able to follow the air stream and impacts into the airway wall usually at airway bifurcations. Larger particles traveling at high velocities are more likely to impact in the upper airway than smaller particles at slower velocities. Particles less than 5 μm can also settle by gravitational sedimentation in more distal airways. Deposition by sedimentation is critically time dependent, with breath-holding for example significantly increasing the number of particles deposited (Bennett 1991). In healthy subjects, both the regional distribution as well as the absolute number of inhaled particles that deposit in the lung are primarily determined by the size of the particles and by the breathing pattern. Conversely, in disease states, the geometry of the airways and changes in regional ventilation may be the dominant influences (Smaldone & Messina 1985a & b).

Strategies to optimize deposition of therapeutic aerosols

combined with particle inertial effects and the geometry of the oropharynx result in upper airway deposition ranging from 30 to 90% of the total deposition in the patient for typical commercial delivery systems. Only nebulizers producing particles with particularly small MMAD (e.g., AeroTech II, MMAD 1.0 μm) bypass the upper airways (5% oropharyngeal deposition in adults) (Smaldone et al. 1998a). In children, the smaller oropharyngeal airways make the task more difficult (Diot et al. 1997). Thus, the demands placed on traditional devices can be overwhelming if the drug must efficiently bypass the upper airways.

Control of breathing pattern and aerosol deposition In normal subjects, the pattern of breathing is the most important factor affecting aerosol delivery and deposition. How the patient breathes affects the device (Smaldone et al. 1991), the penetration of particles past the oropharynx, and deposition within the parenchyma. Earlier studies suggested that much of the variation in parenchymal deposition was related to differences in airway geometry between subjects. However, variability in deposition between subjects appears well controlled if the pattern of breathing is controlled (Bennett & Smaldone 1987). Figure 34.1 depicts data from 11 subjects inhaling 2.6–μm monodisperse particles. The fraction of inhaled particles depositing in the lung (DF) can be closely related to the period of breathing. In simplified form, points near the origin of the horizontal axis represent normal tidal volumes and frequencies. As tidal volume increases and breathing frequency decreases, the time of inspiration is prolonged

The determinants of aerosol deposition apply to both therapeutic and environmental inhaled particles. To optimize deposition of therapeutic aerosols, a number of strategies have been developed based on these principles.

0.6

Getting particles past the oropharynx

0.4

In clinical studies, predicting penetration of aerosol beyond the oropharynx and subsequent lung deposition is a major criterion for device selection. Deposition estimations are often based on particle size measurements defined by in vitro characterization of the aerosol produced by a given device. During tidal breathing, most investigators would expect aerosols below 5 μm to be deposited primarily in the lungs (the fine particle fraction or FPF) (Smaldone & Messina 1985a & b; Smaldone et al. 1998a). However, in a recent clinical study, we found that the so-called FPF for wet nebulizers might be closer to 2.5 μm (Sangwan et al. 2003). Our data for wet nebulizers was similar to that for other traditional devices when data defining FPF were critically analyzed. Most of the reported data from previous studies as well as our data were obtained in patients receiving their aerosol during tidal breathing, rapid inhalation, or metered-dose inhaler (MDI) aerosol delivery. In all cases particle size effects

Aerosol Delivery Systems

DF

0.5 SLOW & DEEP

0.3 TIDAL BREATHING 0.2 Controlled breathing measurements in one subject Spontaneous breathing measurements in each of 10 patients

0.1 0.0 0

5

10

15

VT/f ′2 20

Fig. 34.1 Deposition (defined as deposition fraction or DF, the fraction of particles inhaled that are deposited) of 2.6-mm monodisperse particles versus breathing pattern. Filled circles, data from one subject during controlled breathing; open circles, data from 10 patients during spontaneous breathing. Breathing pattern was defined by a relationship that represents a measure of the period of breathing (tidal volume divided by breathing frequency squared or VT/f ′2). Typical “tidal breathing” parameters found near origin, “slow and deep” inspirations found away from the origin. (From Bennett & Smaldone 1987, with permission.)

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10 L/s L 2 (a)

GD

WM

RH

KS

ON

5 L/s L 1 (b)

SS

AT

RP

BH

HM

HS

Fig. 34.2 Tracings of maximal flow and tidal flow vs. volume. (a) Normal subjects. (b) Patients with severe obstructive lung disease. Note the change in the sensitivity of the axes (ordinate: flow range from 0 to 10 or 0–5 L/s; abscissa: volume range from 0 to 2 or 0–1 L respectively). The patients’ tidal loops are superimposed on maximal flow–volume curves. (From Smaldone et al. 1993, with permission.)

(e.g., slow and deep inspiration). The curve depicted in Fig. 34.1 represents maximum deposition with a slow and deep inspiration for particles of 2.6 μm. The curve would be shifted upwards with deposition approaching 100% for larger particles.

Expiration and problems with aerosol deposition In normal subjects, those particles that do not deposit during inspiration are largely exhaled completely. Particles that pass through the oropharynx during inspiration enter the central airways and traverse them without difficulty because lobar and segmental bronchi are generally widely patent during inspiration. Then the particles enter alveoli with a few depositing by sedimentation and, like cigarette smoke, the bulk of the aerosol begins to be exhaled. Deposition is controlled by sedimentation in small airways, influenced by local geo-

(a)

770

(b)

metry and to a strong degree by the residence time (period of breathing). In obstructive lung disease, maximal expiratory flows are diminished. With moderate disease, maximal flows can be superimposed on tidal breathing, and as the disease progresses maximal flows can be reduced even further (Smaldone 1997) Therefore, it is common to observe that patients are often breathing on their maximal expiratory flow–volume curves even during quiet tidal breathing (Fig. 34.2) (Smaldone et al. 1993). In these patients, flow-limiting segments exist in the same airways found in normal subjects during forced expiration but, for these patients, they form during every tidal breath. In patients with obstructive lung disease the promotion of deposition in the peripheral lung poses a significant challenge as deposition of aerosol is enhanced during expiration at sites of flow limitation (Fig. 34.3) (Smaldone & Messina 1985b; Smaldone 2001). Based on these physiologic considerations peripheral deposition of aerosol in these subjects would be favored using a system that provides a slow prolonged inspiration (to promote deposition by settling) with a breath long enough to minimize the particles available to the airways during expiration.

Slow and deep breathing For many aerosol applications inhaling particles slowly and deeply solves the problems outlined above. A slow inhalation will minimize oropharyngeal deposition. “Slow” inspiration reduces particle inertia and allows inhalation of particles larger than the 2.5–μm cutoff for the FPF that defines deposition in the oropharynx during tidal breathing. This concept has been exploited in physiologic studies of mucociliary clearance in the distal airways utilizing particles as large as 6 μm (Anderson et al. 1995). More recently commercial applications of these principles have combined slow and deep inspiration with direct mechanical feedback to the patient for particles of relatively large MMAD. This is discussed in more detail in a later section.

Fig. 34.3 (a) Sites of flow-limiting segments in humans. (b) Corresponding deposition image in patient with severe chronic obstructive pulmonary disease (posterior view) with maximal expiratory flow–volume curve superimposed on tidal loop (as shown for patients in lower part of Fig. 34.2). Particle deposition occurs in sites of flow-limiting segments. (From Smaldone 2001, with permission.)

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Interface between device and patient and drug delivery In spontaneously breathing adult patients, during quiet tidal breathing, aerosol is inhaled via a mouthpiece. In young children and adults with coordination problems, the situation is more complex as the patient is often interfaced to the device via a facemask and the impact of a facemask on deposition is discussed in a later section.

Measurements of particle size There are two principal approaches to the measurement of particle size: cascade impaction and the use of methods (Baron 2001) based on light scattering. We briefly discuss both of these methods and the limitations of in vitro measurements when extrapolated to the in vivo setting.

Cascade impaction A cascade impactor consists of a series of stages with differentsized orifices that fix local linear velocities of the carrier gas of the aerosol as it passes through the device. After particles pass through the orifice of a given stage, they must turn at 90° around an obstruction or “baffle” or impact on the stage. The ability of a particle to negotiate the baffles downstream from each orifice is a function of the aerodynamic particle diameter (Baron 2001).

Light scattering The scattering of light can be used to measure particle size and has been reviewed elsewhere (Baron 2001). Originally developed to measure monodisperse aerosols, newer devices can measure polydisperse aerosols. Small particles produced by a pressurized MDI (pMDI), whose dimensions are measured by a light-scattering device, may have high velocity and can thus have the same inertial properties as larger particles at slower velocities.

deposition within the lungs. The latter measurement is performed in vivo and is time-consuming, costly, and involves some degree of risk and uncertainty to the patient. The other components of the aerosol delivery process can be well characterized and studied in vitro. The field of aerosol delivery has advanced significantly in the last 10 years such that device characteristics and aerosol behavior can be significantly optimized on the bench before exposure to patients (O’Riordan et al. 1994)

Inhaled mass Figure 34.4 depicts a simple in vitro set-up for measuring the quantity of aerosol produced by a nebulizer (Smaldone 1991). This system does not require an understanding of nebulizer function from first principles. An absolute filter (i.e., a filter that captures all the particles in an aerosol) has replaced the mouthpiece. Because the nebulizer is attached to a breathing device (Harvard pump, Harvard Apparatus, South Natick, MA), the conditions of delivery such as routine tidal breathing can be duplicated. The quantity of drug captured on the filter represents the amount that passes the lips of the patient. To distinguish this quantity from a “dose” or deposited drug the term “inhaled mass” has been coined and thus the filter can be called the inhaled mass filter. The inhaled mass represents “delivery” of drug to the patient, constrained by conditions that should mimic actual clinical delivery.

Aerosol A cascade impactor can be inserted into the circuit depicted in Fig. 34.4 and provide information regarding the aerodynamic distribution of a given aerosol (MMAD and σg). Depending on circumstances, knowledge of MMAD and σg can predict the behavior of particles in the lungs. Figure 34.5

Inhaled mass filter

Expiration

Application of in vitro measurements of particle size to clinical studies Particle size data obtained with different techniques are not necessarily interchangeable. Meaningful comparisons of the sizes of clinical aerosols, should be compared only if obtained with identical techniques. Nevertheless, despite the technical difficulties encountered in measuring the size of polydisperse clinical aerosols, some investigators have established that when used with appropriate caution, in vitro measurement of particle size does provide useful predictive data for subsequent clinical studies (Newman 1996).

Principles of assessment of aerosol delivery systems Assessing effects of an aerosolized drug requires the understanding of three major factors: the aerosol delivery system, the quality of the aerosol produced, and the quantification of

Inspiration

1 2 3 4 5 6

Compressed air

7 8

Harvard pump 10-stage cascade impactor

9 10

Fig. 34.4 Technique for bench measurements of inhaled mass and particle distribution: breathing pattern defined by settings on Harvard pump. Particles presented to “patient” are captured on the inhaled mass filter. In separate experiments, the cascade impactor measures inspired aerosol. (From Smaldone 1991, with permission.)

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Fig. 34.5 Deposition scans of MistyNeb (top) and AeroEclipse (bottom) for three subjects 1, 2 and 3 (left to right) receiving aerosolized interferon-g for tuberculosis. Subject 2 has only the right lung. The lung outlines (133Xe equilibrium scan, central, peripheral regions and horizontal line separates upper from lower lung regions) and stomach outlines for patients 1 and 2 are shown. Deposition in the oropharynx is reduced with AeroEclipse. (From Sangwan et al. 2003, with permission.)

depicts deposition images for three subjects following inhalation of interferon (IFN)-γ aerosols generation by MistyNeb (Allegiance, McGraw Park, IL) and AeroEclipse (Trudell Medical International, Canada) nebulizers. All three patients show an increased deposition in the lungs for the AeroEclipse nebulizer as evidenced by increased activity in the lung fields with reduced deposition in the oropharynx (reduced stomach activity). For MistyNeb, lung deposition varied from 28 to 32% (mean ± SD, 30.9 ± 0.03%) of the total aerosol deposited in the patients. AeroEclipse deposited 59– 73% (68.1 ± 0.08%) of total deposition (Sangwan et al. 2003). In Table 34.1 the distribution of particles between the oropharynx or upper airways and the deep lung are shown in bold for both devices. The aerodynamic distributions from cascade impaction measurements for the different nebulizers are also listed. By

Table 34.1 In vitro assessment of interferon-g aerosol. Aerosol distribution determined by cascade impaction listed for the test nebulizers. All data in this table are from the in vitro cascade studies but bold numbers represent the cascade stages representing mean lung deposition (see text) measured from deposition images in vivo for the three subjects. (From Sangwan et al. 2003, with permission.) Nebulizer

Misty-Neb

AeroEclipse

MMAD (mm) Particles < 6 mm Particles < 3 mm Particles < 2 mm

3.10 55% 49% 30%

2.20 77% 73% 53%

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inspection, one can see that a cutoff of approximately 2.5 μm defines particles that pass the oropharynx and deposit in the lung. For the Misty-Neb approximately 30% of the particles are 2.5 μm or less and this corresponds to the lung depositions seen in the images for the three patients studied. For the AeroEclipse, which produces a smaller particle distribution, the 2.5–μm cutoff predicts that approximately 70% of the particles will deposit in the lung versus the upper airways. Our results are a strong function of the design and control of the experimental set-up. However, for certain devices, knowledge of the MMAD measured on the bench can assist in the design of appropriate aerosol delivery systems prior to in vivo testing.

Deposition The term “deposition” begins to imply a “dose” to the patient. The term deposition needs to be further refined in a given situation, for example oropharyngeal versus parenchymal deposition, or central versus peripheral deposition within the lung. Each of these terms may be important depending on the disease entity to be treated. Obviously the measurement of deposition requires an in vivo experiment. However, deposition can be related to parameters that are measured in vitro as shown in equation (1): Deposition = aerosol inhaled − aerosol exhaled

(1)

Because the term “aerosol” is a little vague with respect to drug activity, equation (1) can be rewritten as: Deposition = inhaled mass − exhaled mass

(2)

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(a)

Fig. 34.6 In vitro set-up: (a) constant flow experiment and (b) breathing simulator for each inhalation device (nebulizer, valved holding chamber). The connection with the flow-generating apparatus was made with a flat plate (sealed configuration) or a face (face configuration). (From Smaldone et al. 2005, with permission.)


Plate (sealed) or face (not-sealed)

Gast vacuum pump

VHC + MDI or nebulizer + compressor

Inhaled mass filter

Computer with control card (b)

MIMIC breathing emulator

Many experiments can be performed on the bench to define the parameters that define the inhaled mass for different devices and experimental conditions (Smaldone 1991).

Measurement of inhaled mass Pediatric in vitro models Figure 34.6 represents a more complex situation than that of Fig. 34.4. An MDI and a valved holding chamber (VHC) are connected in series. For many patients the VHC utilizes a mouthpiece, but younger patients require a facemask. Measurement of inhaled mass requires a strategically placed filter. Reported studies to date place the inhaled mass filter on the VHC and capture particles using a suction device or a breathing machine (Fig. 34.6a). However, the principles illustrated above require the inhaled mass filter to mimic par-

ticles that actually pass the lips. To complete the in vitro model, therefore, the facemask must be placed on a face. Finally, the face must “breathe” with a breathing pattern that is representative of the patient population to be treated. As depicted in Fig. 34.6b, the correct filter location is shown between the breathing emulator and the face facsimile. Thus, all the components of the aerosol delivery system including the MDI, VHC, facemask, and pattern of breathing will be reflected in the measured inhaled mass. Table 34.2 illustrates summary data from a recent study comparing MDIs and nebulizers using patterns of breathing appropriate for pediatric patients using facemasks (Smaldone et al. 2005). The effects of the breathing pattern, the VHC employed, the facemask and different nebulizers were tested. VHCs were also washed with detergent to eliminate static charge. The influence of VHC

Table 34.2 Inhaled mass (percent of label dose) (mean ± SD) as a function of breathing pattern, VHC condition, and presence of facemask for budesonide (nebulizers) and fluticasone propionate (pMDI VHC). (From Smaldone et al. 2005, with permission.) VT Nebulizer (drug) Hudson Updraft II (budesonide) Pari LC plus (budesonide)

VHC (drug) pMDI AeroChamber (fluticasone) pMDI OptiChamber (fluticasone)

Sealed configuration

24.3 ± 3.1 9.6 ± 0.7 18.7 ± 1.9 10.0 ± 1.1

207 75 207 75

207 75 207 75

Face configuraton

Unwashed 7.2 ± 2.0a 2.9 ± 1.5a 7.7 ± 1.6a 0.7 ± 0.5a

19.3 ± 2.3 4.1 ± 0.8 11.7 ± 1.1 9.2 ± 1.9 Washed 53.3 ± 6.2b 30.5 ± 3.2b 50.2 ± 1.2b 27.2 ± 1.4b

Unwashed 2.4 ± 0.7a 3.1 ± 2.4a 2.9 ± 0.3a 1.0 ± 0.2a

Washed 13.6 ± 2.7b 4.7 ± 0.7b 28.6 ± 2.5b 4.0 ± 1.6b

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(a)

(b)

conditioning combined with effects of breathing pattern resulted in inhaled mass ranging from 0.7 ± 0.5 to 53.3 ± 6.2% (mean ± SD). Nebulizers were less variable (9.6 ± 0.7 to 24.3 ± 3.1%). Detergent coating of VHC markedly increased inhaled mass and reproducibility of drug delivery (27.2 ± 1.4 to 53.3 ± 6.2%) for pMDI/VHC combinations but these effects were lost in the presence of facemasks. Using pediatric patterns of breathing, nebulizer/facemask combinations delivered 4.1 ± 0.8 to 19.3 ± 2.3% of the labeled dose, while pMDI and detergent-coated VHC delivered 4.0 ± 1.6 to 28.6 ± 2.5%. Facemask seal was a key factor in drug delivery. Leaks around the facemask reduced drug delivery and for pMDI/VHCs negated effects of detergent coating (Smaldone et al. 2005).

Fig. 34.7 (a) Preferential eye deposition with Laerdal and Pari LC Plus nebulizer (b) Diffuse facial pattern with Salter mask and AeroTech II nebulizer. (From Sangwan et al. 2004, with permission.)

leaks near the bridge of the nose is reduced. An example is shown in Fig. 34.8, which consists of a deposition image created under the same conditions as for Fig. 34.7a using a modified mask design. Deposition on the eyes and face was markedly reduced, while drug delivery to the patient (inhaled mass) was preserved (Smaldone 2004).

Facemasks and facial deposition For pMDI/VHC, leaks around the facemask limit the exchange of tidal air with air in the VHC, reducing the inhalation of aerosol by the patient. For nebulizers operated with compressors the facemask can be kept filled with particles in spite of leaks because the compressor flow can exceed the minute ventilation of the child. However, the very leaks that may preserve delivery to nebulizers result in deposition of drug on the face and in the eyes. Figure 34.7 demonstrates characteristic deposition patterns following nebulizer therapy on the face using a pediatric model of aerosol delivery with a tightly fitted facemask (left) and a straight-in nebulizer and a commercially fitted mask with a straight-up nebulizer (right) (Sangwan et al. 2004). Recent experiments have indicated that deposition on the face and particularly in the eyes can be minimized if masks are designed such that linear velocity in the region of the

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Fig. 34.8 Reduced facial and eye deposition with Pari nebulizer and prototype facemask designed to reduce particle acceleration in the region of the eyes. (From Smaldone 2004, with permission.)

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Fig. 34.9 Lung images from seven patients following inhalation of 99mTc-HSA-labeled cyclosporin A aerosol. n indicates native lungs (see text). There is considerable variation in regional particle deposition. (From Smaldone 2005, with permission.)

Deposition and dose versus response

50 40 ΔFEV1% Day 0

For conventional bronchodilators and steroids, the dose and response are not critical for clinical efficacy because most delivery systems provide drug to the patient on the flat portion of the dose–response curve. Safety of most preparations is enhanced because of the high potency of most drugs and a high threshold for toxicity. The situation for bronchodilators and steroids may not carry over to newer drugs. For example, aerosolized cyclosporin A used in the treatment of lung transplant rejection has recently been shown to significantly reduce mortality. In vivo measurements of deposition have been related to clinical effects (dose vs. response). For example, Fig. 34.9 shows gamma camera images of individual patients from an early study assessing the effects of inhalation of aerosolized cyclosporin A aerosol (Iacono et al. 1997). Analysis of cyclosporin A deposition was related to clinical effects as shown in Fig. 34.10. After institution of aerosolized cyclosporin A therapy in patients with persistent acute rejection, pulmonary function improved (after 3 months of therapy) as illustrated by increases in forced expiratory volume in 1 s (FEV1). However, there was a suggestion of a dose–response relationship, with those patients receiving less than 20 mg of drug per allograft having minimal response. A similar situation will exist for systemically absorbed drugs such as insulin where the dose to the lung parenchyma will be critical in

30 20 10 0 –10 0

10

20

30

40

CSA deposition in transplanted lung(s) Fig. 34.10 Changes in spirometry assessed by change in FEV1 (ΔFEV1 Day 0) from the initiation of aerosol cyclosporin A (CSA) to approximately 200 days of therapy, as a function of cyclosporin A deposited in transplanted lung(s). (From Iacono et al. 1997, with permission.)

patient management. While clinical studies have shown that aerosolized cyclosporin A appears to be effective overall in a population of patients, data from the individual patients shown in Fig. 34.10 suggest that conventional aerosol delivery systems leave some patients at risk for inadequate dosing, while others may be overdosed and exposed to potential toxicity.

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Generation of aerosols

Oropharynx

Oropharynx Rondo

To generate an aerosol, energy must be applied to solid or liquid material in order to disperse the material and cause it to be suspended in a carrier gas which, for the purpose of this chapter, will nearly always be air. Lungs

Pressurized metered-dose inhalers pMDIs consist of a pressurized metal can that contains a mixture of propellants (freon) which, when in equilibrium, are both in liquid and gaseous form at room temperature (Morâen 1993). Suspended in the liquid, the agonist usually consists of solid particles that have been milled into an aerodynamically respirable distribution of diameters. In addition to the medication and propellant, many preparations contain a surfactant or dispersal agent such as lecithin or oleic acid because surface tension affects aerosol behavior. In addition, ethanol is used to enhance the solubility of some formulations. This two-phase suspension is evenly distributed by shaking and then a “metered dose” is released into the atmosphere via the valving system when the canister is compressed and triggered. The metering valve carefully controls the volume of liquid released into the atmosphere. At that point, the freon instantly evaporates, imparting a relatively high kinetic energy to the solid particles. Thus, the metering valve provides precise control of the triggered amount of drug. The volume of drug released per actuation is a function of the size of the metering valve. As the metering volume is increased, a greater amount of each spray will be deposited on the actuator mouthpiece. In addition, however, the proportion of particles depositing in the central airways relative to the lung periphery increases proportionate to metering volume. In contrast, warming of the MDI canister has been reported to increase drug deposition in the distal parts of the lung. An increase in concentration of propellant loaded into the canister elicits a finer aerosol at a higher ejection velocity (as discussed earlier, a high ejection velocity will increase the inertia of the particle with important clinical consequences). The milled size of the drug powder will be another limiting factor determining the particle size of the MDI aerosol: the coarser the drug powder, the larger the aerosol. If the pMDI is actuated within 60 s of a prior actuation, the dose of drug released may decrease significantly. Concern about the effects of subtle changes in the formulation of pMDI medications on aerosol delivery has led regulatory agencies to demand strict requirements that the manufacturers of generic formulations document clinical “equivalence” with existing products (Food & Drug Administration 1998).

Use of accessory devices with pMDIs There are practical difficulties encountered when administering drugs by pMDI. There is a need to coordinate actuation

776

Stomach

Fig. 34.11 Influence of intrinsic particle inertia on deposition pattern. (Left) A patient inhaling particles generated by a metered-dose inhaler without a spacer. (Right) The same patient inhaling the same aerosol but after modification by a Rondo spacer device. (From Newman et al. 1991, with permission.)

with respiration. Even with the use of optimal inhaler technique, significant extrapulmonary deposition occurs because while the particles are of respirable diameters, there is high inertia. Recognition of these problems has led to important attempts to modify the behavior of the aerosol as it is generated. Spacer devices have had a significant influence on drug delivery. As shown in the deposition study illustrated in Fig. 34.11, the spacer device absorbs those particles that have high inertia and which would, in the absence of the spacer, be likely to deposit on the pharynx and larynx (Newman et al. 1991). Significant losses occur in the spacer as particles impact on the walls but the available data in human studies indicate that the parenchymal deposition of drug within the lung is similar with and without the spacer, resulting in decreased exposure of the larynx and pharynx to the inhaled agonist. In addition some spacer devices have been designed to provide coordination control for the patient. Rather than relying on the patient to inhale at the instant of MDI triggering, the spacer serves as a reservoir from which particles remaining in the gas phase can be inhaled more conveniently. The presence of one-way valves within some spacer devices facilitates this maneuver (hence the term “valved holding chamber”; see below). In summary, the use of spacer devices reduces extrapulmonary deposition. This is especially important in preventing toxicity when doses of inhaled medications in excess of the usual maintenance doses are used. Pharyngeal deposition of high doses of inhaled corticosteroid agents, which are designed to be poorly absorbed, can cause local toxicity such as pharyngitis and Candida infection. Pharyngeal deposition of high doses of inhaled β-sympathetic agonists can cause systemic toxicity because these agents are swallowed

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and absorbed through the gastrointestinal tract. Controversy exists as to whether the use of spacer devices improves airway deposition of inhaled medications in situations where optimum inhaler technique is used. However, because patients’ pMDI techniques in “real world” clinical practice are frequently suboptimal, the use of spacers most likely does result in increased airway drug deposition for many patients (Moren 1982). Commercial holding chambers vary in design, size, and construction materials. Some are designed for use with only one drug formulation but most are marketed to be used with most MDIs. The simplest chambers are simple tubes, which should be at least 10 cm in length and 3 cm in diameter (Nelson & Loffert 1994; Newman & Newhouse 1996). These devices decrease oropharyngeal deposition and compensate in part for delay in inhalation after actuation of the device. If the patient exhales during actuation, simple tube spacers are not effective. While chambers of 150 mL volume are efficient, larger chambers up to 750 mL may provide enhanced drug delivery, albeit at a cost of reduced portability and convenience. The effects of poor patient coordination can be reduced by the addition of one-way valves (VHC) that prevent patients from exhaling into the device. A variety of valve designs have been patented. For portability, spacers and VHC may be collapsible. In addition, some devices provide sonic feedback if the patient’s inhalation flow rate is too rapid. It has also been noted that electrostatic interactions between aerosol and plastic chambers may reduce drug delivery (Wildhaber et al. 1996). As a result some manufacturers now include more detailed cleaning instructions (i.e., wash plastic chamber with detergent and leave to dry without wiping) while others have produced chambers that have less electrostatic interactions.

(Goldin et al. 1999): the development of an aerosol with both different aerosol properties and clinical effects (Busse et al. 1999). Compared with the CFC formulation, HFA BDP particles are significantly smaller in size, resulting in reduced extrapulmonary deposition in the oropharynx (which is universally accepted to be an advantage). On the other hand, penetration of the aerosol into the peripheral regions of the lung in enhanced. The benefits of the latter are more controversial and are discussed below. The pace of introduction of new HFA products and dry powder inhalers (DPIs) has been slower in the USA than in Europe, due in part to stricter regulatory requirements in the USA. The US Food and Drug Administration has been reluctant to approve products that do not meet the quality standards that apply to existing CFC formulations in terms of fine particle mass (dose in particles < 5 μm in diameter), dose-to-dose variability, and stability of the formulation over a 12–month storage time (Food & Drug Administration 1998). In addition, extensive clinical programs have been required for regulatory approval of these products because in vitro aerosol measurements are not viewed as acceptable surrogates for clinical data. The changeover from CFC to HFA has therefore been costly. Producing new HFA formulations equivalent in clinical efficacy to CFC formulations is difficult on a cost-of-goods basis when compared with the manufacture of generic CFC formulations. Governments will therefore consider banning the sale of generic CFC products when reliable supplies of the HFA versions of these products become available. Therefore one effect of the initiative to eliminate CFCs will be increased costs to consumers and insurers because of the extension of patent protection to these new HFA formulations.

Reformulation pMDIs without chlorofluorocarbons

Dry powder inhaler devices

Environmental pressures have led to the development of aerosol dispensers free of chlorofluorocarbons (CFCs), the production of which ceased in 1996 (although manufacture of therapeutic aerosols with CFC continues from reserved supplies). Such research has centered on dry powder systems (see below) and pMDIs free of CFCs. For example, the hydrofluoralkane (HFA) 1,1,1,2–tetrafluorethane (HFA 134a) is a propellant that does not affect the ozone layer and is found in the majority of reformulated pMDIs. All existing CFC MDIs will be replaced by HFA and/or dry powder formulations. As a result of this ruling clinical trials for the purpose of registering new products no longer involve CFC formulations. In replacing CFC formulations with HFA formulations, the pharmaceutical industry has adopted two strategies. The first replaces existing CFC formulations with HFA products designed to be clinically equivalent to each other. For example, Proventil (albuterol) HFA and Proventil CFC are very similar in terms of their in vitro aerosol characteristics and clinical effects. The reformulation of beclomethasone dipropionate (BDP) from CFC to HFA has involved a different strategy

Dry powder devices store the drug as either premeasured quantities of powder in capsules or blistered foil strips, from which a single dose of powdered drug is released into an inhalation chamber by opening or puncturing the capsule/ foil blister (Newman 1991; Nerbrink et al. 1994; Thorsson et al. 1994; Fuller 1995; Bisgaard et al. 1998). Alternatively, the powdered drug is stored in a multidose reservoir from which single doses are forced into the inhalation chamber (e.g., Turbuhaler, Astra-Zeneca, Lund, Sweden). To suspend the powder in air, the required energy is provided by the patient. This is usually accomplished via a rapid inhalation through the device and turbulence, created by a series of baffles, blends the powder into a respirable distribution, which is inhaled from the device. By modifying the design features, there is precise control of the released dose, but these systems are flow dependent to a varying degree and, in a manner similar to the MDI, the inhaled material of necessity leaves the device at relatively high velocity. Some DPIs contain only drug particles (e.g., Pulmicort Turbuhaler) whereas other DPIs (e.g., Salmeterol Diskus)

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also contain large particles of an inert dispersal agent such as lactose. Because they require the patient’s own rapid inhalation to generate the aerosol, DPIs are by definition coordinated with the patient’s breathing. Nevertheless, significant extrapulmonary deposition occurs because of the high inspiratory flow rates needed to disperse the powder. Therefore, while dry powder devices may be preferable to a pMDI in patients with suboptimal coordination who are unwilling to use a spacer, the high extrapulmonary deposition raises safety considerations in using higher than usual doses of corticosteroids or bronchodilators. A further reservation concerning these devices is that a threshold inspiratory flow rate is required to actuate these devices and patients with acute exacerbations of asthma may not be able to reach the required flow rates. Because of this concern, manufacturers are endeavoring to reduce the required threshold flow rates of their respective devices. An alternative to lowering the required flow rate is to lower the resistance of the device, so that an equivalent flow could be generated with less respiratory effort.

Nebulizers Nebulizers generate aerosols usually from liquids. Energy can be transferred to the liquid surface by either a jet as the liquid phase is forced through a narrow orifice at high velocity (Newman 1991; Nerbrink et al. 1994) or by sheer forces at the surface generated by ultrasonic waves. Usually, the agonist is dissolved in the solvent, but it is also possible to nebulize suspensions, although the efficiency of some nebulizers may be less with suspensions than with solvents. In terms of the basics of aerosol generation, the major difference between nebulizers and the metered systems (MDIs and DPIs) is that the velocity of particles leaving the generator is really a function of the patient’s method of breathing. Commonly, a patient quietly breathing through a nebulizer will inhale particles whose inertia is a function of the aerodynamic diameter and the local convective flows of the physiologic situation defined by the breathing pattern rather than the rapid evaporation of freon in the MDI or the high flow rates necessary via rapid inhalation in a dry powder device. Therefore, even though particles may have the same aerodynamic characteristics as described by cascade impaction, the nature of the aerosol generator significantly influences the actual aerosol inhaled (aerosol being defined as the distribution of particles and the carrier gas, the carrier gas imparting its own velocity to the particles) and the subsequent deposition patterns are, as described above, significantly affected. While MDIs and DPIs are approved for use by regulatory agencies in concert with a specific medication and are therefore highly regulated, there is much more variability and nonuniformity in the manufacture of nebulizers. Aerosols emitted from nebulizers are modified primarily by baffling systems that are either internal or external to the device.

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Nebulizer delivery systems must be assessed with the tubing and mouthpieces with which they are marketed. In addition, the efficiency of the nebulizer itself will affect the quantity of aerosol delivered in concert with the patient’s breathing pattern (Devadason et al. 1997).

New developments in aerosol delivery systems Significant enhancements in drug delivery by nebulizers are possible by coordinating nebulization with inspiration (e.g., breath actuation) that essentially turns the nebulizer off during expiration. Another improvement in efficiency is called “breath enhancement,” which utilizes the patient’s inspiratory flow through the nebulizer to increase drug delivery (e.g., LC Star Pari, Germany; Ventstream, Medicaid, Bognor Regis, UK) (Devadason et al. 1997). Recently the design of aerosol delivery systems has combined slow and deep inspiration with direct mechanical feedback to the patient for particles of relatively large MMAD. As discussed earlier, a “slow” inspiration reduces particle inertia and allows inhalation of particles larger than the 2.5–μm cutoff for the FPF that defines deposition in the oropharynx during tidal breathing and will thus minimize oropharyngeal deposition Adaptive aerosol delivery (AAD) technology analyzes parameters of inspiration and provides feedback to guide the patient’s inspiratory maneuvers. The I-neb AAD system (Respironics Inc., Cedar Grove, NJ), a hand-held device, combines the latest AAD technology with an optimized form of vibrating mesh technology. The I-neb system can deliver aerosol via two modes of inspiration: (i) the tidal breathing mode (TBM), which sets the device to deliver aerosol in the first 50% of the patient’s inspiration (Fig. 34.12); and (ii) a new algorithm that guides the patient to a slow and deep inspiration, the target inhalation mode (TIM) (Fig. 34.13). Testing of I-neb prototypes has demonstrated that TIM is capable of delivering between 19 and 20 times as much drug per breath as simple tidal (TBM) breathing. In addition, the slow and deep breath provided by the TIM pattern of breathing enhances deposition as much as 2.5–fold. Therefore, when compared with tidal breathing, in vivo measurements of deposition have indicated that TIM can be up to 51 times more efficient per breath in depositing particles in the lungs (Smaldone & Nikander 2004).

Application of principles in aerosol technology to treatment strategies for allergic disease Asthma Inhaled corticosteroids are the mainstay of maintenance therapy of asthma (Brutsche et al. 2000; Selman et al. 2001). One of the most important features of commercial formulations of inhaled corticosteroids (ICS) is reduced oral bioavailability (Derendorf et al. 1998). With many delivery systems

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Pulses aerosol into subsequent breaths

Exhalation flow

Inhalation flow

Monitors the 1st three breaths


Time

Fig. 34.12 The I-neb adaptive aerosol delivery system can be set to two patterns of breathing (Figs 34.4 and 34.5). Tidal breathing mode (TBM), or tidal breathing, sets the device to deliver aerosol in the first 50% of the patient’s inspiration. The patient inhales spontaneously during tidal breathing. The device measures “flow” (vertical axis) and time on the horizontal axis. After the device determines the maxima and minima of three breaths, the software defines the “start” of the next

breath (zero flow) and then injects aerosol into the inhaled gas as a series of pulses or boluses over 50% of the time of inhalation. For each succeeding breath the aerosol pulse is adjusted for the average of the three preceding breaths. The inhaled mass is determined by the sum of the aerosol pulses. (Copyright of Profile Therapeutics, a Respironics Company.) (From Smaldone 2005, with permission.)

for ICS having high levels or oropharyngeal deposition (up to 80% with pMDIs and DPIs), it is crucial that when this oropharyngeally deposited drug is subsequently swallowed that its systemic exposure be kept as low as possible. The oral bioavailability of BDP, one of the oldest ICS, is about 20%, fluticasone is about 1%, and momethasone less than 1% (Derendorf et al. 1998). A newer drug, ciclesanide, does not become activated until it deposits in lung tissue (Humbert 2004). However even if there is no systemic bioavailability, systemic exposure can still occur because of ICS being absorbed through lung deposition. It appears that alveolar deposition may give rise to more systemic exposure because particles depositing in ciliated airways are subject to mucociliary clearance and because the barrier to diffusion may be more permeable in the alveoli than the airway. There is some pharmacodynamic data suggesting that when fluticasone is administered to normal subjects and asthmatics, the normal subjects are more susceptible to hypothalamic–pituitary– adrenal axis suppression (Brutsche et al. 2000). This finding is most likely due to the more proximal deposition in the asthmatic subjects because of reduced airway caliber (Fuller 1995). Most pharmaceutical manufacturers have therefore tried to target airway deposition over alveolar deposition. This appears reasonable because asthma is thought to be an airways disease. However some investigators have suggested that there may be an alveolar inflammatory component in asthma, thus suggesting that alveolar deposition may be beneficial, although this remains a minority viewpoint (Kraft et al. 2001). Targeting of the airways in asthma is complicated by the polydisperse nature of therapeutic aerosols. Making average

aerosol diameter smaller reduces oropharyngeal deposition but increases the amount of alveolar deposition. A low oral bioavailability is more important for polydisperse aerosols with larger MMADs so as to minimize systemic exposure from swallowed drug. While the search continues for the ideal method delivering inhaled steroids to patients with asthma, advances in drug design, formulation, and delivery systems now provide a wider array of options for the clinician. However, no delivery system can be considered to be intrinsically superior to all others. The delivery system should be judged instead by its ability to optimize the pharmacokinetic properties of the drug, most notably oral bioavailability, and by its suitability for the target subpopulation of asthmatics.

Aerosolized drug delivery in infants and young children For children less than 4 years old, inhaled medications can be delivered by a nebulizer and facemask or using a pMDI with holding chamber and mask. DPIs can be used from age 4 years and up. The breath-actuated feature of these devices makes them preferable to pMDIs (without a holding chamber) in children aged 4–6 years. After age 6 years children can be taught how to use a pMDI without a spacer, although the use of spacers should continue to be encouraged (Everard 1996). For young children with asthma, nebulized treatment with a suspension of budesonide is an effective and well-tolerated alternative (Szefler 1999). It should be noted that nebulizers can be less effective at aerosolizing suspensions than solutions

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TRAINING BREATH 20 Inhalation flow (L/min) 0

1

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5

6

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9

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6

7

8

9

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6

7

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SUBSEQUENT BREATHS 20 Inhalation flow (L/min) 0

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Target inhalation time Target inhalation time Extension criteria point

5

Aerosol generation time 2 seconds Inhalation gap

Fig. 34.13 Target inhalation mode (TIM), or slow and deep breathing, a new algorithm for the I-neb that guides the patient to a slow and deep inspiration. Typical inspiratory flows are reduced to approximately 20 L/min via a high-resistance mouthpiece (inhalation is upward). With each breath the system trains the patient to lengthen inspiration via a vibration feedback. As shown in the top panel of the figure, the patient inhales the first training breath and after 3 s the system vibrates, signaling the patient to exhale. A bolus of aerosol was delivered for the first second of the breath. Because inspiration exceeded the so-called “target inhalation time” (vertical mark on horizontal axis), the device automatically extends the target inhalation time in subsequent training breaths (middle panel) and the time of inspiration is gradually lengthened. After approximately 30 breaths the process is complete and the time of inspiration is set at approximately 9 s (the maximum set by the device, lower panel). In this mode the aerosol pulse (darkly shaded area) begins with inspiration and lasts for 7 s. The device stops extending the target inhalation time at this point because the length of the patient’s inspiration is now approximately the same as the target inhalation time. The device “remembers” this pattern and gives the vibratory feedback at the same point in each succeeding breath and for all future treatments. (Copyright of Profile Therapeutics, a Respironics Company.) (From Smaldone 2005, with permission.)

and clinicians are advised to prescribe budesonide only with the brand of nebulizers used in the clinical trials of this product (Smaldone et al. 1998b). Masks are used with holding chambers and nebulizers because young children breathe nasally and because they

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Aerosol delivery during mechanical ventilation Aerosolized β-sympathomimetic agents are an essential component of the treatment of status asthmaticus. Such delivery is complicated if the patient is undergoing mechanical ventilation. However, recent studies have identified ways in which such delivery can be optimized. The use of a pMDI is feasible provided certain conditions are met (Dhand & Tobin 1997). For example, it is necessary to use a spacer/holding chamber when using a pMDI in this setting. Not all holding chambers are equivalent in efficiency and different brands are not necessarily interchangeable. In contrast to the treatment of spontaneously breathing patients, delivery during mechanical ventilation must be synchronized with respiration. It is essential that a dose escalation protocol be employed because doses far in excess of those used for maintenance therapy may be needed. Hence, efforts to obtain objective evidence on response to treatment (e.g., peak airway pressure, dynamic compliance) and toxicity (tachycardia, arrhythmias) should be sought. In endeavoring to maximize delivered doses, however, it needs to be remembered that the efficiency of pMDI delivery decreases significantly if the interval between serial actuations is less than 1 min and if the synchronization with the respiratory cycle is suboptimal. The efficiency of jet nebulizers in delivering aerosols in the setting of mechanical ventilation is very variable and affected by several factors (O’Riordan et al., 1994). Different brands of commercial nebulizers vary in terms of their output per minute and the duration of treatment. Breath actuation by mechanical ventilators (nebulization takes place only during inspiration) may prolong treatment times. Humidification may cause “rain out” of aerosol in tubing and reduce aerosol delivery by 50%. The use of helium/oxygen mixtures has been shown to affect aerosol delivery from MDIs and jet nebulizers in a model of mechanical ventilation, and could result in either enhanced or impaired delivery, depending on how the system was configured (O’Riordan et al. 1992, 1994). In conclusion, once technical factors have been identified and optimized, efficient delivery of aerosolized medications to patients undergoing mechanical ventilation is readily attainable.

Delivery of therapeutic aerosols to the nasal mucosa Among an increasing list of aerosolized medications being used to treat diseases of the nasal mucosa are corticosteroids, cromolyn, anticholinergics (ipratroprium bromide), saline, and decongestants. The particle size of nasal aerosols tends to be larger than pulmonary inhalers. Manual pumps that produce large, relatively low velocity particles are being used as an alternative to high-velocity freon-based pMDIs. There

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are isolated reports of nasal perforation occurring with highvelocity inhalers (Soderberg-Warner 1984) and patients should be advised to direct the spray in the direction of the ipsilateral ear and away from the nasal septum. In severe allergic sinusitis, the mucosa may be so congested that a short course of systemic corticosteroids may be needed to allow penetration of aerosolized therapy. Prolonged use of topical decongestant sprays may lead to rebound hyperemia and intractable nasal congestion and systemic administration of decongestants may be preferable. Intranasal corticosteroids are an effective therapy for seasonal and perennial rhinitis. While regular use of such an agent has been shown to lead to short-term growth suppression in children (Skoner et al. 2000), it appears that this complication can be prevented by the use of a formulation with low oral bioavailability (e.g., mometasone) (Schenkel et al. 2000).

References Agnew, J. (1984) Physical properties and mechanisms of deposition of aerosols (2 ed.). London; Boston: Butterworths. Anderson, M., Philipson, K., Svartengren, M. & Camner, P. (1995) Human deposition and clearance of 6-micron particles inhaled with an extremely low flow rate. Exp Lung Res, 21(1), 187–195. Baron, P.A. (2001) Measurement of airborne fibers: a review. Ind Health, 39(2), 39–50. Bennett, W.D. (1991) Aerosolized drug delivery: fractional deposition of inhaled particles. J Aerosol Med, 4(3), 223–227. Bennett, W.D. & Smaldone, G.C. (1987) Human variation in the peripheral air-space deposition of inhaled particles. J Appl Physiol, 62(4), 1603–1610. Bisgaard, H., Klug, B., Sumby, B.S. & Burnell, P.K. (1998) Fine particle mass from the Diskus inhaler and Turbuhaler inhaler in children with asthma. Eur Respir J, 11(5), 1111–1115. Brutsche, M.H., Brutsche, I.C., Munawar, M., Langley, S.J., Masterson, C.M., Daley-Yates, P.T. et al. (2000) Comparison of pharmacokinetics and systemic effects of inhaled fluticasone propionate in patients with asthma and healthy volunteers: a randomised crossover study. Lancet, 356(9229), 556–561. Busse, W.W., Brazinsky, S., Jacobson, K., Strieker, W., Schmitt, K., Vanden Burgt, J. et al. (1999) Efficacy response of inhaled beclomethasone dipropionate in asthma is proportional to dose and is improved by formulation with a new propellant. J Allergy Clin Immunol, 104(6), 1215–1222. Derendorf, H., Hochhaus, G., Meibohm, B., Mollmann, H. & Barth, J. (1998) Pharmacokinetics and pharmacodynamics of inhaled corticosteroids. J Allergy Clin Immunol, 101(4 Pt 2), S440–446. Devadason, S.G., Everard, M.L., Linto, J.M. & Le Souef, P.N. (1997) Comparison of drug delivery from conventional versus “Venturi” nebulizers. Eur Respir J, 10(11), 2479–2483. Dhand, R. & Tobin, M.J. (1997) Inhaled bronchodilator therapy in mechanically ventilated patients. Am J Respir Crit Care Med, 156(1), 3–10. Diot, P., Palmer, L.B., Smaldone, A., DeCelie-Germana, J., Grimson, R. & Smaldone, G.C. (1997) RhDNase I aerosol deposition and


related factors in cystic fibrosis. Am J Respir Crit Care Med, 156(5), 1662–1668. Everard, M.L. (1996) Aerosol delivery in infants and young children. J Aerosol Med, 9(1), 71–77. Food and Drug Administration. (1998) Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Drug Products Chemistry, Manufacturing, and Controls Documentation. Center for Drug Evaluation and Research (CDER), http://www. fda.gov/cder/guidance/2180dft.htm. Fuller, R. (1995) The Diskus: a new multi-dose powder device– efficacy and comparison with Turbuhaler. J Aerosol Med, 8 Suppl 2, S11–17. Goldin, J.G., Tashkin, D.P., Kleerup, E.C., Greaser, L.E., Haywood, U.M., Sayre, J.W. et al. (1999) Comparative effects of hydrofluoroalkane and chlorofluorocarbon beclomethasone dipropionate inhalation on small airways: assessment with functional helical thin-section computed tomography. J Allergy Clin Immunol, 104(6), S258–267. Humbert, M. (2004) Ciclesonide: a novel inhaled corticosteroid. Expert Opin Investig Drugs, 13(10), 1349–1360. Iacono, A.T., Smaldone, G.C., Keenan, R.J., Diot, P., Dauber, J.H., Zeevi, A. et al. (1997) Dose-related reversal of acute lung rejection by aerosolized cyclosporine. Am J Respir Crit Care Med, 155(5), 1690–1698. Kraft, M., Pak, J., Martin, R.J., Kaminsky, D. & Irvin, C.G. (2001) Distal lung dysfunction at night in nocturnal asthma. Am J Respir Crit Care Med, 163(7), 1551–1556. Morâen, F. (1993) Aerosol dosage forms and formulations (2nd, rev. ed.). Amsterdam; New York: Elsevier. Moren, F. (1982) Drug deposition of pressurized inhalation aerosols. Eur J Respir Dis Suppl, 119, 51–55. Nelson, H.S. & Loffert, D.T. (1994) Comparison of the bronchodilator response to albuterol administered by the OptiHaler, the AeroChamber, or by metered dose inhaler alone. Ann Allergy, 72(4), 337–340. Nerbrink, O., Dahlback, M. & Hansson, H.C. (1994) Why do medical nebulizers differ in their output and particle size characteristics? J Aerosol Med, 7(3), 259–276. Newman, S.P. (1991) Aerosol generators and delivery systems. Respir Care, 36(9), 939–951. Newman, S.P. (1996) Characteristics of radiolabelled versus unlabelled inhaler formulations. J Aerosol Med, 9 Suppl 1, S37–47. Newman, S.P. & Newhouse, M.T. (1996) Effect of add-on devices for aerosol drug delivery: deposition studies and clinical aspects. J Aerosol Med, 9(1), 55–70. Newman, S.P., Talae, N. & Clarke, S.W. (1991) Pressurized aerosol in man with the Rondo spacer. Acta Therapeutica, 17, 49–58. O’Riordan, T.G., Greco, M.J., Perry, R.J. & Smaldone, G.C. (1992) Nebulizer function during mechanical ventilation. Am Rev Respir Dis, 145(5), 1117–1122. O’Riordan, T.G., Palmer, L.B. & Smaldone, G.C. (1994) Aerosol deposition in mechanically ventilated patients. Optimizing nebulizer delivery. Am J Respir Crit Care Med, 149(1), 214–219. Sangwan, S., Condos, R. & Smaldone, G.C. (2003) Lung deposition and respirable mass during wet nebulization. J Aerosol Med, 16(4), 379–386. Sangwan, S., Gurses, B.K. & Smaldone, G.C. (2004) Facemasks and facial deposition of aerosols. Pediatr Pulmonol, 37(5), 447–452. Schenkel, E.J., Skoner, D.P., Bronsky, E.A., Miller, S.D., Pearlman, D.S., Rooklin, A. et al. (2000) Absence of growth retardation in

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children with perennial allergic rhinitis after one year of treatment with mometasone furoate aqueous nasal spray. Pediatrics, 105(2), E22. Selman, M., King, T.E. & Pardo, A. (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med, 134(2), 136–151. Skoner, D.P., Rachelefsky, G.S., Meltzer, E.O., Chervinsky, P., Morris, R.M., Seltzer, J.M. et al. (2000) Detection of growth suppression in children during treatment with intranasal beclomethasone dipropionate. Pediatrics, 105(2), E23. Smaldone, G. (1997) Respiratory physiology in asthma: pulmonary function testing bronchoprovocation, and mucociliary clearance (2nd ed.). Philadelphia: Saunders. Smaldone, G.C. (1991) Drug delivery via aerosol systems: concept of “aerosol inhaled”. J Aerosol Med, 4(3), 229–235. Smaldone, G.C. (2001) Deposition and clearance: unique problems in the proximal airways and oral cavity in the young and elderly. Respir Physiol, 128(1), 33–38. Smaldone, G.C. (2004) Respiratory physiology and disease effects on aerosol deposition. Respir Drug Delivery IX, 1, 179–186. Smaldone, G.C. (2005) Assessing new technologies: patient-device interactions and deposition. Respir Care, 50(9), 1151–1160. Smaldone, G.C., Berg, E. & Nikander, K. (2005) Variation in pediatric aerosol delivery: importance of facemask. J Aerosol Med, 18(3), 354–363. Smaldone, G.C., Cruz-Rivera, M. & Nikander, K. (1998) In vitro determination of inhaled mass and particle distribution for Budesonide nebulizing suspension. J Aerosol Med 11, 113–125. Smaldone, G.C., Diot, P., Groth, M. & llowite, J. (1998) Respirable mass: vague and indefinable in disease. J Aerosol Med, 11 Suppl 1, S105–111. Smaldone, G.C., Foster, W.M., O’Riordan, T.G., Messina, M.S., Perry, R.J. & Langenback, E.G. (1993) Regional impairment of mucociliary clearance in chronic obstructive pulmonary disease. Chest, 103(5), 1390–1396.

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Smaldone, G.C., Fuhrer, J., Steigbigel, R.T. & McPeck, M. (1991) Factors determining pulmonary deposition of aerosolized pentamidine in patients with human immunodeficiency virus infection. Am Rev Respir Dis, 143(4 Pt 1), 727–737. Smaldone, G.C., Itoh, H., Swift, D.L., Kaplan, J., Florek, R., Wells, W. et al. (1983) Production of pharmacologic monodisperse aerosols. J Appl Physiol, 54(2), 393–399. Smaldone, G.C. & Messina, M.S. (1985a) Enhancement of particle deposition by flow-limiting segments in humans. J Appl Physiol, 59(2), 509–514. Smaldone, G.C. & Messina, M.S. (1985b) Flow limitation, cough, and patterns of aerosol deposition in humans. J Appl Physiol, 59(2), 515–520. Smaldone, G.C. & Nikander, K. (2004) Bench performance and in vivo deposition efficiency of the I-neb adaptive aerosol delivery system (AAD) during tidal versus slow and deep breathing. Proceedings of the Aerosol Society, DDL15, 17–20. Soderberg-Warner, M.L. (1984) Nasal septal perforation associated with topical corticosteroid therapy. J Pediatr, 105(5), 840–841. Stahlhofen, W., Gebhart, J. & Heyder, J. (1980) Experimental determination of the regional deposition of aerosol particles in the human respiratory tract. Am Ind Hyg Assoc J, 41(6), 385–398a. Szefler, S.J. (1999) Pharmacodynamics and pharmacokinetics of budesonide: a new nebulized corticosteroid. J Allergy Clin Immunol, 104(4 Pt 2), 175–183. Thorsson, L., Edsbacker, S. & Conradson, T.B. (1994) Lung deposition of budesonide from Turbuhaler is twice that from a pressurized metered-dose inhaler P-MDI. Eur Respir J, 7(10), 1839–1844. Wildhaber, J.H., Devadason, S.G., Eber, E., Hayden, M.J., Everard, M.L., Summers, Q.A. et al. (1996) Effect of electrostatic charge, flow, delay and multiple actuations on the in vitro delivery of salbutamol from different small volume spacers for infants. Thorax, 51(10), 985–988.

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Bronchial Hyperresponsiveness Guy F. Joos

Environmental risk factors (causes)

Summary Measures of bronchial responsiveness are widely used for diagnosis and monitoring of asthma. A wide array of nonspecific bronchoconstrictor stimuli is available. Methacholine and histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Indirect challenges (adenosine, exercise, hypertonic saline) induce airflow limitation by an action on cells other than smooth muscle cells. A variety of cells, mediators, and receptors are involved in the airway narrowing caused by indirect airway challenges. Whereas bronchial responsiveness to a direct stimulus is only weakly related to airway inflammation, indirect airway challenges might better reflect active airway inflammation. Both direct and indirect airway challenges are useful outcome parameters in clinical studies of asthma. For example, an indirect challenge responds within hours to days to treatment with inhaled steroids, while improvement in direct responsiveness may take months to years. Bronchial challenges are also an essential step in the development of some of the potential new antiasthma treatments, such as adenosine or tachykinin receptor antagonists.

Introduction Bronchial hyperresponsiveness (BHR) is one of the hallmarks of bronchial asthma. Other important characteristics include variable airflow obstruction, airway inflammation, and airway remodeling (Bousquet et al. 2000; Global Initiative for Asthma 2006) (Fig. 35.1). Measures of bronchial responsiveness are widely used for diagnosis and monitoring of asthma, in epidemiologic studies, and in clinical trials. They are important outcome parameters in studies evaluating treatment of asthma and are nowadays often combined with noninvasive


INFLAMMATION

Airflow limitation

Airway hyperresponsiveness Triggers

Symptoms Fig. 35.1 Relationship between airway inflammation, bronchial hyperresponsiveness and airflow limitation. (Redrawn from GINA 2006, with permission.) (See CD-ROM for color version.)

measures of airway inflammation such as induction of sputum or measurement of exhaled nitric oxide (NO) (American Thoracic Society 2000; Jayaram et al. 2000; Kharitonov & Barnes 2000; Sterk et al. 1993). Bronchial responsiveness is measured as the change in airway caliber occurring after inhalation of a bronchoconstrictor agent (Lötvall et al. 2000). The development of methods to demonstrate and quantify BHR has greatly contributed to the understanding of the pathogenesis of asthma and its diagnosis and treatment. Studies on BHR have also highlighted the complexity of mechanisms involved in the increased airway responses in asthma (Joos & O’Connor 2003). BHR is also observed in diseases other than asthma and in some apparently healthy subjects. For instance, BHR to methacholine is present in a majority of patients with mild to moderate chronic obstructive pulmonary disease (COPD) (Tashkin et al. 1992). Moreover, the severity of BHR predicts the response to inhaled corticosteroids in patients with asthma (Juniper et al. 1981) and the progression of airflow limitation in patients with COPD (Tashkin et al. 1992). An array of different pharmacologic and physical stimuli is available to measure bronchial responsiveness (Table 35.1). They are called nonspecific bronchoconstrictor stimuli, and differ from specific stimuli, such as allergen or aspirin, which cause bronchoconstriction only in a specific subset of asthmatic patients.

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Table 35.1 Nonspecific bronchoconstrictor stimuli. (Modified from Joos & O’Connor 2003.) Direct stimuli Cholinergic agonists: pilocarpine, acetylcholine, methacholine, carbachol Histamine Prostaglandin D2 Cysteinyl leukotrienes: LTC4, LTD4, LTE4 Indirect stimuli Physical stimuli Exercise Nonisotonic aerosols (hypertonic and hypotonic aerosols, distilled water, mannitol) Eucapnic voluntary hyperpnea of dry air Pharmacologic stimuli Adenosine Tachykinins (substance P, neurokinin A) Bradykinin Metabisulfite/SO2 Propranolol Endotoxin (lipopolysaccharide) Platelet-activating factor Ozone

Historical background In 1921, Alexander and Paddock found that asthmatic subjects developed asthma-like symptoms following subcutaneous administration of pilocarpine in doses that had no effect on normal subjects. In 1932, Weiss et al. showed that asthmatics were hyperresponsive to intravenous histamine. Curry (1947) reported that inhalation of small quantities of inhaled histamine induced bronchoconstriction. Bronchoconstriction induced in asthmatics by acetylcholine and histamine was also described by Tiffeneau & Beauvallet (1945), Herxheimer (1951), and De Vries et al. (1962). In the 1970s and 1980s, methods for bronchial provocation testing with nonspecific stimuli were standardized (Chai et al. 1975; Cockcroft et al. 1977). The number of stimuli that were demonstrated to cause an increased bronchial response in asthma compared with healthy subjects gradually enlarged, as well the understanding that mechanisms involved in BHR to these different stimuli were multiple and complex (Pauwels et al. 1988).

refers to a comparison with the airway response to the same agonist, using the same method to measure the airflow limitation, in a group of healthy subjects. The wording “airflow limitation” is chosen because it encompasses the different mechanisms that can lead to a decrease in the parameters of airflow used in studies on BHR, such as forced expiratory volume in 1 s (FEV1) or airway resistance (Raw). A decrease in FEV1 or an increase in Raw following exposure to a stimulus can reflect constriction of airway smooth muscle, edema of the airway wall, increased amounts of fluid or sputum in the airway wall, or a decrease in the elastic recoil pressure on the airways. The phenomenon of BHR is sometimes referred to as “nonspecific,” i.e., that most patients with active asthma will react to these stimuli. The stimuli used to reveal it, however, act by highly specific mechanisms. As multiple pathophysiologic pathways are involved, it is therefore no surprise that the results of the different challenge tests are only weakly correlated and not mutually interchangeable. Bronchoconstrictor stimuli may be classified into direct and indirect, according to the main mechanism through which they induce airflow limitation (Table 35.1). Combinations of mechanisms are possible. Direct stimuli induce airflow limitation through direct action on the effector cells involved in the airflow limitation. These comprise in the first place airway smooth muscle cells, but bronchial vascular endothelial cells and/or mucus-producing cells may also be involved. Indirect stimuli, on the other hand, act on intermediary cells such as inflammatory cells, bronchial epithelial cells and/or neuronal cells; the proinflammatory mediators and/or neurotransmitters liberated by these cells will then interact with the effector cells to cause airflow limitation (Fig. 35.2) (Van Schoor et al. 2000). It has become clear that several agonists can be used to study bronchial responsiveness. The most widely used pharmacologic agents to asses bronchial responsiveness are methacholine and histamine. Methacholine and histamine cause airflow limitation predominantly through a direct effect on airway smooth muscle and are therefore called direct broncoconstrictor agents.

Direct stimulus

Indirect stimulus

Effector cells:

Intermediary cells:

– Airway smooth muscle cells – Bronchial endothelial cells – Mucus-producing cells

– Inflammatory cells – Neuronal cells

Definition Airflow limitation

Bronchial or airway hyperresponsiveness can be defined as an abnormal increase in airflow limitation following the exposure to a stimulus. In this definition the word “abnormal”

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Fig. 35.2 Cellular pathways involved in the bronchoconstriction induced by direct and indirect stimuli. (After Van Schoor et al. 2000, with permission.) (See CD-ROM for color version.)

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FEV1 (% fall) Methacholine Histamine

50 ASTHMA

40 30 20

COPD SEVERE

MODERATE

MILD

SLIGHT

NORMAL 10 0 0.01

0.1

1.0 10 Dose (µmol)

100

Fig. 35.3 Dose–response curves for methacholine and histamine in patients with asthma, chronic obstructive pulmonary disease (COPD), and normal airways. (From Woolcock et al. 1991, with permission.) (See CD-ROM for color version.)

Methods for measuring bronchial responsiveness Bronchial responsiveness is measured as the change in airway caliber occurring after inhalation of a bronchoconstrictor agent. Typically, a hyperresponsive pattern is observed in patients with asthma: the dose –response curve to a bronchoconstrictor agent displays a leftward shift, an increased slope, and an enhanced maximal response (Fig. 35.3). Thus, the optimal way to measure bronchial responsiveness to one or another agonist is to perform a dose–response curve to the selected agonist and to quantify the response.

Methods for delivering the stimulus Various routes of delivery of stimuli have been used to demonstrate BHR in patients with asthma, including intravenous, subcutaneous, and oral administration, but most stimuli used in studies on BHR have been given via inhalation for reasons of safety and specificity. By use of aerosol, lower doses of the bronchoconstrictor substances are administered and unpleasant systemic side effects are avoided. Aerosols can be produced from solutions either by atomization with compressed air (jet nebulizers) or by the vibration of piezoelectric crystals at ultrasonic frequencies (ultrasonic nebulizers). Three types of nebulizer delivering methods are in use: the tidal breathing, the reservoir, and the dosimeter methods. With the tidal breathing method the aerosol is generated continuously by a nebulizer and inhaled by the subject via a facemask or a mouthpiece, with a nose-clip occluding the nose. The aerosol is inhaled for a set time during tidal breathing (e.g., 2 min) (Cockcroft et al. 1977). This system has the advantage that it is a well-tried method and does not need sophisticated equipment. However, although the output of the

Bronchial Hyperresponsiveness

nebulizer can be measured, the dose of the aerosol inhaled is not exactly known and depends on the minute volume of the subjects (Eiser et al. 1983). The reservoir and dosimeter methods deliver standardized doses of the aerosol during inspiration only. In the reservoir method a storage bell is filled with aerosol, freshly generated from a nebulizer. The subject then inhales a fixed volume from the bell via the mouthpiece and breath-holds for 4 s before exhaling in a balloon. In the dosimeter method, the dose of the inhaled aerosol is standardized via a breath-actuated dosimeter (e.g., Rosenthal-French dosimeter). In comparative studies the results obtained with the tidal breathing method compared well with those obtained with the dosimeter method (Beaupré & Malo 1979; Ryan et al. 1981). In the past few years, a novel osmotic bronchial challenge using mannitol dry powder has been developed as an alternative for the wet aerosol of hypertonic saline. From a practical point of view, it does not require a nebulizer and can be performed outside the laboratory setting using a simple hand-held inhaler device, the mannitol being contained in gelatin capsules (Anderson et al. 1997). The output of the nebulizer is a major determinant of the airway response to bronchial challenge. For instance, a threefold increase in the output of the nebulizer resulted in a threefold lower PC20 (provocative concentration causing a 20% decrease in FEV1) for methacholine (Ryan et al. 1981). Standardization of the droplet size and the flow characteristics and output of the nebulizers are all relevant to this issue, but a discussion of these important parameters of administration via nebulizers is outside the scope of this chapter and readers are referred to other sources for more technical details (Sterk et al. 1993; American Thoracic Society 2000; Anderson & Brannan 2003; Joos & O’Connor 2003). The important message is that the nebulizer should be well characterized so that results of bronchial challenges with the same stimulus can be compared within the same laboratory or lung function department and between investigations. This interinvestigator reproducibility is especially important in the settings of epidemiologic studies and multicenter therapeutic studies.

Methods for measuring the airway response The changes in airway caliber occurring after inhalation of the bronchoconstrictor stimulus can be estimated by measuring changes in airflow during a forced expiratory maneuver or by measuring changes in airways resistance (Raw) and conductance (Gaw). The tests of forced expiration are FEV1, peak expiratory flow rate (PEFR), and flow rates at different levels of the maximal or partial expiratory flow–volume curve (Table 35.2). The choice of which parameter to use is almost always a trade-off between the reproducibilty and the sensitivity of the parameter. Measurement of FEV1 is simple to perform, reproducible (intrasubject coefficient of variation 0–8%) (Eiser et al. 1983), and can be achieved with simple equipment. The FEV1 is relative insensitive at detecting changes in

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Table 35.2 Bronchial provocation testing: measurement of the airway response. Peak expiratory flow rate Spirometry Forced inspiratory maneuvers (if upper airway involvement is suspected) Body plethysmography Transcutaneous oxygen saturation Forced oscillometry

airway caliber, in comparison to Raw and specific (s)Gaw. The measurement of FEV1 involves a deep inspiration. In normal subjects, a deep inspiration may overcome a small induced increase in airway resistance. In asthmatics a deep inspiration may induce bronchoconstriction (Fish & Kelly 1979; Orehek et al. 1981). The measurement of Raw and sGaw more accurately reflect airway patency, but is more affected by changes in large than in smaller airways. It is more sensitive to airway caliber changes, but its reproducibility is less good than for FEV1 (intrasubject coefficient of variation for Raw 10–20%, for sGaw 9%; see Eiser et al. 1983). It does not involve a full inspiratory maneuver and hence does not affect airway tone.

Quantification of BHR Bronchial responsiveness is usually quantified by performing a dose–response study with the agonist, and expressing the result as a dose or quantity of the agonist that causes a predetermined change in the measured airway parameter (Fig. 35.3). The most frequently used measures are the PC20 FEV1 or the PD20 FEV1, respectively the concentration and dose of the agonist that cause a fall in FEV1 of 20% from baseline. To determine the PC20 or PD20, the dose–response curve is plotted on a semilogarithmic scale and the PC20 or PD20 calculated by interpolation. Similar procedures can be used for other parameters, such as the PC35 or PD35 sGaw (concentration or dose that decreases the sGaw by 35% from baseline) or the PC100 or PD100 Raw (concentration or dose that doubles the airway resistance). A dose–response curve to histamine or methacholine is characterized by the threshold concentration, the slope, and the maximal bronchoconstriction that can be reached. The threshold concentration, also called the sensitivity, is the dose at which the curve changes from baseline. The threshold can be defined as the concentration where a change of lung function of twice the coefficient of variation of the baseline measurement has occurred. The slope, also called the reactivity, is obtained by linear regression of the data points from threshold, using the formula y = mx + b (m = slope). Normal individuals have limited bronchoconstriction even if the amount of histamine or methacholine administered is further increased (Sterk et al. 1985). This plateau level is not observed in patients with moderate to severe asthma.

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Mechanisms and receptors involved in BHR Direct stimuli Histamine causes bronchoconstriction by binding to the histamine H1 receptor on airway smooth muscle cells. The cholinergic agonists cause bronchoconstriction by directly acting on the airway smooth muscle cell via the M3 muscarinic receptor. The leukotrienes (LT)C4, LTD4 or LTE4 cause airway narrowing by interaction with the CysLT1 receptor.

Indirect stimuli Several cells are involved in the airway narrowing caused by indirect stimuli. These include epithelial cells, inflammatory cells (including mast cells), nerve cells, and vascular smooth muscle cells. Upon application of a constrictor agent, a wide array of mediators are released: histamine, cysteinyl leukotrienes, prostaglandins, adenosine, acetylcholine, substance P (SP), neurokinin A (NKA), and nitric oxide (NO). Moreover, the nature and amount of mediator released can vary from one indirect stimulus to another (Van Schoor et al. 2000). Adenosine causes bronchoconstriction in asthma by interaction with mast cells; histamine has been shown to contribute to this reaction (Polosa et al. 2002). Using the experimental 5-lipoxygenase inhibitor ABT-761, a role for leukotrienes in the bronchoconstriction induced by inhaled adenosine was suggested (Van Schoor et al. 1997). In addition, Rorke et al. (2002) have produced direct evidence for involvement of cysteinyl leukotrienes in adenosine-induced bronchoconstriction in asthma by using the CysLT1 receptor antagonist montelukast. Adenosine exerts its effect on human cells through interaction with specific adenosine (P1) receptors, of which four subtypes (A1, A2A, A2B and A3) have been described. The A1, A2B and A3 receptors have been shown to be involved in various animal and human models of inflammation (Joos & Pauwels 1996). In particular, the potential role of A2B receptors is being increasingly recognized (Polosa 2002). Airway effects of the tachykinins SP and NKA are mediated through tachykinin NK1 and NK2 receptors (Joos et al. 2001). In vitro, tachykinins constrict human airway smooth muscle through NK2 receptors; in small- and medium-sized bronchi, NK1 receptors are also involved. In vivo inhaled NKA causes bronchoconstriction by indirect mechanisms (via interaction with cholinergic nerves and inflammatory cells); both NK1 and NK2 receptors are involved in this bronchoconstrictor effect (Joos et al. 2004). Exercise causes airway narrowing by the loss of water through evaporation from the airway surface. The mechanism whereby the loss of water causes airways to narrow is thought to relate to thermal (cooling and rewarming) and osmotic (increase in airway osmolarity) effects of dehydration (Anderson & Daviskas 2000). Mediators involved in exercise-induced bronchoconstriction include histamine,

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prostaglandins, leukotrienes, and tachykinins. In addition, an increase in plasma adenosine concentration during exercise was reported: in patients with asthma, a relationship was observed between the increase in adenosine concentration and the level of exercise-induced bronchoconstriction (Vizi et al. 2002). The mechanisms involved in the bronchoconstriction induced by other physical stimuli are similar to those described for exercise. The reader is referred to reviews on this topic (Joos & O’Connor 2003; Van Schoor et al. 2005).

BHR in asthma: relation to airway inflammation and airway remodeling Bronchial responsiveness to a direct stimulus is only weakly related to airway inflammation It is frequently assumed that measures of bronchial responsiveness and airway inflammation can be used interchangeably. However, this does not seem to be the case. Correlations between markers of airway inflammation (bronchoalveolar lavage, bronchial biopsy and/or sputum) and BHR at baseline (i.e., before any intervention) have been examined in a large number of studies. In some instances weak correlations have been found between one or more inflammatory markers and the degree of BHR. However, an almost equal number of studies did not find significant correlations between airway inflammation and BHR (reviewed in Brusasco et al. 1998; Rosi & Scano 2000). When asthmatic patients are examined during the course of their disease, bronchial responsiveness and markers of airway inflammation cannot be considered as overlapping dimensions. This has been demonstrated by the use of factor analysis. The statistical method of factor analysis allows the many parameters that characterize the disease to be reduced to a few independent factors, each factor grouping associated parameters. In a study on 99 stable asthmatics using factor analysis it was concluded that baseline BHR (measured by histamine challenge), airway function, and airway inflammation are to be considered separate dimensions in the description of chronic asthma, thus supporting the utility of routine measurement of all these dimensions (Rosi et al. 1999). A subsequent study, which performed factor analysis on 66 stable asthmatics, assessed whether the relationship between BHR (measured by methacholine challenge), noninvasive markers of airway inflammation, and baseline lung function depended on the duration of the disease; the results of the analysis suggested that with shorter duration of the disease, BHR is associated with airway inflammation, whereas with a longer duration it is associated with impaired lung function, suggesting that in longstanding asthma ongoing alterations become the primary determinant of functional characteristics (Gronke et al. 2002). The distribution of sputum eosinophil count and its relationship with BHR to methacholine has been studied in mild


to moderate steroid-naive asthmatic people. In this study, 118 patients with asthma and 44 healthy people were examined; 69% of the asthma group had a significantly raised sputum eosinophil count (i.e., > 2%). Although the sputum eosinophil count was significantly associated with bronchial responsiveness to methacholine, the relationship was found to be weak (Louis et al. 2002a). The cross-sectional relationships between parameters of airway physiology, inflammation, and remodeling was studied in a group of patients with mild symptomatic asthma; 40% of the variability in BHR could be explained by multiple regression containing remodeling and inflammatory components (reticular basement membrane thickness accounting for 20%, epithelial cells for 11%, and eosinophils for 8%). Thus, while inflammation was an important predictor for BHR, remodeling represented a stronger predictor (Ward et al. 2002). The relation with remodeling might even be stronger in patients with asthma of longer duration (Gronke et al. 2002).

Caliber of the airways and bronchial responsiveness Airway caliber in vivo is the result of a balance between the force generated by the airway smooth muscle and a number of opposing factors. The latter are mainly presented by autonomic mechanisms that tend to limit the airway smooth muscle tone and by mechanical factors that oppose airway smooth muscle shortening (Brusasco et al. 1998). Patients with nonasthmatic fixed airflow limitation also demonstrate BHR to histamine and methacholine. The characteristics are somewhat different in that there is a strong linear relationship between BHR and the reduction in FEV1 in subjects with chronic airflow limitation (Joos & O’Connor 2003). It is difficult to interprete the meaning of a low PC20 in a patient with baseline airway obstruction. For instance, most patients with smoking-related COPD and mild to moderate baseline airway obstruction have BHR, but most have no acute or chronic bronchodilator response or symptoms of asthma (Mullen et al. 1986; American Thoracic Society 2000).

Indirect airway challenges might better reflect active airway inflammation It has been suggested that indirect markers of airway responsiveness are a better reflection of active airway inflammation. One of the best-studied indirect-acting bronchoconstrictors is adenosine monophosphate (AMP) (Holgate 2002a). Indirect challenges are thought to better reflect ongoing inflammation than direct challenges; this hypothesis is based on data obtained from studies in which indirect and direct challenges have been compared head-to-head. This has been most extensively studied in the case of adenosine challenge (van den Berge et al. 2002). Allergen avoidance at high altitude led to a significant diminution of eosinophils and eosinophil cationic protein in blood; this was accompanied by a significant

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decrease in BHR to AMP (2.1 doubling concentrations), BHR to methacholine remaining unchanged (van Velzen et al. 1996). In a group of 120 patients with atopic asthma, the concentrations of AMP causing the FEV1 to drop by more than 20% (PC20 AMP) was found to be more closely associated with inflammatory parameters than PC20 methacholine. In this study PC20 methacholine was predominantly predicted by baseline lung function (FEV1 in percent of predicted value), while PC20 AMP was predominantly predicted by the percentage of eosinophils in sputum. So, PC20 AMP reflects more closely the extent of airway inflammation due to asthma than PC20 methacholine (van den Berge et al. 2001a). Even in patients with clinical remission of asthma, elevated levels of exhaled NO and increased bronchial responsiveness to AMP and methacholine are found. It is interesting to note that in these patients a significant correlation between exhaled NO and responsiveness to AMP, but not methacholine, has been reported (van Den Toorn et al. 2001).

Asymptomatic BHR BHR can be shown in virtually all patients who experience symptoms of asthma. Moreover, a significant proportion of individuals with no past or present history of asthma or of other respiratory diseases, no current symptoms, and no respiratory medication show an increased physiologic airway response (fall in expiratory flows) to agents such as histamine or methacholine or to stimuli such as exercise (reviewed in Boulet 2003). Most studies in adults and children report a prevalence of asymptomatic BHR under 15% (Salome et al. 1987; Rasmussen et al. 1999; Boulet 2003). Subjects with asymptomatic BHR may show a more rapid decline in lung function, and this might be associated with an increased risk of developing asthma (American Thoracic Society 2000; Boulet 2003). Some patients with asthma can evolve toward asymptomatic bronchial hyperreponsiveness or in some cases even normalize bronchial responsiveness (Boulet 2003). Adolescents in clinical remission of atopic asthma are less responsive to the bronchial stimuli adenosine monophosphate and methacholine, in comparison to subjects with current asthma; however they are still more responsive than healthy controls (Van den Toorn et al. 2000, Van den Toorn et al. 2001).

BHR and antiasthma drugs Use of bronchial challenges to evaluate new antiasthma drugs Challenges with specific neurotransmitter (e.g., a cholinergic antagonist) or a specific mediator (e.g., LTD4) have proved useful in the development of new neurotransmitter antagon-

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ists (such as the long-acting anticholinergic agent tiotropium bromide) or mediator antagonists (such as the CysLT1 receptor antagonists) (Pauwels et al. 1995; O’Connor et al. 1996). The tachykinins SP and NKA are able to mimic various pathophysiologic features of asthma and COPD. There is an interest in developing tachykinin receptor antagonists as possible treatment for obstructive airway diseases (Joos & Pauwels 2001). Inhaled NKA induces bronchoconstriction in patients with asthma. A shift in the dose–response curve to NKA suggests airway activity of the compound. Using this approach the potent tachykinin NK2 receptor antagonist SR 48968 was found to have an only limited protective activity in patients with asthma (Van Schoor et al. 1998). Using a similar approach a dual tachykinin NK1/NK2 receptor antagonist, DNK333, and a triple tachykinin NK1/NK2/NK3 antagonist were able to prevent NKA- induced bronchoconstriction (Joos et al. 2004; Schelfhout et al. 2006). Similar approaches are being considered for adenosine receptor antagonists, where challenge with AMP is a logical first step in testing potential airway activity of an adenosine receptor antagonist (Holgate 2002b).

Direct and indirect bronchial responsiveness are useful outcome measures in clinical studies and provide different information The monitoring of symptoms, airflow obstruction, and exacerbations is essential to asthma management. Regular monitoring by physicians improves health outcomes provided it includes monitoring of control of asthma, medication and skills at regular intervals (Gibson 2000). Measures of airway responsiveness and airway inflammation (e.g., sputum induction and exhaled air) can be assessed at regular clinic visits and are increasingly included in studies evaluating control of asthma (Sont et al. 1999; Joos 2001; Green et al. 2002; Smith et al. 2005). It is well known that treatment with inhaled steroids slowly improves the bronchial responsiveness to a direct stimulus (Woolcock et al. 1988). In a prospective longitudinal study on the effect of an inhaled steroid, fluticasone, various markers of airway physiology, inflammation, and remodeling were incorporated. A 1-year treatment with an inhaled steroid was shown to improve airway caliber, improve direct bronchial responsiveness, and decrease inflammatory cells in bronchoalveolar lavage (BAL) fluid, as well as reduce thickness of the reticular basement membrane (a measure of airway remodeling). Whereas the improvement in airway caliber and the decrease in inflammatory cells recovered from BAL reached a plateau after 3 months of treatment, BHR to methacholine (PD20) continued to improve over the same time period as changes in airway remodeling, with improvements in both reticular basement membrane thickness and PD20 occurring with 12 months of treatment. It is of interest to note that in this study one-third of the improvement in BHR with fluticasone propionate was associated

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with early changes in inflammation, but the more progressive and larger improvement was associated with the later improvement in airway remodeling (Ward et al. 2002). Recent findings indicate that using direct BHR as a parameter might be of practical clinical relevance in the optimization of the long-term maintenance treatment of asthma. Commonly used clinical indices of asthma severity, such as symptoms, β2-agonist use, FEV1, and peak flow variabilility, were shown not to be significantly correlated with inflammatory cell counts in the lamina propria of the airways in asthmatics not treated with inhaled corticosteroids (ICS) (Djukanovic et al. 1992) as well as in those receiving ICS (Sont et al. 1996). In addition, infiltration with inflammatory cells in the lamina propria seems to persist despite regular treatment with ICS; the number of infiltrating leukocytes appears to be correlated with direct BHR, but not with symptoms or lung function. In many patients whose disease is considered clinically controlled, it appears that BHR and airways inflammation persist (Sont et al. 1996). According to international guidelines, the level and adjustment of antiinflammatory treatment is based solely on symptoms and lung function, and in the light of the above one could question whether such an approach really leads to the optimal long-term outcome of asthma. Researchers from the Leiden group have postulated that, in addition to the current guidelines, asthma treatment should also be directed toward reducing BHR. They have shown in a subsequent 2-year prospective parallel-group trial (AMPUL study) that a treatment strategy with stepwise adjustments of ICS doses, aimed at reducing BHR on top of optimizing symptoms and lung function, indeed leads to more effective asthma control (significantly greater improvements in FEV1, significant 1.8fold decrease in mild exacerbation rate) and greater improvement of chronic airways remodeling (significantly greater reduction in subepithelial reticular layer thickness), when compared with a “reference” strategy based on current treatment guidelines (Sont et al. 1999). Whereas the steroid-induced improvement in direct bronchial responsiveness may take months or even years, indirect challenges may respond more rapidly (Van Schoor et al. 2002; Reynolds et al. 2002). In a study on the effects of two doses of the inhaled steroid fluticasone propionate, the severity of exercise-induced bronchoconstriction decreased significantly as compared to placebo within 3 weeks. In contrast, responsiveness to methacholine improved during the first 6 weeks of the treatment with fluticasone propionate, and steadily increased during the 24 weeks of treatment (Hofstra et al. 2000). In a study on the effect of 2 weeks’ treatment with oral or inhaled steroids in adult asthmatic patients, PC20 AMP was found to be more sensitive to changes in acute airway inflammation compared with PC20 methacholine (van den Berge et al. 2001b). So, BHR to inhaled AMP is an early and sensitive indicator of the beneficial antiinflammatory effects of topical steroids (Prosperini et al. 2002).


Specific uses of direct and indirect bronchial challenges We now have at our disposal a broad and heterogeneous array of stimuli that can be used for bronchial challenge testing in asthmatic patients. How we should best measure BHR is as yet uncertain. The choice of stimulus depends at least in part on the reason for performing the challenge and on the type of information one wants to obtain. Moreover, one should be aware of the fact that various stimuli can temporarily increase BHR (Table 35.3). In general, BHR to direct stimuli is extremely sensitive for clinically current asthma, and a negative test permits one to exclude with reasonable certainty the diagnosis of asthma in symptomatic patients. However, these tests lack specificity both in differentiating asthma from normal and asthma from COPD (Tables 35.4 and 35.5); moreover, they perform less well in the epidemiologic setting. Parameters of direct BHR appear to be better correlated with the long-term structural alterations of remodeling than with indices of inflammation. In contrast, tests of indirect BHR are both more specific (but less sensitive) for asthma and correlate better with the airway inflammation of the disease. Bronchial responsiveness to indirect stimuli may be preferred for confirming a diagnosis of asthma and for monitoring disease activity and the antiinflammatory effects of antiasthma therapy in the short and medium term, while direct stimuli may be preferred for

Table 35.3 Factors that increase bronchial responsiveness. (From American Thoracic Society 2000, with permission.) Factor

Duration of effect

Exposure to environmental antigens Occupational sensitizers Respiratory infection Air pollutants Cigarette smoke Chemical irritants

1–3 weeks Months 3–6 weeks 1 week Few hours Days to months

Table 35.4 Diseases manifesting bronchial hyperresponsiveness on testing with histamine or methacholine. Asthma COPD Congestive heart failure Cystic fibrosis Bronchiectasis Viral respiratory infections Allergic rhinitis Sarcoidosis

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Table 35.5 Value of direct and indirect bronchial challenges in the diagnosis and monitoring of asthma. (From Anderson & Brannan 2003; American Thoracic Society 2000.) Direct challenge

Indirect challenge

Diagnosis

Not specific for asthma High negative predictive value (approaching 100% for clinically active asthma)

Specific for asthma Less sensitive than direct challenge

Monitoring

Progressive improvement on treatment with inhaled steroid (months to years)

Rapid response to antiinflammatory treatment (days to weeks)

excluding asthma and optimizing antiinflammatory treatment in the long term (Cockcroft 2001; Joos & O’Connor 2003). Both types of challenges thus appear to have their value in the assessment of the different pathophysiologic components of asthma.

Pharmacologic stimuli Adenosine occupies a unique place in the group of indirect stimuli, as it is the challenge which, to date, is the best reflection of Th2-mediated inflammation, dominated by activated mast cells and eosinophils (Holgate 2002a). This means that BHR to AMP can also be present in other conditions characterized by mast cell priming, such as allergic rhinitis and a subgroup of patients with COPD. Adenosine challenge may be a useful tool in making the differential diagnosis of asthma and COPD in cases where the diagnosis is uncertain. This is especially the case in nonsmokers, as smokers with COPD may show AMP responsiveness as well (Polosa et al. 2002; van den Berge et al. 2002). The role of the mast cell in COPD is currently poorly defined. It has been shown that the number of epithelial mast cells in the bronchioli of smokers with COPD is increased (Grashoff et al. 1997). Interestingly, a subset of COPD patients has been described characterized by sputum eosinophilia and mast cell activation; the hypothesis has been raised that responders to steroids have both eosinophil- and mast-cell driven disease, regardless of whether they have asthma or COPD (Louis et al. 2002b). Concerning the association of BHR to AMP with upper airway allergic inflammation, an increasing dose–response slope along the symptom axis from asymptomatic controls to allergic rhinitis/conjunctivitis to allergic asthma has been demonstrated, which was in addition stronger than for methacholine (De Meer et al. 2002). In summary, the specificity of AMP challenge for asthma, together with its high repeatability, could be useful for epidemiologic purposes. AMP challenge testing will also become increasingly used for pharmacologic testing, once specific adenosine receptor antagonists become available for use in humans. Such investigations will allow determination of whether adenosine is a clinically important mediator of asthma,

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as well as identifying which adenosine receptor subtypes play a main role in asthma. Adenosine is inexpensive, and the challenge is technically easy to perform (Joos & O’Connor 2003). Tachykinins are of potential importance as mediators of asthma. Performing bronchial challenges with tachykinins is mainly of pathophysiologic importance, in order to elucidate the actions of the different tachykinins and to study the role of airway tachykinin receptors. Challenge tests with SP and NKA are currently employed to evaluate newly developed tachykinin receptor antagonists. However, the high cost of these peptides will probably limit their use to fundamental and clinical research purposes (Joos & Pauwels 2000). The other pharmacologic stimuli have been much less studied. To date, specific clinical uses have not been identified and these challenges remain essentially of pathophysiologic interest in the laboratory setting .

Physical and physicochemical stimuli This group of challenges better mimics the stimuli that asthmatic patients are likely to encounter in their everyday life. In this respect, exercise challenge is considered to be the most physiologic of all stimuli used for challenge testing. Several international guidelines have described the indications for exercise testing, and these include (i) making a diagnosis of exercise-induced bronchoconstriction (EIB) in asthmatic patients with a history of breathlessness during or after exertion; (ii) evaluating the ability to perform demanding or lifesaving work (e.g., military, police, fire fighting) in persons with a history suggesting asthma; and (iii) determining the effectiveness and optimal dosing of medications prescribed to prevent EIB and evaluating the effects of antiinflammatory treatment (Sterk et al. 1993; American Thoracic Society 2000). Eucapnic voluntary hyperpnea of dry air mimics exercise and has the same clinical significance. In contrast to exercise challenge, dose–response curves can be constructed. Hypertonic saline challenge is easy to perform and allows construction of a dose–response curve. The principal indications for this challenge are to identify BHR consistent with active asthma and to evaluate bronchial responsiveness that will respond to treatment with antiinflammatory drugs. A

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hypertonic saline challenge is an alternative to exercise or hyperventilation for identifying patients with EIB (Anderson & Brannan 2003). It can also be used in the assessment of patients with a history of asthma who wish to scuba-dive (British Thoracic Society 2003). Hypertonic saline challenge has gained widespread use for inducing sputum, and this procedure has become an important research tool for assessing acute and chronic airway inflammation in a noninvasive fashion. The mechanisms by which sputum is induced are not known, but different mechanisms are thought to be involved, such as increased vascular permeability, stimulation of mucus production, humidification of airway mucus, and stimulation of mucociliary clearance and of the cough reflex (Djukanovic et al. 2002). So, hypertonic saline challenge is unique as a challenge tool in that it permits documentation of BHR at the same time as collecting sputum. Recently, hypertonic saline challenge has found a novel application in the noninvasive assessment of microvascular leakage in the monitoring of airway inflammation, via the model of “dual induction” (first leakage, then sputum). In this model, pharmacologic agents that induce leakage (e.g., tachykinins) are inhaled in a first step; this is then followed by sputum induction through a hypertonic saline challenge, allowing quantification of macromolecules which serve as markers of leakage (Van Rensen et al. 2002). Another hypertonic challenge is the recently developed inhalation of hypertonic mannitol dry powder, the results of which show good correlation with the other physical challenges exercise, hypertonic saline, and eucapnic voluntary hyperpnea. A major advantage of this procedure is its technical simplicity as there is no need for an energy source, nor for sophisticated machinery to aerosolize the molecule; the system is portable, which could make it very useful for epidemiologic purposes. The efficacy (in comparison to hypertonic saline) and the safety of this challenge has been demonstrated in a large population of asthmatic and nonasthmatic subjects (Brannan et al. 2005).

Conclusions BHR is a major characteristic of asthma. The methods to measure it have evolved greatly and have revealed the complexity of the airway response in patients with asthma. Measures of bronchial responsiveness are widely used for diagnosis and monitoring of asthma (Table 35.5). A wide array of nonspecific bronchoconstrictor stimuli is available. Methacholine and histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Indirect challenges (adenosine, exercise, hypertonic saline, etc.) induce airflow limitation by an action on cells other than smooth muscle cells. Whereas bronchial responsiveness to a direct stimulus is only weakly related to airway inflammation, indirect airway challenges might better reflect active


airway inflammation. Both direct and indirect airway challenges are useful outcome parameters in clinical studies of asthma. Bronchial challenges are also an essential step in the development of some of the potential new antiasthma treatments, such as adenosine or tachykinin receptor antagonists.

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in asthma, allergic rhinitis and chronic obstructive pulmonary disease. Clin Exp Allergy 32, 824–30. van den Toorn, L.M., Prins, J.B., Overbeek, S.E., Hoogsteden, H.C. & de Jongste, J.C. (2000) Adolescents in clinical remission of atopic asthma have elevated exhaled nitric oxide levels and bronchial hyperresponsiveness. Am J Respir Crit Care Med 162, 953–7. van den Toorn, L.M., Overbeek, S.E., de Jongste, J.C., Leman, K., Hoogsteden, H.C. & Prins, J.B. (2001) Airway inflammation is present during clinical remission of atopic asthma. Am J Respir Crit Care Med 164, 2107–13. Van Rensen, E.L., Hiemstra, P.S., Rabe, K.F. & Sterk, P.J. (2002) Assessment of microvascular leakage via sputum induction: the role of substance P and neurokinin A in patients with asthma. Am J Respir Crit Care Med 165, 1275–9. Van Schoor, J., Joos, G.F., Kips, J.C., Drajesk, J.F., Carpentier, P.J. & Pauwels, R.A. (1997) The effect of ABT-761, a novel 5–lipoxygenase inhibitor, on exercise- and adenosine-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med 155, 875–80. Van Schoor, J., Joos, G.F., Chasson, B.L., Brouard, R.J. & Pauwels, R.A. (1998) The effect of the NK2 tachykinin receptor antagonist SR 48968 (saredutant) on neurokinin A-induced bronchoconstriction in asthmatics. Eur Respir J 12, 17–23. Van Schoor, J., Joos, G.F. & Pauwels, R.A. (2000) Indirect bronchial hyperresponsiveness in asthma: mechanisms, pharmacology and implications for clinical research. Eur Respir J 16, 514–33. Van Schoor, J., Joos, G. & Pauwels, R.A. (2002) Effect of inhaled fluticasone propionate on bronchial responsiveness to neurokinin A in asthma. Eur Respir J 18, 997–1002. Van Schoor, J., Pauwels, R. & Joos, G. (2005) Indirect bronchial hyperresponsiveness: the coming of age of a specific group of bronchial challenges. Clin Exp Allergy 35, 250–61. van Velzen, E., van den Bos, J.W., Benckhuijsen, J.A., van Essel, T., de Bruijn, R. & Aalbers, R. (1996) Effect of allergen avoidance at high altitude on direct and indirect bronchial hyperresponsiveness and markers of inflammation in children with allergic asthma. Thorax 51, 582–4. Vizi, E., Huszar, E., Csoma, Z. et al. (2002) Plasma adenosine concentration increases during exercise: a possible contributing factor in exercise-induced bronchoconstriction in asthma. J Allergy Clin Immunol 109, 446–8. Ward, C., Pais, M., Bish, R. et al. (2002) Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma. Thorax 57, 309–16. Weiss, S., Robb, G.P. & Ellis, L.B. (1932) The systemic effects of histamine in man with special reference to the cardiovascular system. Arch Intern Med 49, 360–9. Woolcock, A.J., Yan, K. & Salome, C.M. (1988) Effect of therapy on bronchial hyperresponsiveness in the long-term management of asthma. Clin Allergy 18, 165–76. Woolcock, A.J., Anderson, S.D., Peat, J.K. et al. (1991) Characteristics of bronchial hyperresponsiveness in chronic obstructive pulmonary disease and in asthma. Am Rev Respir Dis 143, 1438–43.

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Exercise-induced Bronchoconstriction: Animal Models Arthur N. Freed and Sandra D. Anderson

Summary Hyperventilation with cool dry air damages bronchial mucosa and increases airway resistance in all mammalian species examined to date. Although hyperventilation-induced bronchoconstriction (HIB) appears to be qualitatively similar in most animal models and closely mimics exercise-induced asthma in human subjects, species-specific differences do exist. These differences must be taken into account when extrapolating model-derived data to the human condition. Data from animal models confirm that hyperventilation with dry air stimulates the production and release of numerous bronchoactive mediators including leukotrienes, prostanoids, and tachykinins. These mediators clearly modulate the development of HIB. Airway cooling may counterbalance the constrictor effect of airway drying by inhibiting either mediator release or the neuronal activity that accompanies HIB. Various animal models have shown that HIB and bronchovascular hyperpermeability develop simultaneously, making it likely that they also co-occur in humans. However, animal and human studies indicate that bronchovascular leakage and HIB result from independent mechanisms. Although the role of airway edema formation in the development of HIB remains unclear, the former may amplify the effects of smooth muscle contraction. Animal model data confirm human studies implicating the existence of a hyperventilation-induced late-phase response. Delayed airway obstruction develops as a direct result of acute airway injury and mediator release. Both airway hyperosmolarity and airway cooling appear necessary for all aspects of this late response to develop. Canine model data also reveal that an acute hyperventilation-induced mucosal injury is likely to heal quickly, whereas repeated hyperventilation with cold dry air impairs the repair process, and results in persistent inflammation. Chronic inflammation promotes airways remodeling in this model, and results in an asthma-like condition. Thus, repetitive exposure to cold dry air may be one of many environmental factors that contribute to the development of asthma in normal human subjects.


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Animal models of exercise-induced bronchoconstriction Isocapnic hyperventilation-induced bronchoconstriction (HIB) occurs in all mammalian species examined to date (Jammes et al. 1983; Chapman & Danko 1985; Freed et al. 1985; Biagini et al. 1991; Koyama et al. 1992; Yang et al. 1999a), reflecting a conserved physiologic mechanism for limiting access of environmental irritants to the distal lung. Smooth muscle contraction enhances airway barrier function by reducing transepithelial conductance (Freed & Croxton 1993). This mechanism may provide additional protection to subepithelial tissues from environmental insult. Hyperventilation with cold dry air elicits qualitatively similar responses from normal and asthmatic subjects (O’Cain et al. 1980; Therminarias et al. 1998) (Fig. 36.1a). This suggests that the mechanisms directly contributing to the development of HIB are present in the normal population. Although HIB in normal individuals involves very small changes in forced expiratory volume in 1 s (FEV1) (4% on average), the exaggerated response measured in asthmatic individuals probably reflects smooth muscle sensitivity to inflammatory mediators released in response to hyperpnea. Whole-lung animal models are those in which dry air or a dry gas mixture bypasses the upper airway and directly enters the lower trachea via a tube. Guinea pigs are the most commonly used species (Chapman & Danko 1985; Ray et al. 1988; Nagase et al. 1996; Lai & Lee 2000), although rats (Yang et al. 1999a), rabbits (Koyama et al. 1992; Yuan & Nail 1995; Högman et al. 1997), cats (Jammes et al. 1983), and monkeys (Biagini et al. 1991) have also been studied with varying levels of success. The main criticisms of these models are that (i) they are anesthetized and mechanically ventilated during hyperventilation, (ii) the condition of the air as it passes from the tracheal tube to the lung is not the same as if it passed over the upper airway mucosa, and (iii) the tidal volumes and frequencies used during hyperventilation do not truly mimic natural breathing conditions. In addition to whole-lung models (Fig. 36.1b,c), a sublobar airway model is used in which canine peripheral airways are isolated and hyperventilated via a bronchoscope (Freed et al.

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same magnitude of respiratory heat and water loss as smaller increases in ventilation with cold air. Finally, racehorses and sled dogs are two natural models that can be used to study HIB. Racehorses routinely increase minute ventilation 20-fold or greater during strenuous exercise, often under subfreezing conditions (Davis et al. 2002a). Racing sled dogs (Davis et al. 2002b), which can sustain speeds as high as 25 km/hour (Van Citters & Franklin 1969), are capable of running great distances in extremely cold and desiccating environments. Both species exhibit hyperventilationinduced mucosal injury and inflammation similar to that seen in asthmatic subjects.

6 12 18 24 30

Fig. 36.1 Hyperventilation-induced bronchoconstriction in mammals. (a) Asthmatic (closed circles) (Finnerty et al. 1992) and normal nonatopic (O’Cain et al. 1980) humans (open circles). Asthmatic subjects hyperventilated with room temperature air. Normal subjects hyperventilated with either saturated room temperature air at 123 L/min (1), dry subfreezing air at 61 L/min (2), or dry subfreezing air at 123 L/min (3). (b) Guinea pigs: hyperventilated with dry gas containing 95% O2 (closed circles) (data from Ray et al. 1988); hyperventilated with dry gas containing 21% O2 (open circles) (data from Chapman & Danko 1985). (c) Rabbits: sensitized to ovalbumin (closed circles); non-sensitized (open circles) (data from Koyama et al. 1992). (d) Dogs: hyperventilated with 2000 mL/min for 5 min (closed circles) (data from Freed & Adkinson 1990); hyperventilated with 1500 mL/min for 2 min (open circles) (data from Freed et al. 1994b). RL, pulmonary resistance; Rp, peripheral airway resistance; FEV1, forced expiratory volume in 1 s; Vmax 30% VC, maximum flow at 30% of vital capacity. Unless stated, isocapnic hyperventilation was done with either dry room air or 5% CO2 and dry air.

1985) (Fig. 36.1d). In this model, peripheral airway resistance typically increases between 50 and 200% above baseline, and the severity of HIB increases with increasing rates of ventilation (Freed et al. 1987a; Freed 1989). A major criticism of this model is that airflow is unidirectional, eliminating a primary heat and water recovery mechanism that functions under normal conditions in all mammals including humans. However, unidirectional hyperventilation simply increases the evaporative cooling and water loss at any given location in the airway when compared to normal ventilation (Daviskas et al. 1991). This is analogous to increasing tidal volume in whole-lung models to increase the penetration of dry air into the distal lung (Ray et al. 1990). In fact, air (or airway) temperatures in human airways recorded during and after normal hyperventilation with subfreezing air are similar to those recorded in similar-sized canine bronchi exposed to unidirectional hyperventilation with room temperature gas (McFadden et al. 1985; Freed et al. 1987a). Thus local heat and water fluxes created by these two modes of hyperventilation can be similar. Equivalent losses can be generated by brief periods of hyperventilation with cold air and prolonged periods of hyperventilation with warm air. Similarly, big increases in ventilation with warm air can approximate the

Early response to hyperventilation with dry air HIB usually develops and resolves within 30–60 min. Maximal narrowing typically occurs 2–10 min after hyperpnea ends in rats (Yang et al. 1999a), guinea pigs (Ray et al. 1991), rabbits (Koyama et al. 1992), cats (Jammes et al. 1983), dogs (Freed et al. 1989), and human (Blackie et al. 1990) (see Fig. 36.1). This delay may result from either thermal (Freed et al. 1987b, 1991) or mechanical (Blackie et al. 1990; Ray et al. 1991) mechanisms that modulate mediator release during hyperventilation (Freed et al. 1999). Studies in humans and dogs show that HIB is attenuated when airway cooling is sustained into the recovery period. Bronchoconstriction develops slowly and mediator release is delayed during exposure to cool dry air, as opposed to warm humid air, suggesting that cooling affects mediator release during hyperpnea (Freed et al. 1999). These events can account for the slow rise in airway resistance that characterizes this early response. Thus, exercise-induced bronchoconstriction (EIB) may result from an imbalance between two opposing stimuli: evaporative water loss that stimulates and evaporative cooling which inhibits the development of airway obstruction (Freed et al. 1987b). The time over which HIB develops may be species- or even strain-dependent. Of all the animal models examined to date, the rabbit appears to be less sensitive to hyperpnea than guinea pigs, dogs, and asthmatic humans (Fig. 36.1), whereas recovery appears to occur more rapidly in cats (Jammes et al. 1983). The one study examining the effect of cold dry air in monkeys suggests that obstruction develops slowly, with maximal changes in pulmonary resistance appearing about 25 min after the challenge (Biagini et al. 1991). Brown Norway rats appear to require even longer periods for obstruction to develop, whereas August-Copenhagen Irish (ACI) rats exhibit an unusual biphasic peak in airway obstruction (Yang et al. 1999a). However, full recovery of normal lung function was not reported for rat or monkey models, and the unusually long recovery times may result from mechanisms unrelated to HIB. HIB in normal human subjects is associated with mild central airway narrowing (O’Cain et al. 1980), whereas EIB is associated with severe peripheral airway narrowing (Wessling & Wouters 1992). Mild hyperpnea in guinea pigs elicits central

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airway narrowing, whereas more severe levels produce peripheral airway narrowing (Ray et al. 1990; Nagase et al. 1994). Thus, penetration of dry air into the distal lung is a key determinant of HIB in all mammals, regardless of disease state. Studies in dogs (Freed et al. 1985) and in human subjects (Kaminsky et al. 1995, 2000) confirm the direct involvement of small airways in HIB. Both smooth muscle contraction and airway edema have been proposed as the mechanism for the airway narrowing of HIB. β2-Adrenoreceptor agonists inhibit or abolish HIB in guinea pigs (Chapman & Danko 1985), rabbits (Horie et al. 1992), dogs (Teeter & Freed 1991; Wang et al. 1992), and humans (Anderson & Schoeffel 1982). They do so by inhibiting mediator release and reducing smooth muscle responsiveness, and by enhancing water replacement to the airway and its surface during hyperpnea in guinea pigs and dogs (Wang et al. 1992; Yang et al. 1999b). The efficacy of β2 agonists in preventing HIB and the speed with which the airway obstruction develops and subsides (Freed et al. 1985; Blackie et al. 1990; Ray et al. 1991; Koyama et al. 1992) (Fig. 36.1) favors mediator-induced smooth muscle contraction over airway edema as the primary cause of HIB. The parasympathetic nervous system modulates HIB in some but not all asthmatic humans (Finnerty & Holgate 1993). Although parasympathetic activity plays no role in the guinea-pig model (Chapman & Danko 1985; Ray et al. 1988), it does so to varying degrees in rabbits (Horie et al. 1992; Yuan & Nail 1995), cats (Jammes et al. 1983), and dogs (Freed et al. 1987a, 2000; Tang & Freed 1992). Blocking parasympathetic activity reduces HIB in dogs and abolishes it in rabbits and cats, making the latter two models ideal for studying the vagal component of this phenomenon. Engorgement and leakage of bronchial vessels are also likely to contribute to HIB (McFadden 1990). Hyperpnea with dry air causes vasodilation in dogs (Omori et al. 1995a) and sheep (Parsons et al. 1989); vascular hyperpermeability in rats (Yang et al. 1999a), guinea pigs (Fig. 36.2a,b), and dogs (Fig. 36.2c,d); and airway edema in rabbits (Högman et al. 1997). These phenomena are also likely to occur in human subjects. Although animal data reveal that airway edema and not bronchovascular engorgement causes airway obstruction in response to volume loading (Tang & Freed 1994), neither directly contributes to the development of HIB. HIB and bronchovascular hyperpermeability develop simultaneously (Garland et al. 1991; Freed et al. 1994a; Yang et al. 1997), and the latter persists for at least 24 hours in the canine model (Omori et al. 1995b). It is unlikely that this prolonged vascular response is responsible for HIB. Bronchial hyperpermeability induced by hyperpnea is unaffected by eicosanoid and tachykinin antagonists in guinea pigs, or by β2-receptor agonists in dogs despite their ability to inhibit HIB (Garland et al. 1993; Solway et al. 1993; Omori et al. 1995a; Yang et al. 1997; Lai & Lee 1999). Leukotriene and tachykinin antagonists do inhibit bronchial hyperpermeability

(a)

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Fig. 36.2 Bronchovascular leakage in unchallenged control animals (Cont, open bar) and animals hyperventilated with either warm moist air (WAC, light blue bar) or cool dry air (DAC, dark blue bar). (a) Guinea pig main bronchi; (b) guinea pig lower bronchi (data from Garland et al. 1991). (c) Canine peripheral airways (regardless of size); (d) canine peripheral airways (as a function of airway diameter) (data from Freed et al. 1994a). Bronchovascular leakage is expressed as extravasation of either Evans blue dye, monastral blue, or carbon black. The latter two colloids are “vascular labels.” **, P < 0.01 compared to control.

in rats, but this effect is likely to be strain dependent (Yang et al. 1999a). Morphometric analysis of guinea-pig airways confirmed that smooth muscle contraction and not airway edema was responsible for HIB (Nagase et al. 1994). Finally, ligation of the bronchial artery prior to hyperpnea abolishes microvascular leakage in the canine model without affecting bronchoconstriction (Freed et al. 1995), demonstrating that the bronchial circulation plays little if any role in HIB. Bronchovascular leakage may protect airway mucosa from desiccation injury (Persson et al. 1991), the latter resulting in mucus secretion, goblet cell degranulation, microvascular hyperpermeability, and smooth muscle contraction in canine airways (Freed et al. 1994a; Omori et al. 1995b) (Fig. 36.3). Smooth muscle contraction may provide protection by narrowing the bronchial lumen, inhibiting cold air penetration, and decreasing the mucosal surface exposed to desiccation. Microvascular leakage may account for the enhanced mucociliary clearance reported in normal and asthmatic subjects (Daviskas et al. 1995, 1996), and in turn may enhance the removal of bronchoactive mediators (Kelly et al. 1986; Wagner & Mitzner 1990). Finally, the fact that furosemide, norepinephrine, and methoxamine each enhance bronchovascular

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Normal epithelium

Mucus

Vagal afferent

Injured epithelium

IL-2 IL-4 IL-6 IL-8 IL-16 NO LTB4 EO GM-CSF RANTES

CI Goblet

Def-1 Def-2 CAP

CNS

IL-4 IL-5 CD4+ IL-9 IL-10

Axon reflex? PGs LTs Endo

Heat/H2O Bronchial vessel

PGs NO Eos b-agonists VIP

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Squamous epithelium

PGs Mast LTs IL-4 IL-5 IL-8 IL-16 Tryptase SP

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Repairing epithelium

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a-agonists

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MMP-9 TGF-b IL-4 IL-16 GM-CSF LTs PGs

IL-1 GM-CSF IL-6 TGF-b IL-8 TNF-a IL-10 IL-12

Ach NANC efferent

Macro

SP

Smooth muscle (constricted)

PGs IL-8 GM-CSF TGF-b

Fig. 36.3 Schematic of proposed cascade of events that produce hyperventilation-induced bronchoconstriction, late-phase airway obstruction, and airway remodeling. Ach, acetylcholine; ASL, airway surface liquid; CAP, CAP37/azurocidin; CGRP, calcitonin gene-related peptide; Ciliated, ciliated epithelium; CNS, central nervous system; Def-1 and Def-2, defensins HNP-1 and HNP-2; Endo, endothelin; EO, eotaxin; Eos, eosinophil; Goblet, goblet cell; GM-CSF, granulocyte–macrophage colonystimulating factor; Heat/H2O, loss of heat and water from airway surface

and vasculature; IL, interleukin; LTs, leukotrienes; Mast, mast cell; Macro, macrophage; MMP-9, matrix metalloproteinase-9; NO, nitric oxide; PGs, prostaglandins; PMN, neutrophil; RANTES, a chemokine “Regulated by Activation, Normal T-cell Expressed and Secreted”; NANC, nonadrenergic noncholinergic nervous system; NKA, neurokinin A; SP, substance P; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-a; Tx, thromboxane; VIP, vasoactive intestinal polypeptide. (See CD-ROM for color version.)

leakage induced by hyperpnea in dogs (Freed et al. 1996, 1997) suggests that they may serve to counterbalance the effects of evaporative water loss. Although unknown, even small changes in microvascular permeability may be sufficient to compensate for water loss during exercise in cold environments.

ments in dogs revealed that ASL osmolarity of the peripheral airways does increase during hyperventilation with dry air (Freed & Davis 1999). This increase in osmolarity is thought to initiate the synthesis and release of bronchoactive mediators (Fig. 36.4). The extent of bronchoconstriction produced by hyperventilation correlates with that produced by hypertonic saline in asthmatic humans (Belcher et al. 1989; Smith & Anderson 1989) and dogs (Freed et al. 1989), and supports the hypothesis that ASL hyperosmolarity initiates HIB (Anderson et al. 1982). Hypertonicity triggers mediator release in vitro from mast cells (Eggleston et al. 1987) and tachykinin release from cultured C-fiber neurons (Garland et al. 1995). Hyperventilation increases in vivo eicosanoid metabolism in guinea pigs, dogs, and asthmatic subjects (Fig. 36.4). Dietary salt loading in guinea pigs (Mickleborough et al. 2001) and asthmatic subjects (Mickleborough et al. 2005) also elevates mediator activity and enhances HIB, confirming the modulatory role of hypertonicity in the development of this response.

Role of airway surface liquid osmolarity Indirect estimates of hyperventilation- or exercise-induced airway water flux and airway surface liquid (ASL) osmolarity have provided contradictory results (Anderson et al. 1989; McFadden et al. 1999). Although subfreezing air penetrates to at least the level of fifth-generation bronchi during hyperventilation (McFadden et al. 1985), direct measurement of ASL osmolarity failed to detect any postchallenge changes in either normal (Kotaru et al. 2002) or asthmatic (Kotaru et al. 2003) human subjects. Unfortunately, ASL samples were obtained from the trachea, and as such are inappropriate to detect desiccation of the peripheral airways. However experi-

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* *

210 * 60 30 0

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Hyperventilation-induced airway injury

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Fig. 36.4 Mediators in bronchoalveolar lavage fluid recovered from unchallenged control animals (open bar) and from animals hyperventilated with either warm moist air (light blue bar) or cool dry air (dark blue bar). (a) Guinea pigs (data from Ingenito et al. 1990, 1992). (b) Guinea pigs (data from Lai & Lee 1999). (c) Canine peripheral airways (data from Freed et al. 1999). (d) Asthmatic human subjects (data from Pliss et al. 1990). LTB4, leukotriene B4; LTE4, leukotriene E4; LTC4-E4, leukotrienes C4, D4, and E4; PGF2a, prostaglandin F2a; SP, substance P; TxB2, thromboxane B2. *, P < 0.05; **, P < 0.01 compared to control.

50 Epithelial cells (%)

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Mucosal injury following hyperventilation was originally documented using bronchoalveolar lavage fluid cell analyses in guinea pigs, dogs, horses, and asthmatic subjects (Fig. 36.5), and more recently using similar analyses of sputum induced after exercise challenge in human subjects with EIB (Hallstrand et al. 2005). Morphometric analyses of hyperventilated canine airways confirmed that these sampling techniques accurately reflect injury of the bronchial mucosa (Freed et al. 1994a; Omori et al. 1995b). Warming and humidifying the air reduces bronchoconstriction in rats (Yang et al. 1999a), guinea pigs (Ray et al. 1988; Yang et al. 1997), rabbits (Ohtsuka et al. 1993), dogs (Freed et al. 1985, 1987a), and human (Anderson et al. 1982; Stensrud et al. 2006), and nearly eliminates all evidence of airway mucosal injury (Freed et al. 1987c, 1994a, 1999). β2 agonists also protect the bronchial mucosa from injury (Omori et al. 1995a). In contrast to fully or partially conditioned air that reduces the strength of the stimulus, β2 agonists probably decrease desiccation and injury by increasing tramucosal fluid flux (Davis et al. 1979; Wang et al. 1992). Mucosal injury appears to initiate airway narrowing by the release of mediators, and the magnitude of mediator release is directly related to the severity of mucosal injury (Omori et al. 1995a,b; Freed et al. 1999). In canine airways the injury occurs before the release of eicosanoids. This release occurs during or soon after hyperventilation ends (Freed et al. 1999). Thus endogenous release of mediators is unlikely to cause mucosal injury during hyperventilation, although it is possible that mediators do contribute to the mucosal injury. Because hyperventilation with fully conditioned air precludes mucosal injury and inhibits bronchoconstriction (Freed et al.

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Fig. 36.5 Epithelial cells in bronchoalveolar lavage fluid recovered from unchallenged control animals (Cont, open bar) and animals hyperventilated with either warm moist air (WAC, light blue bar) or cool dry air (DAC, dark blue bar). (a) Guinea pig (data from Ingenito et al. 1990). (b) Canine peripheral airways (data from Freed et al. 1999). (c) Horse (data from Davis et al. 2002a). (d) Asthmatic human (data from Pliss et al. 1990). *, P < 0.05; **, P < 0.01 compared to control.

1985, 1994a, 1999), it is unlikely that the mechanical stress developed during hyperventilation causes mucosal injury. Finally, hypertonicity per se does not cause mucosal injury in either dogs or asthmatic subjects (Freed et al. 1994b; Makker

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et al. 1994). Thus the mechanism that results in mucosal injury remains unknown.

Hyperventilation-induced mediator production and release Hyperventilation in dogs (Omori et al. 1995b) and exercise in humans (Crimi et al. 1992) causes mast cell degranulation, and represents direct evidence that mediators contribute to the development of HIB (Fig. 36.3). Mediator release typically occurs 2–5 min after hyperventilation stops (Freed et al. 1999). Although this delay may simply reflect de novo biosynthesis, the temperature change that occurs during hyperventilation may retard mediator release. While hyperventilation with warm humid air inhibits HIB in dogs, the residual obstruction develops earlier, a finding consistent with the reduced amount of cooling that occurs during that challenge (Freed et al. 1999). The fact that hypertonic saline aerosol produces bronchoconstriction more rapidly than either HIB in dogs (Freed et al. 1991) or HIB and EIB in asthmatic subjects (Boulet & Turcotte 1989; Smith & Anderson 1989) supports the hypothesis that cooling per se retards mediator release. Eicosanoids appear to play a significant role in the development of EIB. Numerous studies have shown that leukotrienes modulate HIB in human subjects (van Schoor et al. 1997; Kemp et al. 1998; Lehnigk et al. 1998) (Fig. 36.4d) and in animal models including rats (Yang et al. 1999a), guinea pigs (Garland et al. 1993; Yang et al. 1997; Lai & Lee 2000; Mickleborough et al. 2001) (Fig. 36.4a,b), and dogs (Omori et al. 1996; Freed et al. 2000) (Fig. 36.4c). By comparison, the role of prostanoids in HIB has received far less attention. Hyperpnea-induced prostanoid activity reported in guinea pig (Garland et al. 1993; Yang et al. 1997; Suman et al. 2000) (Fig. 36.4a) and canine models (Freed et al. 1987a,c, 2000; Omori et al. 1996) (Fig. 36.4c) mirror results from similar human studies (Shimizu et al. 1997; O’Sullivan et al. 1998) (Fig. 36.4d), implicating products of cyclooxygenase activity in the development of HIB. Pharmacologic studies indicate that approximately half of the airway narrowing that occurs in response to hyperventilation can be independently attributed to prostanoid and leukotriene activity in the canine model (Freed et al. 1987a,c; Omori et al. 1996) and in asthmatic human subjects (Adelroth et al. 1997; Reiss et al. 1997; Shimizu et al. 1997; van Schoor et al. 1997). However, inhibition of cyclooxygenase and 5lipoxygenase simultaneously provides no more protection than when blocking either pathway alone (Freed et al. 2000). This suggests that these eicosanoids act in series (i.e., either leukotrienes stimulate prostaglandin activity or vice versa) and not in parallel to produce HIB. Eicosanoid mediators released in response to hyperpnea may act directly or indirectly on airway smooth muscle to initiate airway narrowing. Neurokinin (NK) receptor activity modulates HIB in guinea pigs (Ray et al. 1989; Higashide et al. 1997; Anthes et al. 2002) and dogs (Freed et al. 2003), which in turn stimulates afferent C-fibers to locally release tachykinins


(Fang & Lai 1993). Initially it was proposed that leukotrienes directly mediated HIB via the secondary release of neuropeptides (Garland et al. 1993; Lai & Lee 1999). Later it was suggested that dry gas directly stimulated tachykinin release, which in turn stimulated leukotriene activity (Yang et al. 1997). The fact that leukotriene antagonists inhibit tachykinin release induced by hyperpnea in guinea pigs (Lai & Lee 1999, 2000) and NK1 and NK2 antagonists fail to inhibit eicosanoid production in dogs (Freed et al. 2003) strongly supports the initial hypothesis. However, the use of hyperoxic gas during hyperventilation (Ray et al. 1988; Yang et al. 1999a; Lai & Lee 2000) may exaggerate the role of tachykinins in guinea pig and rat models because reactive oxygen species produced during hyperventilation enhance tachykinin release and bronchoconstriction (Fang & Lai 1993; Lai et al. 2002). This, and the fact that NK receptor antagonists do not abolish HIB in either guinea pigs (Solway et al. 1993) or dogs (Freed et al. 2003), suggests that other pathways are activated during hyperventilation (Fig. 36.3). Nitric oxide (NO) has received considerable attention for its potential role in HIB. NO was reported to have little effect on HIB in guinea pigs (Nogami et al. 1998) and asthmatic subjects during and after exercise (Suman & Beck 2002). Other studies concluded that endogenous NO did not inhibit EIB (De Gouw et al. 2001) and markedly augmented HIB (Kotaru et al. 2001). In contrast to these findings, hyperpnea-induced ASL hyperosmolarity inhibits NO-induced relaxation of airway smooth muscle in rabbits (Hogman et al. 1998), and endogenous NO inhibits HIB in dogs (Suzuki & Freed 2000a) and in normal human subjects (Therminarias et al. 1998). Although further study is clearly necessary before any conclusions can be made, endogenous NO production is likely to modulate hyperventilation-induced responses in animal and human subjects. Finally, other inhibitory mediators appear to be activated in response to hyperventilation (Fig. 36.3). PGE2 is inversely correlated with the magnitude of HIB (Omori et al. 1996), supporting the hypothesis that inhibitory mediators counterbalance the development of HIB. Generation of inhibitory prostanoids in asthmatic subjects during hyperpnea supports this scenario (Pavord et al. 1992; Manning et al. 1993). In addition, hyperpnea stimulates release of vasoactive intestinal peptide (VIP) (Tang & Freed 1992) in dogs and calcitonin gene-related peptide (CGRP) in guinea pigs (Nohr et al. 1995; Nagase et al. 1996), and both may modulate HIB in concert with inhibitory prostanoids.

Late-phase airway obstruction in animals Unlike human subjects (Speelberg et al. 1991; Koh et al. 1994; Chhabra & Ojha 1998), the late response to hyperventilation (which is characterized by leukocyte infiltration, mediator release, airway obstruction, and airway remodeling) is readily

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elicited in the canine model. Isocapnic hyperventilation produces airway obstruction in normal canine bronchi within 5 hours after challenge (Freed & Adkinson 1990) that persists for as long as 24 hours after hyperventilation (Davis & Freed 1999, 2001). The magnitude of this late-phase airway obstruction is approximately the same as the initial early response in dogs (Freed & Adkinson 1990). Hyperventilation with dry air transiently alters airway morphology (Omori et al. 1995b), and the extent of these changes probably determines if late-phase airway obstruction develops. The mucosal injury induced by hyperventilation persists throughout the late phase. Injury of the airway mucosa may result in airway obstruction due to impaired production of PGE2 and NO. Although impaired PGE2 production during the early part of the late phase is not evident (Davis et al. 2002c), endogenous NO clearly modulates latephase airway obstruction and inflammation in dogs (Suzuki & Freed 2000a). The bronchovascular leakage induced by hyperventilation (Fig. 36.2) persists throughout the late phase (Omori et al. 1995b), and may amplify the effects of smooth muscle contraction via edematous thickening of the airway wall (Tang & Freed 1994) (Figs 36.3 & 36.6b,c). Although mucosal injury could cause impaired mucociliary clearance and accumulation of intraluminal debris, data discussed below make it unlikely that impaired mucociliary clearance can account for the late increase in peripheral airway resistance seen in the canine model. Pretreatment with drugs that block either cyclooxygenase or lipoxygenase will inhibit the development of latephase obstruction (Davis et al. 2002c), indicating a role for eicosanoids in the late-phase response. The fact that neither leukotrienes nor prostaglandins increase during the late phase (Freed & Adkinson 1990; Davis et al. 2002c; Suzuki & Freed 2002) suggests that early-phase mediator release initiates late-phase airway obstruction in the canine model.

50 mm

(a)

(b)

Late-phase airway inflammation in dogs Late-phase airway obstruction in dogs is accompanied by an influx of leukocytes (Freed & Adkinson 1990; Omori et al. 1995b; Davis et al. 2002c) (Figs 36.3, 36.6 and 36.7). Leukocyte infiltration is not dependent on eicosanoids released during or after hyperventilation, as blockade of either cyclooxygenase or lipoxygenase immediately prior to the challenge has no effect on this inflammatory response (Davis et al. 2002c). Furthermore, although hypertonic aerosol challenge causes late-phase production of eicosanoids in canine peripheral airways, it does not stimulate inflammatory cell infiltration (Suzuki & Freed 2000b), suggesting that mucosal injury is necessary to initiate this event (Fig. 36.3). Bronchial epithelial cells can produce a wide variety of proinflammatory eicosanoids and cytokines (Knobil & Jacoby 1998; Holgate et al. 2000) (Fig. 36.3), leukotriene (LT)B4 and interleukin (IL)-8 act as chemoattractants for neutrophils, and the latter stimulates neutrophils to release other chemoattractants that

800

(c) Fig. 36.6 Photomicrographs of formalin-fixed, light microscopy sections of canine bronchi. (a) Normal bronchial epithelium and lamina propria (vertical bar, i.e., distance between basement membrane and smooth muscle) from a 3.4-mm diameter airway that received repeated bronchoscopy but no challenge. (b, c) Squamous metaplasia in a 3.2-mm airway 24 hours after repetitive hyperventilation with dry air. Note thickening of the lamina propria in (b) and (c) compared with (a). Note the eosinophilic influx in (c). (Adapted from Davis et al. 2003.) (See CD-ROM for color version.)

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70 1.6

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Fig. 36.7 (a) Baseline resistance in airways repeatedly wedged but not challenged (open circles) and airways repetitively hyperventilated with dry air (closed circles). Data are expressed as mean (± SEM). *, P < 0.05 compared to day 1. (b) Bronchoalveolar lavage fluid (BALF) cell concentrations from control (open bar), repeated wedge (light blue bar), and repeatedly hyperventilated (dark blue bar) airways. Mac, macrophage; Lym, lymphocytes; PMN, neutrophils; Eos, eosinophils; Epi, epithelial cells. *, significantly different from control, P < 0.05; **, significantly different from repeated wedge, P < 0.05. (Data from Davis & Freed 1999.)


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mediate T-lymphocyte infiltration (Taub et al. 1996) (Fig. 36.3). The release of IL-16 from epithelial cells and mast cells (Laberge et al. 1997; Rumsaeng et al. 1997) can selectively recruit CD4+ T cells into the airway wall, although without antigen presentation it seems unlikely that these cells would play any role in the late response. Mast cells degranulate in response to hyperventilation (Freed et al. 1994a; Omori et al. 1995b) and secrete IL-4 (Bradding et al. 1994) and IL-5 (Oliveira et al. 1996; Rumsaeng et al. 1997), the latter being a potent chemoattractant for eosinophils in asthmatic airways (Shi et al. 1997). Given the mediator-producing capability of bronchial epithelial and mast cells, a proinflammatory type-2 cytokine environment could be produced even in the absence of T-cell infiltration. The fact that Th2 cytokine expression is upregulated in horses 5 hours after breathing subfreezing air during exercise supports this hypothesis (Davis et al. 2005). The release of these mediators provides the link between the mucosal injury induced by hyperventilation and the eosinophilic inflammation (Becker & Soukup 1999) that slowly develops in human asthmatics (Crimi et al. 1992) and the canine model (Freed & Adkinson 1990; Suzuki & Freed 2002) (Fig. 36.3).

Late-phase airway hyperreactivity in dogs In dogs, but not humans, isocapnic hyperventilation with dry air and inhalation of hypertonic saline result in airway hyperresponsiveness 5– 24 hours after challenge (Davis et al. 2002c; Suzuki & Freed 2002). This increase in airway responsiveness is accompanied by increased production of eicosanoids (Suzuki & Freed 2000b, 2002; Davis et al. 2002c), which are known to contribute to the development of airway hyperreactivity in mice, dogs, and humans (Becker et al. 1995; Fischer et al. 1995; Kleeberger & Freed 1995; Nagase et al. 1997). Thus, increased eicosanoid metabolism may be responsible for the late-phase increase in airway reactivity that develops after inhalation of hypertonic saline in dogs. In contrast to hyperventilation (Freed et al. 1999; Freed & Adkinson 1990; Davis et al. 2002c), hypertonic saline challenge (Suzuki & Freed 2000b) does not cause either leukocyte infiltration or

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late-phase airway obstruction in dogs. The fact that hypertonic saline does not cause mucosal injury in either dogs or asthmatic subjects (Freed et al. 1994b; Makker et al. 1994) may account for these differences. If the main difference between a hypertonic aerosol and hyperpnea challenge is the lack of temperature change with the former stimulus, and only the latter stimulus produces a late-phase response, then unlike the early response, both airway cooling and hyperosmolarity appear to be necessary for late-phase development. Finally, late-phase airway obstruction after exercise or isocapnic hyperpnea in humans is so rare that most investigators dismiss it as experimental artifact. However, canine model data reveal that the dose–response relationship between challenge severity and HIB is distinctly different from the dose–response relationship between challenge severity and the late-phase response (Davis et al. 2002d). The early response increases at a decreasing rate with higher levels of ventilation (i.e., stimulus strength), whereas late-phase bronchoconstriction only occurs after a minimum threshold stimulus is exceeded. This observation implies that the level of ventilation attained in most human HIB and EIB studies is probably below the threshold stimulus that initiates late-phase airway obstruction. Many late-phase studies limited the maximum level of hyperpnea attained during a trial (Zawadski et al. 1988; Crimi et al. 1992; Hofstra et al. 1996), while others used only room temperature air as a stimulus (Hofstra et al. 1996). The use of subthreshold stimuli to initiate the early response may preclude the development of late-phase airway obstruction. Thus, late-phase airway obstruction may be produced if either (i) asthmatic subjects capable of achieving very high minute ventilations were selected for study and/or (ii) frigid inspired air was used for the challenge. Because the development of late-phase airway obstruction is likely to require a much stronger stimulus than the early response, it still may not be demonstrable in many human subjects. Even though cross-country skiers exercise at up to 200 L/min there are no reports of late responses in this group despite many having diagnosed asthma (Anderson et al. 2003). However, the high prevalence of chronic inflammation reported in winter

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athletes (see below) suggests that hyperventilation-induced airway inflammation is an epiphenomenon that can be readily induced, providing a more reliable indicator for late-phase study. Finally, the recognition of a threshold effect reconciles the apparent discrepancies between human and animal studies regarding the existence of the late-phase response to exercise or isocapnic hyperventilation.

Repetitive airway exposure to cold air in humans Elite athletes who exercise in cold environments exhibit an increased incidence of airway hyperreactivity and asthma (Larsson et al. 1993; Leuppi et al. 1998; Sue-Chu et al. 1999; Karjalainen et al. 2000), without a significant increase in atopy (Larsson et al. 1993; Leuppi et al. 1998; Sue-Chu et al. 1999; Karjalainen et al. 2000). These athletes typically exhibit airway inflammation, characterized by eosinophil, mast cell, and T-leukocyte infiltration, and airway remodeling (SueChu et al. 1999; Karjalainen et al. 2000). The fact that elite cold-weather athletes appear to disproportionately suffer from airway inflammation and airway hyperresponsiveness supports the concept that repeated exposure of the lung periphery to unconditioned air could result in chronic airways disease and asthma (Larsson et al. 1993; Leuppi et al. 1998; Sue-Chu et al. 1999; Karjalainen et al. 2000).

Repetitive hyperventilation-induced airway obstruction in dogs Repetitive hyperventilation over a period of days or weeks results in chronic lower airways disease. Peripheral airways in dogs anesthetized and hyperventilated every other day for 2 weeks exhibited significant obstruction after four challenges, and then stabilized at about 150% of their original airway resistance (Davis & Freed 1999) (Fig. 36.7a). Airway obstruction developed more rapidly when canine peripheral airways were challenged daily. Airway resistance was increased within 24 hours after the first challenge, and then stabilized at approximately 200% of the original value (Davis & Freed 2001).

Repetitive hyperventilation-induced airway inflammation Repeated injury of canine peripheral airways with unconditioned air results in an airway phenotype similar to asthma. Repetitive hyperventilation causes airway inflammation in dogs characterized by mast cell infiltration and the progressive development of neutrophilia and eosinophilia (Davis & Freed 1999, 2001; Davis et al. 2003) (Figs 36.6c and 36.7b). Whether granulocytic inflammation and airway obstruction reflect cause and effect or independent events initiated by a common stimulus remains unknown. Although neutrophil infiltration was correlated with late-phase airway obstruc-

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tion in dogs (Davis & Freed 2001) and has been associated with airway obstruction in response to a variety of noxious stimuli (Fabbri et al. 1984; Seltzer et al. 1984; Cormier et al. 1993), other studies suggest that neutrophils are neither necessary nor sufficient to cause obstruction (O’Byrne et al. 1984; Imai et al. 1990). Thus, neutrophil influx may not be directly related to the development of airways obstruction. However, eosinophil influx was also correlated with the severity of peripheral airway obstruction in dogs (Davis et al. 2003). These granulocytes secrete cationic proteins which can block prejunctional M2 muscarinic receptors, leading to increased tonic secretion of acetylcholine and increased airway resistance (Fryer & Jacoby 1992). They can also damage airway mucosa and connective tissue (Coyle et al. 1994). The former effect could reduce inhibitory prostanoid production and impair mucociliary clearance; the latter would promote edema formation via decreased tissue pressure and increased bronchovascular permeability. Both mucosal injury and lamina propria thickening are present in airways after repeated hyperventilation with unconditioned air (Davis et al. 2003), providing additional evidence for the role of eosinophils in this canine model of EIB.

Repetitive hyperventilation-induced airway remodeling Submucosal edema is believed to contribute to airway obstruction in asthmatic human subjects (Hogg et al. 1987). Data demonstrating a thickened lamina propria in dogs repeatedly hyperventilated with unconditioned air supports this belief (Davis et al. 2003) (Fig. 36.6a vs. Fig. 36.6b,c). Airway wall thickening is in part due to edema that may result from increased bronchovascular permeability, and/or decreased tissue hydrostatic pressure, and/or increased tissue oncotic pressure. A single bout of hyperventilation will produce bronchovascular hyperpermeability that persists for at least 24 hours after acute bronchoconstriction occurs (Omori et al. 1995b). The contribution of other factors, such as increased oncotic pressure (due to leakage and retention of plasma proteins) or increased compliance of the interstitial space (due to the breakdown of interstitial matrix by granulocyte-derived proteases) (Koller et al. 1993) is unknown. However, given the magnitude of granulocyte influx, these cells likely contribute to the development of repetitive hyperventilationinduced airway wall thickening. Repeated hyperventilation also causes the remodeling of normal ciliated epithelium into stratified squamous epithelium. Under normal circumstances, damaged epithelium is quickly repaired by the migration and expansion of adjacent basal cells (Rennard 1996). Undifferentiated cell stratification occurs within 2–3 days, followed by transformation into normal ciliated columnar epithelium. Thus, at the normal rate of repair, airways that were injured one day prior to harvesting would be expected to have only a single layer of cells covering the basement membrane. The appearance of

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multiple layers of squamous epithelium in repetitively injured canine airways (Figs 36.3 and 36.6b,c) suggests that either repair mechanisms are upregulated or the areas of stratification represent a more advanced stage of repair resulting from previous exposures. Repetitive hyperventilation-induced airway mucosal remodeling may reflect functional adaptations that protect against further desiccation-induced injury. The appearance of stratified squamous epithelia is normally associated with protection from desiccation and mechanical injury (Banks 1986). This raises the possibility that squamous metaplasia of the airway mucosa protects the underlying tissues from desiccation better than normal ciliated columnar epithelium. However, it may also impede movement of extravasated fluid into the lumen, resulting in further edema formation and thickening of the airway wall. The airway remodeling observed after repetitive hyperventilation may have other adverse consequences on airway function. Bronchial epithelium is an important source of PGE2, which can inhibit cholinergic neurotransmission (Ito et al. 1990), mast cell degranulation (Kleeberger & Freed 1995), and smooth muscle contraction (Spannhake et al. 1978). Thus, repetitively injured and remodeled airway mucosa may produce less PGE2. In addition, mucociliary clearance is likely to be impaired in repetitively hyperventilated airways due to a decrease in ciliated cell density. Indeed, the deleterious effects of airway remodeling may outweigh any protective benefits the remodeling may provide.

Summary and conclusion Hyperventilation with cool dry air causes airway mucosal injury and increases resistance in all mammals examined to date. Although the functional response to hyperventilation in most mammalian models appears qualitatively similar, species-specific differences exist. Thus caution is required in the extrapolation of data from any given animal model to the human condition. Data from animal models indicate that hyperventilation with dry air increases bronchoactive mediator metabolism. Both human and animal studies suggest that airway cooling counterbalances the constrictor effect of airway drying, and cooling may inhibit either the neuronal or mediator activity that contributes to HIB. Although eicosanoids and neurokinins contribute to the development of airway obstruction, the exact pathways leading to acute airway narrowing are unknown, and the hierarchy of mediator activity may be species-dependent. Less clear is the role for other types of mediators such as NO and VIP, and the contribution of airway edema to airway narrowing. All animal models examined to date exhibit bronchovascular hyperpermeability, suggesting that it also occurs in human. HIB and bronchovascular leakage are products of


independent mechanisms. The former response probably reflects a protective mechanism that minimizes distal lung desiccation and injury, with asthmatic individuals exhibiting an exaggerated version of this normal mammalian response. The latter response appears to further modulate the bronchoconstriction, possibly protecting the airway mucosa from desiccation injury. Although HIB is primarily the result of airway smooth muscle contraction, edema may amplify its effect. Late-phase airway obstruction begins during the early phase and both ASL hyperosmolarity and airway cooling appear to be necessary to produce all aspects of the response. Bronchial epithelial cells and mast cells may generate proinflammatory type-2 cytokines even in the absence of T-cell infiltration, providing a link between acute mucosal injury and the delayed eosinophilic inflammation that develops in response to acute hyperventilation in canine and asthmatic human subjects. Finally, although acute mucosal injury induced by hyperventilation is likely to be quickly repaired in normal individuals, repetitive hyperventilation with cold dry air appears to interfere with mucosal repair and results in chronic inflammation. This persistent inflammation promotes airway remodeling, and may ultimately result in asthma. Thus, repeatedly overwhelming mechanisms that normally protect peripheral airways from desiccation may account for the high incidence of asthma-like symptoms and inflammation reported in winter athletes (Larsson et al. 1993; Karjalainen et al. 2000). In fact, repetitive exposure to dry air may be one of many environmental factors that can contribute to the development of asthma.

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Mickleborough, T.D., Lindley, M.R. & Ray, S. (2005) Dietary salt, airway inflammation, and diffusing capacity in exercise-induced asthma. Med Sci Sports Exerc 37, 904–14. Nagase, T., Dallaire, M.J. & Ludwig, M.S. (1994) Airway and tissue responses during hyperpnea-induced constriction in guinea pigs. Am J Respir Crit Care Med 149, 1342–7. Nagase, T., Ohga, E., Katayama, H. et al. (1996) Roles of calcitonin gene-related peptide (CGRP) in hyperpnea-induced constriction in guinea pigs. Am J Respir Crit Care Med 154, 1551–6. Nagase, T., Ishii, S., Katayama, H., Fukuchi, Y., Ouchi, Y. & Shimizu, T. (1997) Airway responsiveness in transgenic mice overexpressing platelet-activating factor receptor. Roles of thromboxanes and leukotrienes. Am J Respir Crit Care Med 156, 1621– 7. Nogami, H., Umeno, E., Kano, S., Hirose, T. & Nishima, S. (1998) Effect of nitric oxide synthase inhibitor on allergen- and hyperventilation-induced bronchoconstriction in guinea-pigs. Eur Respir J 12, 1318–21. Nohr, D., Eiden, L.E. & Weihe, E. (1995) Coexpression of vasoactive intestinal peptide, calcitonin gene-related peptide and substance P immunoreactivity in parasympathetic neurons of the rhesus monkey lung. Neurosci Lett 199, 25–8. O’Byrne, P.M., Walters, E.H., Aizawa, H., Fabbri, L.M., Holtzman, M.J. & Nadel, J.A. (1984) Indomethacin inhibits the airway hyperresponsiveness but not the neutrophil influx induced by ozone in dogs. Am Rev Respir Dis 130, 220–4. O’Cain, C.F., Dowling, N.B., Slutsky, A.S. et al. (1980) Airway effects of respiratory heat loss in normal subjects. J Appl Physiol 49, 875– 80. Ohtsuka, A., Koyama, S. & Horie, T. (1993) [Influence of airway temperature and humidity of hyperventilation induced bronchoconstriction in rabbits.] Arerugi 42, 864–9. Oliveira, S.H., Faccioli, L.H., Cunha, F.Q. & Ferreira, S.H. (1996) Participation of interleukin-5 and interleukin-8 in the eosinophil migration induced by a large volume of saline. Int Arch Allergy Immunol 111, 244–52. Omori, C., Schofield, B.H., Mitzner, W. & Freed, A.N. (1995a) A β2adrenergic agonist inhibits dry air-induced injury in canine peripheral airways. J Appl Physiol 78, 2169–79. Omori, C., Schofield, B.H., Mitzner, W. & Freed, A.N. (1995b) Hyperpnea with dry air causes time-dependent alterations in mucosal morphology and bronchovascular permeability. J Appl Physiol 78, 1043–51. Omori, C., Tagari, P. & Freed, A.N. (1996) Eicosanoids modulate hyperpnea-induced bronchoconstriction in canine peripheral airways. J Appl Physiol 81, 1255–63. O’Sullivan, S., Roquet, A., Dahlén, B. et al. (1998) Evidence for mast cell activation during exercise-induced bronchoconstriction. Eur Respir J 12, 345–50. Parsons, G.H., Pare, P.D., White, D.A. & Baile, E.M. (1989) Airway blood flow response to eucapnic dry air hyperventilation in sheep. J Appl Physiol 66, 1443–7. Pavord, I., Wisniewski, A. & Tattersfield, A.E. (1992) Inhaled frusemide and exercise induced asthma: evidence of a role for inhibitory prostanoids. Thorax 47, 797–800. Persson, C.G., Erjefalt, J.S., Alkner, U. et al. (1991) Plasma exudation as a first line respiratory mucosal defence. Clin Exp Allergy 21, 17–24. Pliss, L.B., Ingenito, E.P., Ingram, R.H. & Pichurko, B. (1990) Assessment of bronchoalveolar cell and mediator response to isocapnic hyperpnea in asthma. Am Rev Respir Dis 142, 73–8.

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Ray, D.W., Hernandez, C., Munoz, N., Leff, A.R. & Solway, J. (1988) Bronchoconstriction elicited by isocapnic hyperpnea in guinea pigs. J Appl Physiol 65, 934– 9. Ray, D.W., Hernandez, C., Leff, A.R., Drazen, J.M. & Solway, J. (1989) Tachykinins mediate bronchoconstriction elicited by isocapnic hyperpnea in guinea pigs. J Appl Physiol 66, 1108–12. Ray, D.W., Eappen, S., Hernandez, C. et al. (1990) Distribution of airway narrowing during hyperpnea-induced bronchoconstriction in guinea pigs. J Appl Physiol 69, 1323– 9. Ray, D.W., Garland, A., Hernandez, C., Eappen, S., Alger, L. & Solway, J. (1991) Time course of bronchoconstriction induced by dry gas hyperpnea in guinea pigs. J Appl Physiol 70, 504–10. Reiss, T.F., Hill, J.B., Harman, E. et al. (1997) Increased urinary excretion of LTE4 after exercise and attenuation of exercise-induced bronchospasm by montelukast, a cysteinyl leukotriene receptor antagonist. Thorax 52, 1030– 5. Rennard, S.I. (1996) Repair mechanisms in asthma. J Allergy Clin Immunol 98, S278–S286. Rumsaeng, V., Cruikshank, W.W., Foster, B. et al. (1997) Human mast cells produce the CD4+ T lymphocyte chemoattractant factor, IL-16. J Immunol 159, 2904–10. Seltzer, J., Scanlon, P.D., Drazen, J.M., Ingram, R.H. Jr & Reid, L. (1984) Morphologic correlation of physiologic changes caused by SO2-induced bronchitis in dogs. The role of inflammation. Am Rev Respir Dis 129, 790–7. Shi, H., Qin, S., Huang, G. et al. (1997) Infiltration of eosinophils into the asthmatic airways caused by interleukin 5. Am J Respir Cell Mol Biol 16, 220– 4. Shimizu, T., Mochizuki, H., Shigeta, M. & Morikawa, A. (1997) Effect of inhaled indomethacin on exercise-induced bronchoconstriction in children with asthma. Am J Respir Crit Care Med 155, 170–3. Smith, C.M. & Anderson, S.D. (1989) A comparison between the airway response to isocapnic hyperventilation and hypertonic saline in subjects with asthma. Eur Respir J 2, 36– 43. Solway, J., Kao, B.M., Jordan, J.E. et al. (1993) Tachykinin receptor antagonists inhibit hyperpnea-induced bronchoconstriction in guinea pigs. J Clin Invest 92, 315– 23. Spannhake, E.W., Lemen, R.J., Wegmann, M.J., Hyman, A.L. & Kadowitz, P.J. (1978) Effects of arachidonic acid and prostaglandins on lung function in the intact dog. J Appl Physiol 44, 397–405. Speelberg, B., Panis, E.A., Bijl, D., van Herwaarden, C.L. & Bruynzeel, P.L. (1991) Late asthmatic responses after exercise challenge are reproducible. J Allergy Clin Immunol 87, 1128–37. Stensrud, T., Berntsen, S. & Carlsen, K.H. (2006) Humidity influences exercise capacity in subjects with exercise-induced bronchoconstriction (EIB). Respir Med 100, 1633– 41. Sue-Chu, M., Larsson, L., Moen, T., Rennard, S.I. & Bjermer, L. (1999) Bronchoscopy and bronchoalveolar lavage findings in cross-country skiers with and without “ski asthma”. Eur Respir J 13, 626– 32. Suman, O.E. & Beck, K.C. (2002) Role of airway endogenous nitric oxide on lung function during and after exercise in mild asthma. J Appl Physiol 93, 1932– 8. Suman, O.E., Morrow, J.D., O’Malley, K.A. & Beck, K.C. (2000) Airway function after cyclooxygenase inhibition during hyperpneainduced bronchoconstriction in guinea pigs. J Appl Physiol 89, 1971– 8.


Suzuki, R. & Freed, A.N. (2000a) Endogenous nitric oxide modulates acute and late phase response to hyperventilation with dry air in dogs. Eur Respir J 16, A189. Suzuki, R. & Freed, A.N. (2000b) Hypertonic saline aerosol increases airway reactivity in the canine lung periphery. J Appl Physiol 89, 2139–46. Suzuki, R. & Freed, A.N. (2002) Heparin inhibits hyperventilationinduced late-phase hyperreactivity in dogs. Am J Respir Crit Care Med 165, 27–33. Tang, G.J. & Freed, A.N. (1992) The autonomic nervous system modulates dry air-induced constriction in the canine lung periphery. Am Rev Respir Dis 145, 1301–5. Tang, G.J. & Freed, A.N. (1994) The role of submucosal oedema in increased peripheral airway resistance by intravenous volume loading in dogs. Eur Respir J 7, 311–17. Taub, D.D., Anver, M., Oppenheim, J.J., Longo, D.L. & Murphy, W.J. (1996) T lymphocyte recruitment by interleukin-8 (IL-8). IL8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J Clin Invest 97, 1931–41. Teeter, J.G. & Freed, A.N. (1991) Effect of salbutamol on dry air- and acetylcholine-induced bronchoconstriction in the canine lung periphery. Eur Respir J 4, 972– 8. Therminarias, A., Oddou, M.F., Favre-Juvin, A., Flore, P. & Delaire, M. (1998) Bronchial obstruction and exhaled nitric oxide response during exercise in cold air. Eur Respir J 12, 1040–5. Van Citters, R.L. & Franklin, D.L. (1969) Cardiovascular performance of Alaska sled dogs during exercise. Circ Res 24, 33–42. van Schoor, J., Joos, G.F., Kips, J.C., Drajesk, J.F., Carpentier, P.J. & Pauwels, R.A. (1997) The effect of ABT-761, a novel 5-lipoxygenase inhibitor, on exercise- and adenosine-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med 155, 875–80. Wagner, E.M. & Mitzner, W.A. (1990) Bronchial circulatory reversal of methacholine-induced airway constriction. J Appl Physiol 69, 1220–4. Wang, D., Chen, H.I., Chou, C.L., Hsu, K. & Freed, A.N. (1992) Terbutaline acts at multiple sites to inhibit bronchoconstriction induced by dry air in canine peripheral airways. Am Rev Respir Dis 145, 1295–300. Wessling, G.J. & Wouters, E.F.M. (1992) Respiratory impedance measurements in a dose-response study of isocapnic hyperventilation with cold air. Respiration 59, 259–64. Yang, X.X., Powell, W.S., Hojo, M. & Martin, J.G. (1997) Hyperpneainduced bronchoconstriction is dependent on tachykinin-induced cysteinyl leukotriene synthesis. J Appl Physiol 82, 538–44. Yang, X.X., Powell, W.S., Xu, L.J. & Martin, J.G. (1999a) Strain dependence of the airway response to dry-gas hyperpnea challenge in the rat. J Appl Physiol 86, 152–8. Yang, X.X., Ho, G., Xu, L.J., Powell, W.S. & Martin, J.G. (1999b) The beta(2)-agonist salbutamol inhibits bronchoconstriction and leukotriene D(4) synthesis after dry gas hyperpnea in the guinea-pig. Pulm Pharmacol Ther 12, 325–9. Yuan, L. & Nail, B.S. (1995) A differential bronchomotor response to cooling and drying the upper airway. Respir Physiol 101, 121–8. Zawadski, D.K., Lenner, K.A. & McFadden, E.R. Jr (1988) Reexamination of the late asthmatic response to exercise. Am Rev Respir Dis 137, 837–41.

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37

Exercise-induced Bronchoconstriction: Human Models Arthur N. Freed and Sandra D. Anderson

Summary

5

4 FEV1 in litres

Airway narrowing following exercise, usually known as exercise-induced bronchoconstriction (EIB), is common in clinically recognized asthmatics and becoming increasingly reported in asymptomatic healthy subjects, particularly athletes. EIB appears to be an early manifestation of asthma in children and studies are required to determine if this is also the case when it develops in young adults. The stimulus for EIB is the evaporative loss of water in conditioning air during exercise. The mechanism relates to the osmotic and thermal consequences of the evaporation of water. The osmotic consequences include the release of inflammatory mediators that act on bronchial and possibly vascular smooth muscle to cause the airways to narrow. In asthmatics, EIB is associated with airway inflammation and EIB severity may be an indirect marker of disease activity. The severity of EIB is reduced in all asthmatics in response to chronic treatment with inhaled corticosteroids. Less is known about the pathophysiology, repeatability, or response to drugs of EIB that occurs in otherwise asymptomatic healthy subjects. An objective test with hyperpnea of dry air or a hyperosmolar aerosol should still be regarded as important for patients suspected of having EIB and who complain of breathlessness on or after exertion. By documenting the hyperresponsiveness to hyperpnea or a hyperosmolar aerosol, a diagnosis of EIB, and even asthma itself, can be confirmed and the appropriate therapy prescribed.

A diagnosis of EIB is made by measuring a fall in forced expiratory volume in 1 s (FEV1), an indirect measure of airways resistance, after exercise of 10% or more of the value measured immediately before exercise (Sterk et al. 1993; Roca et al. 1997; Task Force on Recognizing and Diagnosing ExerciseRelated Asthma Respiratory and Allergic Disorders in Sports 2005) (Fig. 37.1). Data in children and young adults indicate that a fall in FEV1 of 13% has 94% specificity for identifying those with a clinical diagnosis of asthma (Godfrey et al. 1999). A provisional diagnosis of EIB can be made on history if a person complains of breathlessness that is worse 5–10 min after ceasing exercise compared with during exercise. Vocal cord dysfunction (VCD) (McFadden & Zawadski 1996; Morris et al. 1999), or inspiratory stridor, a symptom of VCD (Rundell & Spiering 2003) or exercise hyperventilation syndrome (Hammo & Weinberger 1999), can be incorrectly diagnosed as EIB. For these disorders the symptoms occur during rather than after exercise.

Introduction Exercise-induced bronchoconstriction (EIB) and exerciseinduced asthma (EIA) are the terms used to describe the transient increase in airways resistance that follows vigorous exercise. While these terms are used interchangeably, EIA is best used only for those with a clinical diagnosis of asthma (Anderson & Henriksen 1999). For the purposes of this chapter the term EIB is used.

Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN 978-1-4051-5720-9.

808

< 10% Normal

< 25% Mild

3

> 25% Moderate 2

> 50% Severe Exercise

1 0

8

14

20

Time (min) Fig. 37.1 Pattern of change in forced expiratory volume in 1 s (FEV1) after 8 min of vigorous exercise inspiring dry air at a ventilation exceeding 50% of maximum in a normal healthy subject without exercise-induced bronchoconstriction (EIB) and in subjects with mild, moderate or severe EIB. The severity of the response is based on the fall in FEV1 after exercise expressed as a percentage of the baseline value. If a subject is taking inhaled corticosteroids on a daily basis, a postexercise fall in FEV1 of 30% or more would be considered as severe.

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37

Exercise-induced Bronchoconstriction: Human Models Arthur N. Freed and Sandra D. Anderson

Summary

5

4 FEV1 in litres

Airway narrowing following exercise, usually known as exercise-induced bronchoconstriction (EIB), is common in clinically recognized asthmatics and becoming increasingly reported in asymptomatic healthy subjects, particularly athletes. EIB appears to be an early manifestation of asthma in children and studies are required to determine if this is also the case when it develops in young adults. The stimulus for EIB is the evaporative loss of water in conditioning air during exercise. The mechanism relates to the osmotic and thermal consequences of the evaporation of water. The osmotic consequences include the release of inflammatory mediators that act on bronchial and possibly vascular smooth muscle to cause the airways to narrow. In asthmatics, EIB is associated with airway inflammation and EIB severity may be an indirect marker of disease activity. The severity of EIB is reduced in all asthmatics in response to chronic treatment with inhaled corticosteroids. Less is known about the pathophysiology, repeatability, or response to drugs of EIB that occurs in otherwise asymptomatic healthy subjects. An objective test with hyperpnea of dry air or a hyperosmolar aerosol should still be regarded as important for patients suspected of having EIB and who complain of breathlessness on or after exertion. By documenting the hyperresponsiveness to hyperpnea or a hyperosmolar aerosol, a diagnosis of EIB, and even asthma itself, can be confirmed and the appropriate therapy prescribed.

A diagnosis of EIB is made by measuring a fall in forced expiratory volume in 1 s (FEV1), an indirect measure of airways resistance, after exercise of 10% or more of the value measured immediately before exercise (Sterk et al. 1993; Roca et al. 1997; Task Force on Recognizing and Diagnosing ExerciseRelated Asthma Respiratory and Allergic Disorders in Sports 2005) (Fig. 37.1). Data in children and young adults indicate that a fall in FEV1 of 13% has 94% specificity for identifying those with a clinical diagnosis of asthma (Godfrey et al. 1999). A provisional diagnosis of EIB can be made on history if a person complains of breathlessness that is worse 5–10 min after ceasing exercise compared with during exercise. Vocal cord dysfunction (VCD) (McFadden & Zawadski 1996; Morris et al. 1999), or inspiratory stridor, a symptom of VCD (Rundell & Spiering 2003) or exercise hyperventilation syndrome (Hammo & Weinberger 1999), can be incorrectly diagnosed as EIB. For these disorders the symptoms occur during rather than after exercise.

Introduction Exercise-induced bronchoconstriction (EIB) and exerciseinduced asthma (EIA) are the terms used to describe the transient increase in airways resistance that follows vigorous exercise. While these terms are used interchangeably, EIA is best used only for those with a clinical diagnosis of asthma (Anderson & Henriksen 1999). For the purposes of this chapter the term EIB is used.


808

< 10% Normal

< 25% Mild

3

> 25% Moderate 2

> 50% Severe Exercise

1 0

8

14

20

Time (min) Fig. 37.1 Pattern of change in forced expiratory volume in 1 s (FEV1) after 8 min of vigorous exercise inspiring dry air at a ventilation exceeding 50% of maximum in a normal healthy subject without exercise-induced bronchoconstriction (EIB) and in subjects with mild, moderate or severe EIB. The severity of the response is based on the fall in FEV1 after exercise expressed as a percentage of the baseline value. If a subject is taking inhaled corticosteroids on a daily basis, a postexercise fall in FEV1 of 30% or more would be considered as severe.

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CHAPTER 37

RESPIRATORY WATER LOSS Mucosal cooling

Mucosal dehydration Increase [Na+] [Cl–] [Ca2+] [K+]

Vasoconstriction

Cough

Increased osmolarity

Mucus

Rapid rewarming of airways Cells shrink and mediators released Vascular engorgement ± edema

Smooth muscle contraction ± edema Exercise-induced bronchoconstriction

Fig. 37.2 Schematic diagram showing the two hypotheses (Anderson & Daviskas 2000; McFadden et al. 1996) that explain the events leading to exercise-induced bronchoconstriction. Respiratory water loss can cause airway cooling and mucosal dehydration. Airway cooling is associated with vasoconstriction, vascular engorgement, and edema. Airway dehydration is associated with an increase in osmolarity of the airway surface, with a consequent release of mediators that act on smooth muscle causing it to contract. An increase in osmolarity also causes cough and mucus independently of airway narrowing.

The stimulus for EIB is the loss of water by evaporation from the airway surface that is the consequence of humidifying large volumes of ambient air in a very short time (Anderson & Daviskas 2000). Thus EIB is most likely to occur in a dry environment in response to an exercise intensity that raises the minute ventilation to a value that is 50% or more of predicted maximum achievable. The mechanism for dehydration causing the airways to narrow relates to the osmotic and thermal effects of evaporation (McFadden et al. 1996; Anderson & Daviskas 2000) (Fig. 37.2). The thermal effect, i.e., airway cooling, is thought to cause vasoconstriction of the bronchial vasculature, an event followed by a reactive hyperemia at the end of exercise when the airways rapidly rewarm (Gilbert & McFadden 1992). These thermal events may be accompanied by an increase in vascular permeability, exudation of plasma, and airway edema that all contribute to airway narrowing (Kanazawa et al. 2002). This vascular hypothesis does not implicate the bronchial smooth muscle in EIB. The osmotic effect, i.e., hyperosmolarity of the airway surface liquid (ASL), is thought to provoke release of mediators that act on specific receptors on bronchial smooth muscle causing it to contract and the airways to narrow (Reiss et al. 1997; O’Sullivan et al. 1998; Hallstrand et al. 2005a). Increased vascular permeability and airway edema may be caused by the same mediators and plasma exudation may also occur in response to an osmotic stimulus (Greiff et al. 2003a,b). The increase in ASL osmolarity may be the signal for the release of nitric oxide (NO) (Smith et al. 1993; Anderson & Daviskas 1999; Högman et al. 2001) and could account for some of the vascular events (Kanazawa et al.

Exercise-induced Bronchoconstriction: Human Models

2002). In asthmatics exercising under conditions of breathing cold dry air, both thermal and osmotic effects are likely to contribute to EIB. Under temperate or hot dry environmental conditions, when significant airway cooling is unlikely to occur, it is the osmotic effects that are the most important (Aitken & Marini 1985; Anderson & Daviskas 1992, 2000; Argyros et al. 1993; Anderson & Holzer 2000; Evans et al. 2005). In elite athletes without a clinical diagnosis of asthma, EIB may be a consequence of epithelial injury and subsequent changes to the contractile properties of bronchial smooth muscle that make it more responsive (Anderson & Kippelen 2005). The injury may lead to an imbalance between the bronchodilator mediator prostaglandin (PG)E2 and the bronchoconstricting cysteinyl leukotrienes, making the muscle appear more hyperresponsive (Hallstrand et al. 2005b). Excessive exposure to cold air, airborne allergens, pollutants or particulate matter, and viral infections may also amplify this problem in athletes (Heir & Larsen 1995; Heir et al. 1995; Rundell et al. 2003; Bjermer & Anderson 2005).

EIB and airway inflammation In asthmatics, EIB is associated with active airway inflammation, and the severity of EIB serves as an indirect marker of the severity of inflammation. Eosinophils, a well-known marker of asthmatic inflammation, are higher in sputum from asthmatic children and adults with EIB relative to those without EIB (Yoshikawa et al. 1998; Hallstrand et al. 2005b). The severity of EIB is also related to airway injury, as reflected by the number of columnar epithelial cells collected in sputum before exercise (Hallstrand et al. 2005b). The severity of EIB is also related to the concentration of NO derivatives in sputum (Kanazawa et al. 2000), and to expired levels of NO (eNO) (Terada et al. 2001; Buchvald et al. 2005). The relationship between eNO and EIB is only significant in those who are atopic (Rouhos et al. 2005). EIB is markedly reduced by drugs that exert an antiinflammatory effect, such as inhaled corticosteroids (ICS) (Hofstra et al. 2000; Jonasson et al. 2000; Subbarao et al. 2006) (Fig. 37.3), and drugs that inhibit the products of 5-lipoxygenase (LTB4, LTC4, LTD4, LTE4) (Meltzer et al. 1996; Lehnigk et al. 1998) or which antagonize the effects of leukotrienes at the receptor.

Mediators and EIB The potential for inflammatory mediators such as histamine, prostaglandins, and leukotrienes to cause EIB has been acknowledged for many years, based on the finding that specific receptor antagonists provide some protection. The precise role of the preformed mediator histamine in EIB remains unclear. Histamine is probably important in contributing to the early fall in FEV1, whereas the prostaglandins

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Physiology

70

70

60

60

50

50 % Fall in FEV1

% Fall in FEV1

PART 4

40 30 20 10

40 30 20 10

10/14 (71%)

0 Pre

9/14 (64%)

0

Post

Pre

Post

100 mg/day for 12 weeks

200 mg/day for 12 weeks

Baseline: FEV1 100 ± 12% Predicted

FEV1 101 ± 10% Predicted

and leukotrienes appear to be more important for sustaining the fall in FEV1. The intensity of the exercise stimulus may be the determining factor for histamine to contribute significantly to the maximal fall in FEV1 (Anderson & Brannan 2002). Histamine receptor antagonists were found effective in inhibiting EIB in some studies (Patel 1984; Baki & Orhan 2002 ) but not others (Dahlén et al. 2002; Peroni et al. 2002). The same histamine antagonist that was reported as effective in one study (Baki & Orhan 2002) was ineffective in another (Dahlén et al. 2002). It is not known if the presence of IgE is important in determining the efficacy of a histamine antagonist. The leukotriene antagonists montelukast and zafirlukast both inhibit and enhance recovery from EIB (Kemp et al. 1998; Pearlman et al. 1999; Villaran et al. 1999; Dahlén et al. 2002) (Fig. 37.4).

Fig. 37.3 Maximum percentage fall in FEV1 from baseline after exercise in two groups of children who received 100 mg or 200 mg of budesonide daily for 2 weeks. The percentage of children who had a response within the normal range after treatment is shown. (Individual data kindly provided by Jonasson et al. 2000.)

There is now direct evidence to implicate mediator release in EIB, mainly due to the availability of sensitive assays for detecting leukotrienes, prostaglandins, and histamine, and the use of induced sputum and urine. Urinary excretion of LTE4 and the metabolite of PGD2 (9α,11β-PGF2) are increased after exercise in both asthmatic (Fig. 37.5) and healthy subjects (Caillaud et al. 2003; Mickleborough et al. 2003). The same findings have been reported following inhalation of the hyperosmolar aerosol mannitol (Brannan et al. 2003). In sputum, histamine, tryptase, and cysteinyl leukotrienes (Hallstrand et al.

100

P < 0.05

P < 0.05

30 min

90 min

5

% Change FEV1 (mean ± SEM)

0 –5 –10 Placebo % fall 26 ± 14 Montelukast % fall 18 ± 13 = 25

–15 –20

9a,11b-PGF2 ng/mmol creatinine

80

60

40

20

–25 0 –30 0

5

10

15

30

45

60

Time after exercise (min) Fig. 37.4 Mean percentage change in FEV1 in children in response to 6 min of running exercise 20–24 hours after 5 mg of montelukast once daily in the evening for 2 days. The maximum fall in FEV1 is also given. Note the rapid return of FEV1 to baseline following exercise in the presence of the montelukast. (Data from Kemp et al. 1998.)

810

Before

Fig. 37.5 Mean (SEM) values for urinary excretion of the mast cell mediator prostaglandin D2 metabolite 9a,11b-PGF2 measured before and after exercise in asthmatic patients who responded or did not respond to challenge by exercise. The values at 30 and 90 min after exercise were significantly higher in the seven responders than in the five nonresponders and the values at 90 min in the responders were significantly increased above baseline. (From O’Sullivan et al. 1998, with permission.)

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CHAPTER 37

Exercise-induced Bronchoconstriction: Human Models

40

10

500

0

Baseline Post-exercise

5

Baseline Post-exercise

2000

4

10

1500 CysLT pg/mL

Tryptase ng/mL

20

3 2

Treatment

0

1000

500

1

Placebo

1000

treatment with oral formulations of histamine and leukotriene antagonists, significantly lower levels of histamine and cysteinyl leukotrienes were found in sputum after exercise (Hallstrand et al. 2005a) (Fig. 37.7). It is not known if this is due to an antagonist action reducing vascular permeability or a pharmacologic effect on mast cells. Enhanced transcription of two genes coding for 5-lipoxygenase (ALOX5) and ALOX5activating protein, together with enhanced levels of LTB4 and LTC4 in plasma, were reported following cycling exercise in healthy subjects (Hilberg et al. 2005). These data provide evidence to connect the putative mediators to the stimulus of exercise. The beneficial effec

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