Allergy And Asthma in Modern Society: A Scientific Approach

Allergy and Asthma in Modern Society: A Scientific Approach

Chemical Immunology and Allergy Vol. 91

Series Editors

Johannes Ring Munich Luciano Adorini Milan Claudia Berek Berlin Kurt Blaser Davos Monique Capron Lille Judah A. Denburg Hamilton Stephen T. Holgate Southampton Gianni Marone Napoli Hirohisa Saito Tokyo

Allergy and Asthma in Modern Society: A Scientific Approach Dedicated to Kurt Blaser Volume Editor

Reto Crameri Davos

29 figures, 3 in color, and 12 tables, 2006

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Chemical Immunology and Allergy Formerly published as ‘Progress in Allergy’ (Founded 1939) continued 1990–2002 as ‘Chemical Immunology’ Edited by Paul Kallos 1939–1988, Byron H. Waksman 1962–2000

Reto Crameri Head Molecular Allergology Swiss Institute of Allergy and Asthma Research (SIAF) Obere Strasse 22 CH–7270 Davos (Switzerland)

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2006 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 1660–2242 ISBN 3–8055–8000–2

Contents

XIII Foreword R. Crameri, Davos Introduction 1 The Magic Mountain of Allergy Research J. Ring, H. Behrendt, München The Environment 3 Environmental Factors Influencing Allergy and Asthma T.A.E. Platts-Mills, E.A. Erwin, J.A. Woodfolk, P.W. Heymann, Charlottesville, Va. 3 Abstract 6 The Cat Paradox: High Exposure to Cat or Dog Allergens Does Not Increase the Prevalence or Titer of IgE ab 7 The American Inner City as a Special Issue 8 The Role of Specific Antibody Responses in Influencing Total Serum IgE, the Prevalence of Asthma and Severity of Asthma 9 Endotoxin: Another Environmental Exposure with a Nonlinear Dose Response 10 Conclusions 12 Acknowledgements 13 References 16 Should Asthma Management Include Sojourns at High Altitude? G. Schultze-Werninghaus, Bochum 16 Abstract

V

16 16 17 18 19 22 25 25 26 27 27

Features of High Mountains’ Climate Air Pollution Allergens Effects of the High Altitude in Bronchial Asthma Laboratory Markers of Airway Inflammation Immunoglobulin E and T-Cell Functions Nonspecific and Specific BHR Lung Function Duration of Effects Conclusions References

30 The Immunological Basis of the Hygiene Hypothesis H. Renz, N. Blümer, S. Virna, S. Sel, H. Garn, Marburg 30 30 37 41 45

Abstract Epidemiological and Human Exposure Studies Animal Models: A Proof-of-Concept Approach Cellular and Molecular Mechanisms of Allergy Protection References The Lung, Eosinophils and Asthma

49 Molecules Involved in the Regulation of Eosinophil Apoptosis H.-U. Simon, Bern 49 51 51 52 52 52 52 53 53 53 55 56 56

Abstract Proapoptotic Extracellular Stimuli Death Receptor Ligands Other Death Triggers: Siglec-8, CD30, CD45 and CD69 Antiapoptotic Extracellular Stimuli Classical Survival Cytokines TNF-Like Molecules Intracellular Regulators Caspases IAP Family Members Bcl-2 Family Members Conclusions References

59 The Role of T Lymphocytes in Asthma A.B. Kay, London 59 60 61 62 63

Abstract The Asthma Phenotypes Effector Mechanisms: How Do T Cells Cause Asthma? Are T Cells Required to Perpetuate Asthma? T-Cell-Derived Cytokines and Cytokines That Act on T Cells

Contents

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64 64 65 66 67 67 69 69 69 70 71

Controls of T-Cell Cytokine Production TReg Cells and Asthma Provoked Asthma under Controlled Clinical Conditions Activation of T Cells in Asthma: Antigen-Presenting Cells CD8
T Cells and  Cells Homing of T Cells to the Airway in Asthma T Cells and Treatment of Asthma Corticosteroids Cyclosporin A Antigen-Directed Targeting of T Cells and TRegs: The Future for Asthma Therapy? References The Skin

76 Allergic Manifestations of Skin Diseases – Atopic Dermatitis K. Breuer, Norderney/Hannover; T. Werfel, A. Kapp, Hannover 76 76 78 80 80 82 84 84

Abstract Atopic Dermatitis – A Chronic Inflammatory Skin Disease Food Allergy in AD Inhalant Allergens and AD The Importance of Staphylococcus aureus as a Trigger Factor of AD New Treatment Options for AD Conclusions References

87 Skin-Homing T Cells in Cutaneous Allergic Inflammation L.F. Santamaría-Babí, Barcelona 87 89 92 92 93 95

Abstract Atopic Dermatitis Allergic Contact Dermatitis Nonimmediate Cutaneous Allergic Reactions to Drugs Features of Circulating CLA
T Cells in Cutaneous Allergic Inflammation References

98 The Role of Sensitization to Malassezia sympodialis in Atopic Eczema P. Schmid-Grendelmeier, Zurich; A. Scheynius, Stockholm; R. Crameri, Davos 98 99 99 100 101 103 104

Abstract Skin Barrier Dysfunction and Its Impact on Microbial Colonization Fungi as Contributing Factors to AE The Special Role of Malassezia Species in Allergic Reactions Related to AE The Allergen Repertoire of M. sympodialis M. sympodialis in the Intrinsic Form (Nonatopic Type) of AE Therapeutic Long-Term Options and Anti-Inflammatory Approaches

Contents

VII

105 106 107 107

Fungal Allergens Leading to Autoreactivity in AE Conclusions Acknowledgements References

110 Allergic Conjunctivitis:The Forgotten Disease S. Bonini, Rome 110 110 111 111 114 116 117 118 119

Abstract Definition Classification Etiopathogenesis Clinical Presentation Diagnosis Treatment Prognosis References Molecular Aspects of Allergy and Asthma

121 Fungal Allergies: A Yet Unsolved Problem R. Crameri, M. Weichel, S. Flückiger, A.G. Glaser, C. Rhyner, Davos 121 122 123 124 125 126 127 130 131 132 132

Abstract Epidemiology Incidence and Clinical Relevance of Fungal Allergies Diagnosis of Fungal Allergy The Dimension of the Problem Recombinant Fungal Allergens Cross-Reactivity Involvement of Fungal Allergens in the Pathogenesis of Severe Atopic Diseases Conclusions Acknowledgements References

134 Structural Features of Allergenic Molecules R.C. Aalberse, Amsterdam 134 Abstract 135 Cross-Reactivity in Relation to Mast Cell Triggering 138 Cross-Reactivity in Relation to B-Cell Activation: Cross-Reactive Priming, Epitope Spreading and ‘The Original Antigenic Sin’ 138 IgE Immunogenicity 139 The ‘Healthy’ Antiallergen Immune Response and Its Relation to the Modified Th2 Response 141 Working Hypothesis: the ‘Nonmodified’ Th2 Response is an Incomplete B-Cell Response Which Fails to Induce Mature Germinal Centers 143 To What Extent Is IgG4 Special in This Context?

Contents

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144 Consequences for the Allergenicity Issue 145 References 147 Regulation of Human T Helper Cell Differentiation by Antigen-Presenting Cells: The Bee Venom Phospholipase A2 Model J.M. Carballido, N. Carballido-Perrig, C. Schwärzler, G. Lametschwandtner, Vienna 147 148 150 151 152 153 154 155 155

Abstract Th-Cell Differentiation Is Largely Controlled by Cytokine Signaling Th Polarizing Signals Generated by DC Are Regulated by Danger Signals Costimulatory Molecules Displayed on DC Surfaces Modulate Th-Cell Differentiation Antigen Presentation by Nonprofessional APC Influences Th-Cell Differentiation Affinity of Interaction between MHC Class II Molecules and Antigen Peptides Might Determine the Type of Immune Response Conclusions Acknowledgements References

159 T Regulatory Cells in Allergy M. Akdis, K. Blaser, C.A. Akdis, Davos 159 Abstract 159 Anergy, Tolerance and Active Suppression Are Not Fully Distinct Events 160 Essential Features of Allergic Inflammation 162 TReg Cells 162 Tr1 Cells 163 Th3 Cells 165 CD4
CD25
TReg Cells 165 Other Regulatory Cells 166 BReg Cells: Do They Exist? 166 Dendritic Cells That May Play a Regulatory Function 167 Other Cells with Possible Regulatory Function 167 Suppression Mechanisms of TReg Cells 169 Clinical Relevance of TReg Cells 170 Conclusions 171 Acknowledgements 171 References 174 The Role of Histamine in Regulation of Immune Responses M. Jutel, Davos/Wroclaw; K. Blaser, C.A. Akdis, Davos 174 175 175 176

Abstract Cellular Sources of Histamine Synthesis and Metabolism of Histamine Histamine Receptors

Contents

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179 179 181 182 183 184 184 184 185 185

Regulation of Immune Response Antigen-Presenting Cells T Cells and Antibody Isotypes Histamine and Chronic Inflammatory Responses Effects of Histamine and Antihistamines on Airway Function Histamine and HR1 in Autoimmunity Histamine Signal in Malignancies Conclusions Acknowledgements References

188 Gene Expression Profiling in Allergy and Asthma C.B. Schmidt-Weber, Davos 188 188 189 190 193 193 193

Abstract The DNA Array Technology Application of DNA Arrays Gene Expression Profiles in Allergy Conclusions Acknowledgements References Immunotherapy

195 Mechanisms of Allergen-Specific Immunotherapy C.A. Akdis, K. Blaser, M. Akdis, Davos 195 Abstract 195 Peripheral T-Cell Tolerance in Allergen-Specific Immunotherapy 197 Peripheral T-Cell Tolerance to Allergens is Associated with Regulation of Antibody Isotypes 198 Suppression of Effector Cells by Allergen-Specific Immunotherapy 199 Mechanism of T-Cell Suppression by IL-10 and Its Relationship to Anergy 200 Conclusions 201 Acknowledgements 201 References 204 Regulation of the IgE Response at the Molecular Level: Impact on the Development of Systemic Anti IgE Therapeutic Strategies G. Achatz, G. Achatz-Straussberger, Salzburg; E. Luger, Berlin; R. Lamers, Freiburg; R. Crameri, Davos 204 204 206 208 209 210

Abstract Overview The Biological Function of the B-Cell Antigen Receptor Regulation of the Membrane Expression of IgE The Biochemical Process of Polyadenylation Alternative Polyadenylation of IgE

Contents

X

211 213 214 215 215

Plasma-Cells – A Therapy Resistant Population of Cells? Pros and Cons of Systemic IgE Therapies Summary Acknowledgements References

218 Author Index 219 Subject Index

Contents

XI

Foreword

Allergic diseases and asthma constitute a growing health care problem, especially in industrialized countries. In spite of marked worldwide variation, the prevalence of symptoms of asthma, eczema and allergic rhinoconjunctivitis is increasing. Although genetic factors defining the atopic background of a population are undoubtedly important, they cannot explain this phenomenon. As the genetic background of a population must be regarded as quite stable over short periods of time, environmental factors must be included to explain the remarkable changes in the prevalence and severity of asthmatic and allergic diseases during the last 40 years. As brilliantly summarized by Platts-Mills et al., environmental factors can influence the spread of these diseases; however, single changes in environmental parameters alone cannot explain the consistency or the scale of the rise in allergy and asthma observed between 1960 and 2000. Our environment is extremely complex, poorly defined and difficult to monitor. However, a direct demonstration of the pivotal influence of environmental factors on the severity of asthma comes from a recent reinvestigation by SchultzeWerninghaus of a very old observation describing the beneficial role of sojourns at high altitude. The therapeutic value of such sojourns for severe bronchial asthma patients is well documented, and there is no scientific reason to doubt it. Obviously, our limited knowledge about host-environment interactions and atopic diseases favored the development of various, more or less attractive hypotheses and theories aiming to explain this phenomenon. Among these, the hygiene hypothesis, discussed by Renz et al., is perhaps the most attractive. According to current scientific views, it tries to integrate the interaction between environmental factors, innate and adaptive immunity into a sophisticated

XIII

model. We must realize that the human body is not an isolated system. To survive, we need a continuous selective exchange with our environment, allowing the uptake of essential biovital elements and excretion of unwanted metabolites, but aiming to avoid offending agents. Physical barriers and an orchestrated primary and secondary line of defense are required to allow survival. Skin and mucosal surfaces represent by far the largest interface between a human being and the environment and from this point of view it is not astonishing that the respiratory tract (Simon, Kay), skin (Breuer et al., Santamaría-Babí, Bonini) and digestive tract determine an individual’s quality of life. However, other diseases, notably conjunctivitis, an often forgotten disease (Bonini), significantly contributes to the health burden of modern society as well. Inappropriate immune responses to normally harmless environmental antigens, following, for example, exposure to fungi (Crameri et al.) still represent an unsolved health care problem although our understanding of the structural basis of allergens (Aalberse) and their role in the pathogenesis of chronic allergic diseases (Schmid-Grendelmeier et al.) is rapidly increasing. Complex mechanisms regulate the healthy immune responses to allergen exposure (Carballido et al., Akdis et al.) and it is the long-neglected study of these responses that recently contributed to a better understanding of the orchestrated cascades resulting either in normal, protective, or abnormal, diseaserelated immune responses. Antigen-antibody interactions at the end of the cascade are relatively easy to access experimentally and, as a consequence, our knowledge about these phenomena is quite advanced. Early, tightly regulated cellular interactions resulting from the complex interplay between cytokines, receptors and small molecules, such as histamine (Jutel et al.), strongly depending on genetic background and environmental influences, determine the immune response initiated and the fate of each single individual. It becomes increasingly evident that such immune responses in allergy and asthma are extremely complex. New global technologies based on gene expression profiling (Schmidt-Weber) and proteomic approaches will be required to integrate our knowledge about molecular and cellular interactions into more complete networks aimed at understanding the pathophysiology of allergy and asthma. However, there is light at the end of the tunnel. The considerable progress in our understanding of molecular and cellular interactions starts to translate into new strategies to combat allergic diseases (Akdis et al., Achatz et al.). Although many drugs are available to control the symptoms of allergy and asthma, immunotherapy is the only treatment currently able to cure these diseases. Several new treatments have been or will be introduced soon for clinical use and will hopefully strongly improve immunotherapy and benefit the patients.

Foreword

XIV

Kurt Blaser, Director of the Swiss Institute of Allergy and Asthma Research, Davos.

Allergy and asthma are very important diseases, and as a consequence, an overwhelming number of original contributions, reviews and books covering the different aspects of these diseases are published every year. Why a book more about this topic? The answer to this justified question can be found in the introduction by Johannes Ring: Kurt Blaser, the director of the Swiss Institute of Allergy and Asthma Research, celebrated his 65th birthday on June 25, 2005. He dedicated all his life to allergy and asthma research and has become one of the most prominent and appreciated global players in this field. I am convinced that, together with me, all authors of this book and many other scientists worldwide are grateful to him for many exceptional scientific contributions and political fights to speed up progress in a field which, in spite of its recognized socio-economic impact, still lacks the political support to mobilize the financial resources required to satisfy its needs. Thank you Kurt! I am especially grateful to Thomas Nold and his team for their excellent cooperation in editing this book, to the industrial sponsors and to Johannes Ring who supported the idea from the beginning. Of course, I am very grateful to the authors, who have spent much time for the preparation, revision and final checking of the manuscripts. Finally, a big thanks goes to Rosalina and Danja, the unlucky members of the family, waiting at home until I am back from work every day. I am not so sure that I would have that much patience. Reto Crameri

Foreword

XV

Introduction Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 1–2

The Magic Mountain of Allergy Research Johannes Ring, Heidrun Behrendt Klinische Kooperationsgruppe Umweltdermatologie und Allergologie GSF/TUM, Klinik und Poliklinik für Dermatologie und Allergologie am Biederstein, Technische Universität München, München, Germany

Davos (Switzerland) is known worldwide for its healthy climate, its high mountains for skiing and mountaineering, but also for its beneficial effects on health. Although Thomas Mann in his famous novel Der Zauberberg (‘The Magic Mountain’) described these effects sometimes rather critically, many patients undoubtedly achieve cure or significant amelioration for their chronic lung or skin diseases, especially allergies, in the healthy climate of Davos. Apart from this, Davos has gained international reputation as a center of science. Maybe, its healthy climate also exerts a positive influence on the researchers. When, at the end of the eighties of the last century, the Swiss government decided to found a Swiss Institute for Allergy and Asthma Research (‘Schweizerisches Institut für Allergie- und Asthma-Forschung’; SIAF) in Davos, nobody could foresee the future. With the choice of Kurt Blaser as the head of this institute, the officials had a very lucky hand. Within 10 years Kurt Blaser turned the SIAF into one of the leading research centers in allergy in the world. Kurt Blaser was born in Thun, on June 25, 1940, and started as a laboratory trainee from 1957–1960 at a chemical company in Switzerland where he obtained his diploma working on the synthesis of heterocyclic compounds. He then attended an engineering school, where he graduated in 1965. From 1965 to 1972, he studied chemistry at the University of Bern and became a research assistant in inorganic and then organic chemistry. He was awarded an industrial grant to work at the BASF Company, while preparing his PhD at the Institute for Immunology of Bern (with Profs. de Weck and Schneider) from 1972 to 1975. After obtaining his PhD, he worked as a post-doctoral fellow at the Massachusetts Institute for Technology in the USA from 1975 to 1977 (with Profs. Eisen and Luria); from there he returned to the University of Bern and worked as an assistant researcher from 1977 to 1988 (with Profs. Riva and de

Weck). In 1983 he received his degree of ‘Privatdozent’ (‘Habilitation’) to teach experimental cell physiology. In 1994 he was awarded the title of ‘Professor’ and in 2002 he was elected ‘Extraordinarius’ at the University of Zürich. Since 1988, Professor Kurt Blaser is the director of the SIAF in Davos. Kurt Blaser was able to attract excellent people. With his pupils R. Crameri, H.U. Simon, J. Carballido, M. Jutel, C.B. Schmidt-Weber, C. and M. Akdis – just to name some of them – the SIAF soon gained international acceptance and representation at all the major congresses in the field. The list of publications of the SIAF contains several ‘citation classics’ and reads like a list of milestones in allergy research. Among the best-known results originating from the SIAF are the works on eosinophil apoptosis (H.U. Simon), the cutaneous lymphocyte antigen (CLA) (L.F. Santamaría-Babí), the mechanism of immunotherapy (C. Akdis), keratinocyte apoptosis in eczema (M. Akdis), allergen characterization (R. Crameri), histamine in T-cell regulation (M. Jutel). The SIAF coordinates the Swiss branch of the Global Asthma and Allergy European Network (GALEN) in the European Centres of Excellence in allergy program. Kurt Blaser served as member of the Council of the Collegium Internationale Allergologicum (CIA) over 6 years and of the Swiss National Research Council. He also was a research advisor for government research programs, especially in Germany, but also in Austria and other European countries. Kurt Blaser has given input in many of the various centers for allergy research in Germany. With his quiet, professional, sometimes visionary mind and his critical but always inoffensive way, he has had a major influence on allergy research in Europe and in the world. Prof. Dr. Dr. Johannes Ring Klinik und Poliklinik für Dermatologie und Allergologie am Biederstein Technische Universität München Biedersteiner Strasse 29 DE–80802 München (Germany) Tel. ⫹49 89 4140 3170, Fax ⫹49 89 4140 3171, E-Mail [email protected]

Ring/Behrendt

2

The Environment Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 3–15

Environmental Factors Influencing Allergy and Asthma Thomas A.E. Platts-Mills, Elizabeth A. Erwin, Judith A. Woodfolk, Peter W. Heymann Asthma and Allergic Disease Center, University of Virginia, Charlottesville, Va., USA

Abstract Despite the strong and consistent association between immediate hypersensitivity, allergy, asthma and exposure, there is still controversy about the role inhaled allergen plays and about the timing of events related to sensitization. However, IgE antibodies are essential for the asthmatic response and the symptoms are much more closely associated with IgE antibodies to perennial than to seasonal allergens. Although there have been many changes in our environment, none of these alone explains the consistency or the scale of the rise in asthma over the 40 years between 1960 and 2000. Copyright © 2006 S. Karger AG, Basel

The central question about the role of environmental factors in asthma is to understand their relevance to the increasing prevalence of disease. There are two questions that we need to ask first. Are the patients involved in the increase in asthma (i.e. those who would not have had symptoms in 1950) allergic? Second, are all allergens involved equally? If, as appears likely, the majority of the patients are allergic and multiple different allergens are involved, then there are three types of explanation for the increase in asthma. Increased exposure due to changes in housing and increased time spent indoors has lead to an increase in the number of allergic patients. The same changes could have led to increased severity of symptoms among allergic individuals. Alternatively it has been suggested that decreased exposure to farm animals, or decreased early childhood infections could have lead to a nonspecific increase in IgE antibody (IgE ab) responses. Similar effects have been ascribed to decreased helminth infection, increased early antibiotic use, or a decline in hepatitis A infection.

If as we believe neither of the first two explanations is adequate to explain the scale or consistency of the increase, there is an alternative. This proposal is that changes in lifestyle including diet or physical activity have lead to the loss of a protective effect against wheezing among allergic individuals. Clearly there are ways in which these three could interact but the key at the moment is that in each of these models the patients who are affected by the changes are allergic to one or more foreign proteins [1]. Exposure to foreign proteins can occur in many ways. These include contact with the skin, commensal organisms or even pathogenic organisms. However, the great bulk of allergic disease involves inhaled allergens. Not all allergens play an important role in asthma and we will address both the nature of the exposure and the reasons why the impact of different allergens is different. The air we breathe contains gases, fine particles e.g. tobacco smoke and particles carrying allergens and infectious agents. The nose is designed to clean the air by collecting particles on the turbinate and most allergens are carried on particles that will be collected very efficiently during nose breathing. There is an important principle here that no allergens are inhaled as molecules in the gaseous phase. Most allergens that have been characterized are proteins or glycoproteins with a molecular weight of 10–50 kDa (see www.allergen.org). Molecules of this size have a saturated vapor pressure close to zero, and thus they can only become airborne on particles. The characteristics of these particles strongly influence the dose of exposure, and do play a significant role in defining the symptoms of allergic disease. The size and shape of the particles determines (1) the quantity of allergen per particle, (2) the ease with which the particles become, or stay airborne, (3) the speed with which allergens are released and (4) the sites of deposition in the respiratory tract. To illustrate these differences we will consider four different allergen sources and their associated particles: dust mite (feces); cat (dander); grasses (pollen grains) and fungi of the genus Aspergillus (spores) (table 1). Pollen grains are designed to release recognition proteins rapidly on contact with a stamen; mite feces which are ⬃30 ␮m in diameter release protein rapidly because the peritrophic membrane is freely permeable to proteins [2]; cat dander particles remain airborne because they have smaller aerodynamic size, which may reflect their shape (i.e. flat flakes) rather than their size but they also release protein rapidly [3]; finally the spores of Aspergillus have a diameter of ⬃2–3 ␮m and are spherical, these spores are designed to resist dehydration and do not release proteins rapidly. Indeed some of the major fungal allergens, e.g. Asp f 1, are not expressed until the spore germinates [4]. Equally important, the volume of an Aspergillus spore is approximately 1/1,000th of the volume of a pollen grain or a mite fecal particle.

Platts-Mills/Erwin/Woodfolk/Heymann

4

Table 1. Allergens: sources, proteins and particles Source

Allergen

MW (kDa)

Properties

Particles

Release of protein

Size (mm)

Relative volume

Dust mite

Der p 1 Der p 2 Fel d 1 Fel d 2 Lop p 1 Asp f 1

29 15 38 – 27 18

Enzyme

Feces

Rapid

10–35

⬃1,000

CCSP1

Dander

Rapid

2–20

20–200

Ribotoxin

Pollen Spores

Rapid Slow2

30 1–3

⬃1,000 1–10

Cat Grass Aspergillus

CCSP ⫽ Clara cell secretory protein (immunomodulatory). Spores germinate and then express proteins within hours.

1 2

Both dust mites and domestic animals are important causes of sensitization and may be strongly associated with asthma. However, there are striking differences in the relationship to symptoms as well as in the effects of high exposure. It is not unusual for a cat-allergic patient to experience nasal or lung symptoms within minutes after entering a house with a cat. In keeping with this allergen can be detected airborne at all times and the quantity inhaled per day has been calculated to be as high as 0.5 ␮g. By contrast mite-allergic patients generally do not experience deterioration in symptoms until they have been in an area of high exposure for a day or more. In keeping with this mite allergen is only detectable airborne during and shortly after disturbance and the total inhaled per day may be as low as 10 ng. Inside homes the time that particles remain airborne is determined very largely by their aerodynamic size. Outdoors the rules are different; although most pollen grains are large, they are designed to remain airborne if there is any significant wind. Nonetheless the calculated exposure to pollen allergens per day is only a few nanograms. When Charles Blackley first calculated the quantity of pollen inhaled in 1873 he was so impressed at how small the quantities that gave him symptoms of hay fever were that he joined the British homeopathic society. As early as 1921 it was recognized that dust obtained from the houses of allergic patients contained allergens other than those recognized at that time. Several groups tried unsuccessfully to identify the house dust atopen without success. In 1967 Voorhorst et al. [5] established that dust mites were the major source of house dust allergens. Once dust mite allergen extracts were available for skin testing, the importance of this allergen was rapidly confirmed in most

Environmental Factors in Allergy and Asthma

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humid areas of the world. Although we will focus on dust mites because of their importance in asthma, it is important to recognize that there are many other insects and acarids that can contribute to house dust. Obvious examples include flies, moths, spiders, beetles, crickets, and the Asian Lady Beetle all of which can contribute foreign proteins to dust. Although some allergens may be ‘better’ than others, it is likely that any source of foreign proteins can sensitize some individuals and give rise to symptoms. Detailed study of any allergen source is difficult without purification of one or more specific allergen. Thus, purification of each of the major allergens has made it possible to measure exposure in dust and airborne, as well as facilitating studies of antibody and cellular immune responses. Having established the identity of a major allergen, it becomes possible to clone and sequence the relevant proteins. For dust mite extensive research has focused on the properties of Der p 1 [6]. This protein is produced in glands alongside the gut [Tovey, PhD thesis], in keeping with the fact that Der p 1 has sequence homology with cysteine proteases and is a potent enzyme. That this activity could be relevant to its role as an allergen is strongly supported by evidence that it can cleave CD23 and CD25 in vitro as well as open tight junctions in respiratory epithelium [7, 8]. Recent work has shown that under conditions of ‘high’ exposure dust mite allergens induce higher titers of IgE antibodies than most other allergens [9]. However, this observation applies to both Der p 1 and Der p 2 [9, 10]. Since Der p 2 is not an enzyme, it is difficult to argue that the enzymatic activity of the individual protein is critical. On the other hand if both Der p 1 and Der p 2 are inhaled on fecal pellets, then it may be that Der p 2 is inhaled ‘in the context’ of a potent enzyme. Similarly, the quantity of endotoxin on the particle may be critical but at present it is difficult to establish how much endotoxin the individual particles carry. What is certain is that exposure to mite allergens can induce IgE antibodies, in up to 35% of the population, and that the titer of IgE ab can increase to mean values as high as 20 IU/ml [9, 10].

The Cat Paradox: High Exposure to Cat or Dog Allergens Does Not Increase the Prevalence or Titer of IgE ab

In 1999 Hesellmar et al. [11] first observed that children raised in a house with a cat were less likely to become sensitized to cat allergens. Since that time at least ten studies have confirmed the general observation. However, the details are not the same. In some studies dogs or cats have been associated with decreased sensitization to allergens in general [12]. In other cases the tolerance that occurs with a cat in the home has been found to be cat specific [9, 13, 14]. The evidence about whether tolerance is persistent or can be reversed if a child

Platts-Mills/Erwin/Woodfolk/Heymann

6

is no longer exposed to a cat is also not consistent. At first it seemed possible that the phenomenon could be an example of reverse causality i.e. that families with a history of allergy would avoid owning a cat. The choice may be relevant in some countries (e.g. Sweden and the Netherlands) but it is not the cause of the effect. In the USA, UK or New Zealand, families do not often make decisions on the basis of family or personal history of allergy [9, 12, 13]. Second, the effect of cats has been seen on a population basis in the European Community Respiratory Health Survey studies. Thus in countries such as Australia or New Zealand where up to 60% of the homes have a cat there is a lower population prevalence of sensitization to cat compared with countries where only 20% of homes have a cat [15]. Most recently detailed studies in a country where most homes have high exposure to both mite and cat allergens have shown that the presence of a cat has no effect on the IgE ab response to mite allergens [9, 10]. In that study among the 140 children who had ever lived in a house with a cat, only 26% of the allergic children had IgE ab to cat. Perhaps more striking, out of 50 children with asthma who were living in a house with a cat, 34 had IgE ab to mite but not to cat. Thus, even when we focus on atopic children the phenomenon of tolerance to cat allergens is very clear. This of course supports the argument that the phenomenon is cat specific and unlikely to be due to a nonspecific effect, such as we would expect with endotoxin. The fact that high exposure to cat allergens does not influence the titer of IgE ab to mite allergens can be seen either from the point of view of the cat or the mite! If tolerance to cats is specific it argues strongly that the milieu of a tolerant response, whatever the mechanism, does not influence other responses. Alternatively, we could argue that the response to dust mite is not only resistant to the normal effects of high dose itself, but that it is also resistant to any bystander effects from the response to another allergen.

The American Inner City as a Special Issue

Children raised in large cities have always suffered from poverty, malnutrition and neglect. However, during the industrial revolution and even up to 1950 this environment was not thought to give rise to asthma. Indeed asthma was still rare in childhood even in the most polluted cities in the world. Since then asthma has risen to become an epidemic with a major impact on school absenteeism, interference with parental schedules, costs of medications, emergency room visits and hospitalization. In the USA this increase has been most severe among children living in poverty in cities [16, 17]. Evaluation of inner city children has consistently identified allergy as a major factor in asthma, particularly

Environmental Factors in Allergy and Asthma

7

among children presenting to hospital [18–21]. Indeed there is considerable evidence that African Americans presenting with acute episodes are more allergic when compared to other patients presenting to the ER [18, 22, 23]. The nature of the allergens that are relevant to asthma in cities can be different from those that are relevant to suburban or rural areas of the same country. The importance of German cockroaches as a source of allergen was recognized as early as 1966 [24]. Subsequently it became clear that these allergens were relevant to both adults and children presenting for emergency treatment and in each case sensitization was paralleled by high levels of allergen exposure [18, 19]. Finally Rosenstreich et al. [20] demonstrated that the combination of sensitization and exposure was a major risk factor for hospitalization of children living in poverty. Given the surprising data on high-dose exposure to cat allergens, we and others have questioned whether tolerance to cockroach allergens could occur with high exposure [25, 26]. The reported levels of cockroach allergens are not as high as those reported for dust mite or cat allergens. At present there is no convincing evidence for tolerance to cockroach and high titer IgE ab to B. germanica allergens does occur [27]. The implication is that the response to cockroach allergens is comparable to the response to dust mite allergens. Rodent infestation is a common problem in large cities and is particularly severe in New York and Chicago, two cities that have figured prominently in the rise in asthma mortality in the USA. In addition it is well established that urine is a major source of allergens for scientists working with rodents. On the other hand studies on animal handlers have suggested that these allergens can give rise to a form of tolerance that includes an IgG/IgG4 antibody response comparable to that seen with cat exposure [28]. Thus, it would be very interesting to understand the dose response to mouse or rat allergen exposure in homes. Current data from National Inner City Asthma Study and from Baltimore suggests that sensitization is common [29, 30]. However most of the data is based on skin tests. Looking at 110 children with asthma in Atlanta we found very little serological evidence for sensitization to mouse or rat urine [31]. Given the evidence about differences between the response to cat and dust mite, each new allergen should be evaluated for both dose response and the effects of high dose on the titer of IgE antibodies.

The Role of Specific Antibody Responses in Influencing Total Serum IgE, the Prevalence of Asthma and Severity of Asthma

Immune responses to foreign proteins are subject to a variety of controls which prevent inappropriate or excessive response. Responses of the IgE

Platts-Mills/Erwin/Woodfolk/Heymann

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isotype are particularly well controlled which may reflect the fact that the conditions of a mature germinal center are not conducive to the survival of IgE B cells [32]. Thus surprisingly the essence of the exposure that gives rise to an IgE antibody response maybe low dose without adjuvants [32]. The effectiveness of the control over IgE responses is clear from the fact that most children and adults are skin test negative and that the mean total serum IgE is generally less than 100 IU or ⬍250 ng/ml. As soon as it became possible to measure IgE in serum, reports started to appear that total IgE was elevated among patients with asthma. By 1980 this was well established; however, in areas where dust mites dominated sensitization the correlation with specific IgE was so strong that total IgE was considered to be a surrogate for specific IgE. By contrast Burrows et al. [33] in Tucson, Arizona, reported that the correlation between total IgE and asthma was not reflected in specific IgE ab or skin tests. They also reported that seasonal rhinitis correlated strongly with skin tests to pollens but not with total IgE. Their results were interpreted as suggesting that IgE had a relationship to asthma not dependant on specific antibodies. Subsequently it became clear that Alternaria was a major allergen in South West USA [34]. However, there was an important implication of those studies, which is that the perennial allergens associated with asthma have more effect on total IgE than the seasonal allergens do. Comparing two population-based studies in countries which have dramatically different climates, we have found a striking difference in the number of sera with high-titer (i.e. ⱖ10 IU/ml) IgE ab [9, 14]. Furthermore, in those studies the mean total IgE in one country, i.e. New Zealand, was higher. Thus, in Sweden compared to New Zealand the mean total IgE of the population was lower among wheezing and nonwheezing children. In addition the prevalence of wheezing in Sweden was 8.5% compared to 21% in New Zealand. We now believe that the dominance of dust mite allergens in New Zealand contributes to the elevated total IgE, higher prevalence and increased severity of asthma in that country [35].

Endotoxin: Another Environmental Exposure with a Nonlinear Dose Response

The term endotoxin describes a family of lipopolysaccharides found in the cell walls of gram-negative bacteria. These substances are known to interact with Toll receptor 4 and can produce biological effects both in vivo and in vitro. For many years it has been known that airborne endotoxin can give rise to symptoms in animal handlers and can contribute to asthma symptoms in miteallergic individuals [36]. On the other hand there is compelling evidence that

Environmental Factors in Allergy and Asthma

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endotoxin or comparable products related to farm animals can inhibit the allergic response in early childhood [37]. However, this appears to require doses that are higher than those encountered in the domestic setting [38]. The possibility that endotoxin has a biphasic effect has been strongly supported by recent studies in mice, showing that the IgE ab response to ovalbumin is enhanced by small quantities of endotoxin but is inhibited by higher quantities [39]. Recently Peden and his colleagues [40] in North Carolina have reported results in human volunteers showing that low-dose endotoxin challenge can enhance IL-13 production while decreasing IFN-␥ production. It is clearly a possibility that the tolerance induced by domestic animals is related to increased endotoxin in homes with animals. However, the published data on endotoxin has been largely related to floor dust samples rather than airborne exposure. Furthermore, the results do not consistently demonstrate higher levels in homes with an animal [41]. Using a novel high-volume technique for sampling airborne particles we have measured endotoxin and allergens in homes [42, 43]. The result shows that endotoxin is measurable in the air of all homes and that overall there is no significant difference between homes with or without animals. On the other hand the levels found in homes with a dog or dogs are higher than in those with a cat and could be relevant to the nonspecific effect reported from Detroit [12]. It is extremely unlikely that the specific tolerance seen with a cat in the home is related to higher exposure to endotoxin.

Conclusions

Antibodies of the IgE isotype are closely associated with prevalence and severity of asthma. However chronic asthma symptoms and bronchial hyperresponsiveness are much more closely associated with IgE antibodies to perennial allergens than to seasonal allergens. The view that the IgE antibodies are essential for the asthmatic response is further supported by two recent findings: (1) the demonstration that injections of anti-IgE are an effective treatment for moderate asthma and can decrease exacerbations [44, 45] and (2) the finding that many children raised with a cat who have made IgG and IgG4 ab to Fel d 1, without IgE, do not have an increased risk of asthma. The evidence taken together strongly supports the view that allergen exposure is essential for both sensitization and ongoing inflammation of the respiratory tract. The air we breathe includes a range of different gases and particles which can contribute to lung pathology. Although there have been many changes in our environment, none of these alone explains the consistency or the scale of the rise in asthma over the 40 years, 1960–2000. Traditional air pollution including SO2 declined

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dramatically in London and Philadelphia over the period when asthma was increasing. Similarly, air pollution in Eastern European industrial cities, such as Katowice in Poland, was very severe but was associated with bronchitis rather than asthma and if anything lower levels of allergy. That dust mite infestation increased in England and New Zealand with the introduction of central heating seems undoubted. However these changes were largely complete by 1975 and asthma continued to increase for a further 20 years. In addition there are areas of the world where asthma has increased but dust mites are not relevant. In this respect it seems very unlikely that exposure to cockroach allergens has increased consistently in New York, Baltimore or Chicago over the period when asthma has been increasing. Certainly it is unlikely that cockroach infestation has increased sufficiently to explain a 20fold increase in hospitalization for asthma [16, 17, 20]. Similarly, a progressive increase in cat exposure cannot be an explanation for the progressive increase in asthma in Sweden or in Los Alamos, New Mexico [14, 25]. Even though cat allergens are the major risk factor for asthma in both of these sites, it is now clear that the dose-response relationship between cat exposure and sensitization is not linear [11–13]. There is an outside possibility that a decline in the number of cats in Sweden has contributed to the increase in allergy, but this is not a conceivable explanation for the scale of the increase in prevalence of asthma in that country. The American inner city poses a severe challenge to all theories about why asthma has increased. Firstly most of the adults who have lived in these cities over the period we are concerned with do not accept that there have been changes in cockroach or rodent infestation, sufficient to explain the increase in asthma. Arguing that cleanliness or hygiene has improved in these areas is not convincing for apartments or even homeless shelters that are overcrowded and infested. On the other hand children in the cities are spending a larger and larger proportion of their time indoors. Neither the reasons for this change nor the consequences are simple; mothers are afraid of the streets and violence, children are seduced by computers or television and communities do not provide safe outdoor environments to play. The consequences include increased snacking/junk food, more time spent sedentary and increasing obesity. The scale of these changes is so great that it seems inevitable that they have had an impact on lung physiology [46]. The evidence that obesity relates to asthma is clear but the actual numbers do not suggest that asthma is the real cause of the epidemic. It seems more likely that obesity is either the cause for a minority of cases or a surrogate for the real culprit. Recently several kinds of evidence have suggested that full expansion of the lungs could provide an important defense mechanism against wheezing. These include the evidence that bronchial smooth muscle requires full extension in vitro and the increasing

Environmental Factors in Allergy and Asthma

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evidence that 30 min without a deep breath will lead to bronchial hyperresponsiveness in nonasthmatic subjects [47, 48]. In addition there is evidence from 6-day monitoring that wheezing children enrolled in a head start program are less active that nonwheezing children, i.e. as early as 4 years of age [49]. The real question at this time is why children raised in an environment where they have more than adequate caloric intake, higher exposure to indoor allergens, such as mite and cockroach, and an increasingly sedentary life have such severe asthma. Although factors such as the details of the diet, immunization policy, air pollution and maternal smoking may all contribute, increases in asthma have occurred without these factors being relevant in different parts of the world. There are some reports suggesting that obesity is a risk factor for wheezing in nonatopic children but the data is still underwhelming and this has not been shown for acute or severe asthma. Thus, both for children living in poverty and those who are not acute, asthma remains very strongly associated with atopy and high total IgE [18, 19, 21, 22]. Recent evidence that some allergens influence total IgE more than others may be critically relevant to the causes of severity [35]. The scale of the increase in asthma is now so great that any attempt to treat the disease which does not address the causes of the increase is unlikely to be successful [1]. Considering the possible causes of the increase, we can attempt to control exposure but it is now clear that avoidance designed to prevent sensitization to mite allergens is extremely difficult in a community where all homes have mites, i.e. New Zealand or Manchester UK. Equally avoidance of exposure to cat allergens is not possible because the allergens are present in schools and homes without a cat. The next possibility is to control or influence Th2 responses. The obvious model is the tolerance that occurs in up to 50% of atopic children who live in a house with a cat. Many of these subjects are making an IgG/IgG4 ab response to Fel d 1 and have circulating T cells which can produce IL-10 and IFN-␥ in vitro [50]. It seems best to consider the increasing severity of asthma in the context of a series of changes: (1) moving to cities, clean water and the decline in enteric infections lead to the appearance of allergic disease; (2) the progressive triumph of indoor entertainment means that children spend many hours per day in an environment with perennial high levels of allergens; (3) finally the rise of sedentary entertainment and the closely associated rise in obesity represents the loss of the protective effect of normal outdoor play [1].

Acknowledgements Grant Support PO1 AI50989, AI20565.

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References 1 2 3

4 5

6 7

8

9

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11 12 13

14

15

16 17

18

19 20

Platts-Mills TAE: Asthma severity and prevalence: The results of an ongoing interaction between exposure, hygiene and lifestyle. PLoS Med 2005;2:e34. Tovey ER, Chapman MD, Platts-Mills TA: Mite faeces are a major source of house dust allergens. Nature 1981;289:592–593. Luczynska CM, Li Y, Chapman MD, Platts-Mills TAE: Airborne concentrations and particle size distribution of allergen derived from domestic cats (Felis domesticus). Measurements using cascade impactor, liquid impinger, and a two-site monoclonal antibody assay for Fel d I. Am Rev Respir Dis 1990;141:361–367. Arruda LK, Platts-Mills TAE, Fox JW, Chapman MD: Aspergillus fumigatus allergen I, a major IgE binding protein. J Exp Med 1990;172:1529–1532. Voorhorst R, Spieksma FTHM, Varekamp H, Leupen MJ, Lykelma AW: The house dust mite (Dermatophagoides pteronyssinus) and the allergens it produces. Identity with the house dust allergen. J Allergy 1967;39:325. Chapman MD, Platts-Mills TAE: Purification and characterization of the major allergen from D pteronyssinus – Antigen P1. J Immunol 1980;125:587–592. Ghaemmaghami AM, Gough L, Sewell HF, Shakib F: The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12. Clin Exp Allergy 2002;32:1468–1475. Wan H, Winton HL, Soeller C, Tovey ER, Gruenert DC, Thompson PJ, Stewart GA, Taylor GW, Garrod DR, Cannell MB, Robinson C: Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions. J Clin Invest 1999;104:123–133. Erwin EA, Wickens K, Custis NJ, Siebers R, Woodfolk J, Barry D, Crane J, Platts-Mills TA: Cat and dust mite sensitivity and tolerance in relation to wheezing among children raised with high exposure to both allergens. J Allergy Clin Immunol 2005;115:74–79. Erwin E, Custis N, Perzanowski M, Woodfolk J, Crane J, Wickens K, Platts-Mills TAE: Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a highcapacity solid phase. J Allergy Clin Immunol 2005;115:1029–1035. Hesselmar B, Aberg B, Eriksson B, Aberg N: Asthma in children: Prevalence, treatment, and sensitization. Ped All Imm 2000;29:611–617. Ownby DR, Johnson CC, Peterson EL: Exposure to dogs and cats in the first year of life and risk to allergic sensitization at 6 to 7 years of age. JAMA 2002;288:963–972. Platts-Mills TAE, Vaughan J, Squillace S, Woodfolk JA, Sporik R: Sensitisation, asthma, and a modified Th2 response in children exposed to cat allergen: A population-based cross-sectional study. Lancet 2001;357:752–756. Perzanowski MS, Ronmark E, Platts-Mills TAE, Lundback B: Effect of cat and dog ownership on sensitization and development of asthma among preteenage children. Am J Resp Crit Care Med 2002;166;696–702. Roost HP, Kunzli N, Schindler C, Jarvis D, Chinn S, Burney P, Wuthrich B: Role of current and childhood exposure to cat and atopic sensitization. European Community Respiratory Health Survey. J Allergy Clin Immunol 1999;104:941–947. Gergen PJ, Weiss KB: Changing patterns of asthma hospitalization among children: 1979 to 1987. JAMA 1990;264:1688–1692. Crater DD, Heise S, Perzanowski M, Herbert R, Morse CG, Platts-Mills TAE: Asthma hospitalization trends in Charleston, South Carolina, from 1956–1997: Twenty-fold increase among African American children over a thirty-year period. Pediatrics 2001;108:1–6. Gelber LE, Seltzer LH, Bouzoukis JK, Pollart SM, Chapman MD, Platts-Mills TAE: Sensitization and exposure to indoor allergens as risk factors for asthma among patients presenting to hospital. Am Rev Respir Dis 1993;147:573–578. Call RS, Smith TF, Morris E, Chapman MD, Platts-Mills TAE: Risk factors for asthma in inner city children. J Pediatr 1992;121:862–866. Rosenstreich DL, Eggleston P, Kattan M, Baker D, Slavin RG, Gergen P, Mitchell H, McNiff-Mortimer K, Lynn H, Ownby D, Malveaux F: The role of cockroach allergy and exposure

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to cockroach allergens in causing morbidity among inner-city children with asthma. N Engl J Med 1997;336:1356–1363. Gruchalla RS, Pongracic J, Plaut M, Evans R, Visness CM, Walter M, Crain EF, Kattan M, Morgan WJ, Steinback S, Stout J, Malindzak G, Smartt E, Mitchell H: Inner city asthma study. J Allergy Clin Immunol 2005;115:584–591. Heymann PW, Carper HT, Murphey DD, Platts-Mills TA, Hatley TK, Chamberlain R: Viral infection in relation to age, atopy and season of admission among children hospitalized for wheezing. J Allergy Clin Immunol 2004;114:239–247. Litonjua A, Celedon J, Hausmann J, Nikolov M, Sredl D, Ryan L, Platts-Mills TAE, Weiss S, Gold D: Variation in total and specific IgE: Effects of ethnicity and socioeconomic status. J Allergy Clin Immunol 2005;115:751–757. Bernton HS, Brown H: Cockroach allergy II: The relation of infestation to sensitization. South Med J 1967;60:852–855. Sporik R, Squillace SP, Ingram JM, Rakes G, Honsinger RW, Platts-Mills TAE: Mite, cat and cockroach exposure, allergen sensitization and asthma in children: A case-control study of three schools. Thorax 1999;54:675–680. Eggleston PA, Rosenstreich D, Lynn H, Gergen P, Baker D, Kattan M, Mortimer KM, Mitchell H, Ownby D, Slavin R, Malveaux F: Relationship of indoor allergen exposure to skin test sensitivity in inner-city children with asthma. J Allergy Clin Immunol 1998;102(4 Pt 1):563–570. Satinover S, Reefer A, Pomes A, Chapman M, Platts-Mills TAE, Woodfolk J: Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 2005;115: 803–809. Platts-Mills TAE, Longbottom J, Edwards J, Cockroft A, Wilkins SR: Occupational asthma and rhinitis related to laboratory rats. J Allergy Clin Immunol 1987;79:505–515. Matsui EC, Krop EJ, Diette GB, Aalberse RC, Smith AL, Effleston PA: Mouse allergen exposure and immunologic responses: IgE-mediated mouse sensitization and mouse specific IgG and IgG4 levels. Ann Allergy Asthma Immunol 2004;93:171–178. Matsui EC, Simons E, Rand C, Butz A, Buckley TJ, Breysse P, Eggleston PA: Airborne mouse allergen in the homes of inner-city children with asthma. J Allergy Clin Immunol 2005;115: 358–363. Carter MC, Perzanowski MS, Raymond A, Platts-Mills TAE: Home intervention in the treatment of asthma among inner-city children. J Allergy Clin Immunol 2001;108:732–737. Aalberse RC, Platts-Mills TA: How do we avoid developing allergy: Modifications of the Th2 response from a B-cell perspective. J Allergy Clin Immunol 2003;113:983–986. Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG: Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989;320:271–277. Halonen M, Stern DA, Wright AL, Taussig LM, Martinez FD: Alternaria as major allergen for asthma in children raised in a desert environment. Am J Respir Crit Care Med 1997;115: 1356–1361. Erwin EA, Ronmark E, Wickens K, Perzanowki M, Barry D, Pollart S, Lundback B, Crane J, Platts-Mills TAE: The IgE antibody response to specific allergens can make an important contribution to total IgE; submitted. Michel O, Kips J, Duchateau J, Vertongen F, Robert L, Collet H, Paulwels R, Sergysels R: Severity of asthma is related to endotoxin in house dust. Am J Respir Crit Care Med 1996;154(6 Pt 1): 1641–1646. Braun-Fahrlander C, Ridler J, Herz U, Eder W: Environmental exposure to endotoxin and it relation to asthma in school age children. N Engl J Med 2002;347:869–877. Gereda JE, Leung DY, Thatayatikom A, Streib JE, Price MR, Klinnert MD, Liu AH: Relation between house-dust endotoxin exposure, type 1 T-cell development and allergen sensitisation in infants at high risk of asthma. Lancet 2000;355:1680–1683. Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K: Lipopolysaccharideenhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002;196:1645–1651. Alexis NE, Lay JC, Almond M, Peden DB: Inhalation of low-dose endotoxin favors local T(H)2 response and primes airway phagocytes in vivo. J Allergy Clin Immunol 2004;114:1116–1123.

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Platts-Mills J, Custis NJ, Woodfolk JA, Platts-Mills TAE: Airborne endotoxin in homes with domestic animals: Implications for specific tolerance. J Allergy Clin Immunol 2005;116:384–389. Custis NJ, Woodfolk JA, Vaughan JW, Platts-Mills TA: Quantitative measurement of airborne allergens from dust mites, dogs, and cats using an ion-charging device. Clin Exp Allergy 2003;33: 986–991. Platts-Mills J, Custis N, Kenney A, Tsay A, Chapman M, Feldman S, Platts-Mills TAE: The effects of cage design on airborne allergens and endotoxin in animal rooms: High-volume measurements with anion-charging device. Contemp Top Lab Anim Sci 2005;44:12–16. Milgrom H, Fick RB Jr, Su JQ, Reimann JD, Bush RK, Watrous ML, Metzger WJ: Treatment of allergic asthma with monoclonal anti-IgE antibody: rhuMA␤-E25 Study Group. N Engl J Med 1999;341:1966–1973. Holgate S, Casale T, Wenzel S, Bousquet J, Deniz Y, Reisner C: The anti-inflammatory effects of omalizumab confirm the central role of IgE in allergic inflammation. J Allergy Clin Immunol 2005;115:459–465. Hark W, Thompson W, McLaughlin T, Wheatley L, Platts-Mills TAE: Spontaneous sigh rates during sedentary activity: Comparison between watching a television screen and reading. Ann Allergy Asthma Immunol 2005;94:247–250. Fredberg JJ: Airway smooth muscle in asthma: Perturbed equilibria of myosin binding. Am J Respir Crit Care Med 2000;161(3 Pt 2):S158–S160. Skloot G, Togias A: Bronchodilation and bronchoprotection by deep inspiration and their relationship to bronchial hyperresponsiveness. Clin Rev Allergy Immunol 2003;24:55–72. Firrincieli V, Keller A, Ehrensberger R, Platts-Mills J, Shuffleberger C, Geldmaker B, Platts-Mills TAE: Activity and wheezing in preschool children. Pediatr Pulmonol 2005; in press. Reefer AJ, Carneiro RM, Custis NJ, Platts-Mills TA, Sung SS, Hammer J, Woodfolk JA: A role for IL-10 mediated HLA-DR7-restricted T cell-dependent events in the modified Th2 response to cat allergen. J Immunol 2004;172:2763–2772.

Prof. Thomas A.E. Platts-Mills Asthma and Allergic Disease Center University of Virginia, PO Box 801355 Charlottesville, VA 22908 (USA) Tel. ⫹1 804 924 5917, Fax ⫹1 804 924 5779, E-Mail [email protected]

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The Environment Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 16–29

Should Asthma Management Include Sojourns at High Altitude? Gerhard Schultze-Werninghaus Berufsgenossenschaftliche Kliniken Bergmannsheil, Bochum, Germany

Abstract Sojourns in the high mountains have been recommended by specialists for patients with asthma since many decades. An inquiry among physicians of the ‘Davoser Ärzteverein’ revealed as early as 1906 that 133/143 patients with bronchial asthma had no or only few asthma attacks during their stay in Davos, and that 81% had a persistent improvement of their disease. These early observations about effects of the alpine climate were, of course, reported at a time, when the spectrum of pharmacotherapy was very limited. However, these observations were consistent and were therefore regarded as proof for the therapeutic value of sojourns under alpine conditions in bronchial asthma. In recent years, however, the indication for asthma treatment in high mountains is increasingly questioned, in particular by health insurance systems. Therefore it is the aim of this contribution to summarize the available data about the effects of a stay of asthmatic patients at 1,500–1,800 m above sea level. It is concluded that the available evidence suggests a significant beneficial effect of high altitude in bronchial asthma, in particular in steroid-dependent patients. Copyright © 2006 S. Karger AG, Basel

Features of High Mountains’ Climate

There are a number of meteorological and climatic changes with increasing height above sea level, which may have influences on human health (table 1). In chronic airway diseases the reduction of allergens and air pollution is of particular interest. Air Pollution Nitric oxide (NO), nitric dioxide (NO2), ozone (O3), sulfur dioxide (SO2), diesel soot and dust particles (PM10) are regarded as risk factors for the origin

Table 1. Changes of meteorological factors with increasing height above sea level [26] Increase or decrease per ⫹100 m Increase

Decrease

Global radiation UV radiation Electromagnetic radiation Snow covering Wind velocity Inhaled allergens Air pressure Air temperature Water vapor pressure Air pollution Oxygen partial pressure

⫹20–25% ⫹20–30%

⫺12% ⫺6⬚C ⫺25% ⫺12%

and the course of allergic diseases. These factors may lead to an increase in asthma symptoms with a deterioration of lung function [1]. In a study on 4,470 children (6–15 years) in 10 Swiss communities, a close correlation was found between the exposure to PM10, NO2, SO2, meteorological data, but not O3, and bronchial symptoms (chronic cough, dry cough at night, bronchitis) [2]. Air pollution with NO2, NO, SO2, dust (TSP, PM10) and also O3 (incidence of concentrations above limits, maximum values) is significantly diminished in high mountains compared with lower areas (Bundesamt für Umwelt, Wald und Landschaft, Switzerland, 2003; table 2). Allergens In high mountains allergen concentrations are significantly reduced in comparison to lower areas, due to differences in temperature, humidity, and vegetation. In particular pollen season is much shorter and clinically relevant pollen concentrations are found only on few days [3] (fig. 1). The nearly total absence of dust and storage mites at and above 1,500 m [4, 34] may be of even greater importance, because these are the most frequent causes of perennial allergic airway diseases. The concentration of mites or mite allergen in house dust samples is below threshold values for allergic symptoms. In a study in Briançon/France (1,326 m) 0.36 ␮g mite allergen/g dust was found, at sea level the concentration was 15.8 ␮g/g [5]. Similarly, in Misurina/ Italy (1,756 m) 0.04 ␮g/g was measured, in contrast to 15 ␮g/g in low areas [6]. In Davos the concentration of dust mite allergens in living and bedrooms is below 0.02 ␮g/g and even 0 ␮g/g under hospital conditions [4].

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Table 2. Selected immission data in 2003 (Bayerisches Landesamt für Umweltschutz, Landesumweltamt Nordrhein-Westfalen/Germany, Bundesamt für Umwelt, Wald und Landschaft, Switzerland)

Threshold values Davos/Switzerland, 1,637 m Basel/Switzerland, 280 m Garmisch-Partenkirchen Germany, Kreuzeckbahnstr., 735 m Garmisch-Partenkirchen Germany, Wankgipfel, 1,776 m Duisburg-Walsum Germany, 80 m München-Stachus Germany, 520 m

Ozone, annual mean value (␮g/m3)

Nitric dioxide, annual mean value (␮g/m3)

Nitric oxide, annual mean value (␮g/m3)

PM10, annual mean value (␮g/m3)

Sulfur dioxide, annual mean value (␮g/m3)

nd 79 53 59

40 4 28 10

30 3 22 2

50 15* 27 nd

20 ⬍1* 5 nd

101

3

0

nd

nd

38

33

19

31

10

38

68

53

46

3

* ⫽ Rigi/Switzerland, 1,700 m above sea level; nd ⫽ no data. Threshold values ⫽ limits for the protection of human health, according to 22nd and 33rd. BlmSchV (Verordnung über Immissionswerte für Schadstoffe in der Luft, Verordnung zur Verminderung von Sommersmog, Versauerung und Nährstoffeinträgen, Germany). Meters (m) are meters above sea level.

Moreover, mould spore concentrations are much lower in high mountains. The number of Cladosporium spp. spores is 25–50% of that of lower areas, Alternaria spp. only 1–3% [3].

Effects of the High Altitude in Bronchial Asthma

The high mountains are apparently of favorable influence on the health of permanent residents: the frequency of mite sensitizations in asymptomatic subjects is much lower than at sea level (Briançon/France, 1,760 m: 10.2%; Marseille/France, 0 m: 27.5%) [5]. The effects of a limited sojourn of a few weeks up to more than one year in high mountains have been studied in asthmatic children and juveniles.

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Birch pollen 2004

1,200 1,000

Basel

Per m3

800

Davos

600 400 200

28.5

24.5

20.5

12.5 2.7

16.5

8.5

4.5

25.6

30.4

26.4

22.4

18.4

I4.4

6.4

10.4

2.4

29.3

25.3

0

Grass pollen 2003

300

Per m3

250 200 150 100 50 30.7

22.7

16.7

9.7

18.6

11.6

4.6

28.5

21.5

14.5

7.5

30.4

21.4

16.4

0

Fig. 1. Pollen counts, Basel and Davos/Switzerland, 2004 (birch pollen) and 2003 (grass pollen) (Source: Meteoschweiz).

These studies have uniformly shown beneficial effects on the disease. In older investigations [7–9] clinical findings, such as attenuation of asthmatic symptoms and reduction or cessation of asthma medication was used as outcome parameters. In recent decades objective parameters were increasingly included, such as lung function, provocation tests of bronchial hyperresponsiveness (BHR), and laboratory markers of airway inflammation. These studies are summarized in this review. The majority of published data stem from hospitals located in Davos/Switzerland, 1,560 m, Misurina/Italy, 1,756 m, and Briançon/ France, 1,760 m. For this review data from investigations above 1,500 m were primarily included, according to the definition of ‘high mountains’ (table 3). Laboratory Markers of Airway Inflammation Activated T lymphocytes and increased concentrations of eosinophil granulocytes in blood and sputum correlate with the severity of bronchial asthma [10]. In the first study about the changes of activity markers of airway

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19

Schultze-Werninghaus

Table 3. Most important clinical studies in asthmatic patients under high altitude conditions Study

Ref. Subjects

Nr.

Nr.

n

Months

Age (y) range (mean ± SEM)

1 2

23 24

31 20

(11.7) 12–17

3

9

42

9–17

9

4

7

14

(11.5 ⫾ 1.7)

8

Controls

Observation n period *) b/r: or time back to low back to area low area remain in (weeks) low area

~3 ⱕ19

24

Medication (Steroids) ↓ reduction ↑ increase = constant a) high b) low

Lung function ↑ improvement ↓ deterioration = constant

Hyperresponsiveness ↓ decrease ↑ increase = constant

Mite specific IgE ↓ decrease ↑ increase = constant

Eosinophils (Blood/Sputum) ↓ decrease ↑ increase = constant

Markers of inflammation ↓ decrease ↑ increase = constant

a) high

a) high b) low

a) high

a) high b) low

a) high b) low

↓ ⭈

↑(14/31) ↑

↓

⭈

b) low

↓

b) low

↓ ↓

↑

↓

5

20

20

(10.6 ⫾ 1.7)

ⱖ3

2

(↓)

(↑)

⫽

⫽

↓

⫽

↓

(↑)

6

19

45

a) (10.6 ⫾ 1.4) b) (10.8 ⫾ 2)

ⱖ9

12

(↓)

(↑)

(⫽)

(⫽)

↓

↑

↓

⫽

7

17

14

8–15 (12.5 ⫾ 2.1)

1.2

8

33

14

a) (14 ⫾ 0.5) b) (15 ⫾ 0.3)

0.5

9

31

12

(11.1 ⫾ 1.1)

3

10

14

16

(10.7 ⫾ 1.6)

3

a) 8/14: b*

⫽

↑

2

⫽

⭈

↓

⭈

↑

3

↓

⫽

⫽

↓

↑

↓

⭈

↓

↓

↓

↓ ↓

20

11

13

16

(14.6 ⫾ 0.2)

1

⫽

↓

12

15

20

8–13

2

⭈

↓

⭈

↓

⭈

↓

⭈

⭈

↑

↓

⭈

↓

⭈

High Altitude and Asthma

13

16

14

a) (15.5 ⫾ 0.6) 2.5 b) (14.0 ⫾ 1.0)

14

28

18

(10.7 ⫾ 1.6)

9

b) 6/14 r*

6

⫽

⫽

↑

↓

2

↓

↑

(↑)

(↓)

↓

Data in brackets ( ): no precise data, information from text or trends. Data a) high: formal study, b) low: formal study or post study observation period of variable duration. Study 2: Data from conference lecture, no formal study. *) Controlled study Nr. 8 (after ⱖ4 weeks): 6/14: continuous stay in high mountains, 8/14: return to low area 2 weeks; *) Controlled study Nr. 13: 8/14 high mountains, then low area; 6/14 remaining in low area.

⫽

↓

↑

↓

(↑)

↓

⫽

21

inflammation by Boner et al. [11, 12] in 12 children with allergic asthma during a 3-month sojourn in Misurina demonstrated that markers of eosinophilic inflammation, such as eosinophil cationic protein and eosinophil protein X decreased significantly. After return into the lower areas of Italy signs of inflammation increased again. A reduction of markers of airway inflammation in asthmatic patients during a sojourn in Davos and Misurina has also been found by other investigators, e.g. a reduction of eosinophilia in blood [13] and induced sputum [14, 15], or of eosinophil products, such as urine eosinophil protein X [16] and serum eosinophil cationic protein [13]. In juveniles, receiving inhaled corticosteroids at a constant dose, Grootendorst et al. [16] confirmed during a 10-week sojourn in Davos a decrease of urine leukotriene E4 and of 9␣, 11␤ prostaglandin F2. The inflammation markers did not return to initial concentrations up to 6 weeks after return of the patients to the Netherlands. This finding demonstrates persistence of the anti-inflammatory effect of high mountains for at least 6 weeks, in juveniles under a high daily dose of inhaled steroids. As a further parameter of airways inflammation, sputum epithelial cells were investigated by Piacentini et al. [15]. Also this parameter, related to BHR and chronicity of asthma, was favorably influenced by a sojourn in Misurina. Thus, a number of investigations uniformly demonstrate anti-inflammatory effects of a sojourn in high mountains, such as eosinophil activation. These effects are not caused by pharmacotherapy, clearly to be excluded at least in some of the studies in which patients were under a constant drug treatment [16, 17]. It is of particular importance that these effects have also been observed in patients who inhaled a high daily corticosteroid dose, thereby demonstrating an additional anti-inflammatory effect of high mountains in asthma [16]. Immunoglobulin E and T-Cell Functions In 42 mite-allergic asthmatic children a significant and important reduction of total and mite-specific immunoglobulin E was found during a 9-month sojourn in Briançon [9, 18] (fig. 2). Other investigators confirmed these results [19, 20]. These data might be interpreted as a result of decreased allergen exposure and could be related to the reduction in airway inflammation. However, the reduction in immunoglobulin E synthesis could also be the consequence of profound changes in the immune system. In 1990, Wüthrich et al. [21] demonstrated in 27 patients with atopic eczema an initial T-cell activation [soluble interleukin-(IL-)2 receptor] that diminished during a sojourn in Davos (mean duration 37 days). Simon et al. [17] found in 14 mite-allergic asthmatic children an initial T-cell activation [CD25 expression (␣-chain of the IL-2 receptor of CD4⫹ T cells) and HLA-DR antigen]. Without any change of medication there was a

Schultze-Werninghaus

22

1,000

*

**

800

***

600

400

0

3

6

Mite-specific lgE (counts per minute) geometric mean⫾ 9

Total lgE (U/ml) geometric mean⫾9

n ⫽ 42 *p⬍ 0.02 **p⬍ 0.01 ***p⬍ 0.001

1,500

n⫽34 *p⬍0.02 **p⬍0.001

2,000

* ** 1,000

**

500 0

9

3

6

9

Months

Months

Fig. 2. Total immunoglobulin E (IgE) and mite-specific IgE during a sojourn in Briançon/France [9].

5

7.5 * * 5

2.5

Positive CD4⫹cells (%)

Eosinophil granulocytes (% of leukocytes)

10

* *

3

CD25 ⫹ HLA-DR⫹

2 1 0

0 2

a

4

21

35

Days

2

b

21

35

Days

Fig. 3. a Blood eosinophilia (% of leukocytes), b T-cell activation marker, CD4⫹ T cells – at the beginning and at 3 and 5 weeks, respectively, of a sojourn in Davos [17].

significant reduction in CD25 expression after 3 weeks of stay in Davos, together with a normalization of blood eosinophilia and an improvement of lung function (fig. 3). Thus, it has been convincingly demonstrated that T-cell activation, a hallmark of allergic inflammation, is significantly diminished during a stay in high mountains. Simultaneously, clinical and laboratory parameters of airway inflammation, such as BHR and sputum or blood eosinophilia, show a significant and

High Altitude and Asthma

23

Allergen intolerance BHR

Sea level UV light Allergens infections

TNF-␣, IL-12 IFN-␥, IL-4, Th1, Th2 Monocyte

UV light Vit.D3, IL-10

T cell IL-10, TGF-␤ Treg

Allergens infections High altitude

Allergen tolerance

Fig. 4. Potential effects of high altitude conditions on some immunological functions related to bronchial asthma [Blaser et al., Davos].

impressive improvement or normalization. Traditionally, these findings were attributed primarily to a reduction of allergen exposure, thereby reducing the proinflammatory signals for Th2-cell activation. In a recent study it was shown that a stay in Davos stimulates IL-10-producing, regulatory CD4⫹ T cells, possibly thereby reducing activation of IL-13 and interferon-␥-producing, proinflammatory CD4⫹ T cells and local airway inflammation (NO synthesis), not only in allergic, but also in intrinsic asthmatic patients [22; Blaser, pers. commun.] (fig. 4). This would mean that a stay at or above 1,500 m above sea level exerts its positive effects in asthmatic patients not only by reduction of allergen exposure, but by profound changes in the immune system. The feature of these changes is in some aspects comparable to that seen after corticosteroid treatment and may therefore from a clinical point of view explain, why a significant reduction or even cessation of corticosteroid treatment was possible in a number of investigations in high altitude [7, 23, 25] (table 3). It has long been discussed that the favorable effects of sojourns in high mountains in allergic diseases could be due to increased cortisol production, in addition to the reduction of air pollution and allergen load [26]. It has been demonstrated in patients with psoriasis during a 4-week stay in Davos that after an initial increase in serum cortisol concentrations, there is a significant decrease between week 1 and 3, and a rise in week 4 [26]. Thus a general increase of cortisol concentrations was not demonstrated. This issue deserves further investigation.

Schultze-Werninghaus

24

Nonspecific and Specific BHR

As early as 1970, Kerrebijn [24] reported a significant and important reduction of BHR over time in asthmatic children during a stay of more than one year in Davos, the outcome in part depending on the initial severity of BHR (fig. 5). For the assessment of BHR a variety of chemical agents are used, the results of which are not in perfect agreement. Several investigators have emphasized a special suitability of provocation tests with adenosine monophosphate (AMP) for the detection of asthmatic BHR. In confirmation of the sensitivity of AMP provocations, Benckhuijsen et al. [27] found in 16 asthmatic children during a 4-week sojourn in Davos under constant medication an improvement of BHR to AMP and physical exercise, but not to methacholine. Grootendorst et al. [16] found a reduction of BHR to AMP and histamine during a 10-week stay in Davos of 10 atopic asthmatic juveniles, using an inhaled steroid dose of 500–2,000 ␮g/day, the effect starting in the third week. Six weeks after return to the Netherlands reduction of BHR was still significant. Also, during a longer stay in high mountains BHR to methacholine is reduced, as shown in 16 asthmatic children after 3 months in Misurina [14]. In 12 allergic asthmatic children a sojourn of 3 months in Misurina resulted in a reduction of peak-flow variability and BHR. Three weeks after return to sea level the effects were not persisting [29]. In 20 mite-allergic asthmatic children in Misurina a reduction of BHR was found on days 40 and 80. Fifteen days after return to sea level the effects began to decrease [20]. In mite-allergic asthmatic children after 3 and 9 months in Misurina a reduction of BHR to methacholine and histamine was found [19], which persisted in part up to 3 months after return to sea level. In summary, a profound reduction of BHR to various stimuli has been demonstrated in multiple investigations, as proof of an attenuation of airway inflammation, dependent on the duration of stay in high mountains. These findings corroborate the findings from the investigations of inflammatory markers in serum, sputum, and urine. The effects cannot be attributed to pharmacological treatment [16, 17]. In addition, Peroni et al. [19] demonstrated a reduction of specific airway reactivity against house dust mite extracts by provocation tests during a stay in Misurina. Lung Function The earliest report of longitudinal lung function tests during a stay in Davos was published by Kerrebijn et al. [23] in 1967. The authors found an improvement in lung function parameters of asthmatic children during a stay of several months, in spite of a reduction of steroid treatment. In 14 mite-allergic

High Altitude and Asthma

25

Histamine concentration needed for a decrease in FEV1 by 20% (PC20)

32 16 8 4 2 1

0

2

4

6

8

10

12

Months spent at Davos/Switzerland 1,560 m above sea level

Fig. 5. Bronchial hyperresponsiveness (BHR) during 12 months in Davos; 10 steroiddependent asthmatic children with a moderately severe BHR at the start of the study [34, data from 24].

asthmatic children a peak expiratory flow (PEF) improvement at rest and after physical exercise was found during a 3-week stay in Davos [17]. In subsequent studies it was shown that asthma-induced changes of lung function may be more sensitively detected by using forced expiratory flows at lower lung volumes (e.g. FEF25–75) or residual volume, than by FEV1 or PEF [28, 29]. In a recent study Grootendorst et al. [16] investigated 10 mite-allergic asthmatic juveniles (12–18 years old), treated with inhaled corticosteroids at a constant dose of 500–2,000 ␮g/day throughout the study. Life quality, lung function, BHR to AMP and histamine, induced sputum, and urine markers were studied during a stay of 10 weeks in Davos and up to 6 weeks after return to sea level. A comparison was made with 8 control patients who remained at sea level. Lung function improved in Davos, as did clinical and laboratory markers of inflammation. The authors conclude that a sojourn of 10 weeks in high mountains improves symptom control in severe juvenile asthma, and that the effects are greater than those of high doses of inhaled corticosteroids [16]. Duration of Effects In 1998 a retrospective study of 860 asthmatic patients was published, who had previously been treated in a hospital in Davos [30]. For the year after the stay in Davos absence of asthma symptoms or a relevant improvement was found in 65.2% of patients.

Schultze-Werninghaus

26

After return to sea level the effects of a sojourn in high mountains decrease [6, 19, 31]. However, it has been shown that the effects may last up to several months [12, 23]. A subsequent drug treatment supports the success of a previous stay in high mountains. In a double-blind study in 30 asthmatic children it could be demonstrated that the effects of the high mountains persist if subsequently an inhaled steroid treatment is performed [32]. Conclusions

The available studies on the effects of a sojourn of patients with bronchial asthma in high mountains uniformly demonstrate a reduction of clinical and laboratory markers of airway inflammation, apparently with increasing efficacy with the duration of the stay. The effects were observed in spite of a reduction or cessation of steroid treatment in many studies. In studies with constant steroid dose the effects were additive to drug therapy. It is not clear, if these effects are due to the very low concentrations of air pollutants and allergens, particularly above 1,500 m, or to other mechanisms. A reduction of the increased T-cell activation in allergic diseases has been demonstrated. This attenuation of proinflammatory immune function, which is associated with an increase in regulatory T cells, has not only been shown in allergic asthmatics, but also in intrinsic asthma. Therefore, high mountains’ climate could act via changes of immune functions that are independent of allergen exposure. Clearly, more data about the clinical effects of high mountains in asthma are warranted. The available studies are small. Only few controlled studies have been published and there is a lack of longitudinal observations. A more global assessment of the effects would be helpful, for example symptom-medication scores and life quality, in particular to better clarify the indications for asthma treatment in high mountains. However, the available evidence is clearly in favor of a positive effect of high mountains climate in asthma, as the data uniformly demonstrate antiasthmatic effects. Therefore, clinically controlled sojourns in high mountains add favorably to asthma management at sea level, in particular in steroid-dependent patients. References 1

Zemp E, Elsasser S, Schindler C, Künzli N, Perruchoud AP, Domenighetti G, Medici T, Ackermann-Liebrich U, Leuenberger P, Monn C, Bolognini G, Bongard JP, Brändli O, Karrer W,

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2

3 4 5

6 7

8 9

10

11

12 13

14

15

16

17

18 19

Keller R, Schöni MH, Tschopp JM, Villiger B, Zellweger JP: Long-term ambient air pollution and respiratory symptoms in adults (SAPALDIA study). The SAPALDIA Team. Am J Respir Crit Care Med 1999;159:1257–1266. Braun-Fahrländer C, Vuille JC, Sennhauser FH, Neu U, Kunzle T, Grize L, Gassner M, Minder C, Schindler C, Varonier HS, Wüthrich B: Respiratory health and long-term exposure to air pollutants in Swiss schoolchildren. SCARPOL Team. Swiss Study on Childhood Allergy and Respiratory Symptoms with Respect to Air Pollution, Climate and Pollen. Am J Respir Crit Care Med 1997;155:1042–1049. Leuschner RM, Boehm W: Pollen and inorganic particles in the air of climatically very different places in Switzerland. Grana 1981;20:161–167. Menz G, Petri E, Lind P, Virchow C: House dust mite in different altitudes of Grisons (Switzerland). Experientia, Advances in Aerobiology 1987;151(suppl):197–202. Charpin D, Birnbaum J, Haddi E, Genard G, Lanteaume A, Toumi M, Faraj F, Van der Brempt X, Vervloet D: Altitude and allergy to house-dust mites: A paradigm of the influence of environmental exposure on allergic sensitization. Am Rev Respir Dis 1991;143:983–986. Piacentini GL, Peterson C, Peroni DG, Bodini A, Boner AL: Allergen avoidance at high altitude and urinary eosinophil protein X. J Allergy Clin Immunol 1999;104:243–244. Boner AL, Niero E, Antolini I, Valletta EA, Gaburro D: Pulmonary function and bronchial hyperreactivity in asthmatic children with house dust mite allergy during prolonged stay in the Italian Alps. Ann Allergy 1985;54:42–45. Morrison-Smith J: The use of high altitude treatment for childhood asthma. Practitioner 1981;225: 1663–1666. Vervloet D, Bongrand P, Arnaud A, Boutin Ch, Charpin J: Données objectives cliniques et immunologiques observées au cours d’une cure d’altitude à Briançon chez des enfants asthmatiques allergiques à la poussière de maison et à Dermatophagoides. Rev Fr Mal Respir 1979;7: 19–27. Walker Chr, Kägi M, Braun P, Blaser K: Activated T-cells and eosinophilia in bronchoalveolar lavages from subjects with asthma correlate with disease severity. J Allergy Clin Immunol 1991; 88:935–942. Boner AL, Peroni DG, Piacentini GL, Venge P: Influence of allergen avoidance at high altitude on serum markers of eosinophil activation in children with allergic asthma. Clin Exp Allergy 1993a; 23:1021–1026. Boner AL, Peroni D, Sette L, Valletta EA, Piacentini G: Effects of allergen exposure-avoidance on inflammation in asthmatic children. Allergy 1993b;48(suppl 17):119–124. van Velzen E, van den Bos JW, Benckhuijsen JAW, van Essel T, de Bruijn R, Aalbers R: Effect of allergen avoidance at high altitude on direct and indirect bronchial hyperresponsiveness and markers of inflammation in children with allergic asthma. Thorax 1996;51:582–584. Piacentini GL, Martinati L, Mingoni St, Boner AL: Influence of allergen avoidance on the eosinophil phase of airway inflammation in children with asthma. J Allergy Clin Immunol 1996; 97:1079–1084. Piacentini GL, Vicentini L, Mazzi P, Chilosi M, Martinati L, Boner AL: Mite-antigen avoidance can reduce bronchial epithelial shedding in allergic asthmatic children. Clin Exp Allergy 1998;18: 561–567. Grootendorst DC, Dahlen SE, Van Den Bos JW, Duiverman EJ, Veselic-Charvat M, Vrijlandt EJ, O’Sullivan S, Kumlin M, Sterk PJ, Roldaan AC: Benefits of high altitude allergen avoidance in atopic adolescents with moderate to severe asthma, over and above treatment with high dose inhaled steroids. Clin Exp Allergy 2001;31:400–408. Simon HU, Grotzer M, Nikolaizik WH, Blaser K, Schöni MH: High altitude climate therapy reduces peripheral blood T lymphocyte activation, eosinophilia, and bronchial obstruction in children with house-dust mite allergic asthma. Pediatr Pulmonol 1994;17:304–311. Vervloet D, Charpin D, Magnan A, Birnbaum J: Relations asthme-allergie. L’altitude: un modèle d’étude. Presse Med 1994;23:1684–1686. Peroni DG, Boner AL, Vallone G, Antolini I, Warner JO: Effective allergen avoidance at high altitude reduces allergen-induced bronchial hyperresponsiveness. Am J Respir Crit Care Med 1994; 149:1442–1446.

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20

21

22

23

24 25

26 27

28

29 30 31

32

33

34

Piacentini GL, Martinati L, Fornari A, Comis A, Carcereri L, Boccagni P, Boner AL: Antigen avoidance in a mountain environment: Influence on basophil releasability in children with allergic asthma. J Allergy Clin Immunol 1993;92:644–650. Wüthrich B, Joller-Jemelka H, Grob P, Späth P, Hasler D, Braun P: Influence of mountain climate on immune parameters in atopic dermatitis, psoriasis and controls. Immunology of Skin and Skin Diseases 1990:344–353. Karagiannidis C, Hense G, Rueckert B, Menz G, Blaser K, Schmidt-Weber CB: High-altitude climate therapy reduces airway inflammation and activation of CD4⫹ T cells (abstract). München, World Allergy Congress, 2005. Kerrebijn KF, de Kroon JPM, Roosenburg JG, Zuidema P: Corticosteroïdbehandeling bij kinderen met CARA. II. Staken van de behandeling in het hooggebergte. Ned Tijdschr Geneeskd 1967;111: 2353–2359. Kerrebijn KF: Endogenous factors in childhood CNSLD; in Orie NGM, Van der Lende R (Hrsg): Bronchitis III. Niederlande, Royal Van Gorcum Assen, 1970, pp 38–48. Speelberg B, Folgering HT, Sterk PJ, van Herwaarden CL: Longfunctie van volwassen patiënten met asthma bronchiale of chronische obstructieve lonaandoening, vóór en na een verblijf van 3 maanden in het Nederlands Asthmacentrum Davos. Ned Tijdschr Geneeskd 1992;136:469–473. Engst R, Vocks E: Hochgebirgsklimatherapie bei Dermatosen und Allergien – Wirkmechanismen, Ergebnisse und Einflüsse auf immunologische Parameter. Rehabilitation 2000;39:215–222. Benckhuijsen J, van den Bos JW, van Velzen E, de Bruijn R, Aalbers R: Differences in the effect of allergen avoidance on bronchial hyperresponsiveness as measured by methacholine, adenosine 5⬘monophosphate, and exercise in asthmatic children. Pediatr Pulmonol 1996;22:147–153. Peroni DG, Piacentini GL, Costella S, Pietrobelli A, Bodini A, Loiacono A, Aralla R, Boner AL: Mite avoidance can reduce air trapping and airway inflammation in allergic asthmatic children. Clin Exp Allergy 2002;32:850–855. Valletta EA, Piacentini GL, Del Col G, Boner AL: FEF25–75 as a marker of airway obstruction in asthmatic children during reduced mite exposure at high altitude. J Asthma 1997;34:127–131. Drzimalla K, Wagner SA, Disch R: Langzeitergebnisse der Hochgebirgsklimatherapie in Davos. Allergologie 1999;22(suppl 1):29–35. Valletta EA, Comis A, Del Col G, Spezia E, Boner AL: Peak expiratory flow variation and bronchial hyperresponsiveness in asthmatic children during periods of antigen avoidance and reexposure. Allergy 1995;50:366–369. Boner AL, Comis A, Schiassi M, Venge P, Piacentini GL: Bronchial reactivity in asthmatic children at high and low altitude: Effect of budesonide. Am J Respir Crit Care Med 1995;151: 1194–1200. Christie PE, Yntema JL, Tagari P, Ysselstijn H, Ford-Hutchinson AW, Lee TH: Effect of altitude on urinary leukotriene (LT) E4 excretion and airway responsiveness to histamine in children with atopic asthma. Eur Respir J 1995;8:357–363. Platts-Mills TAE, Chapman MD: Dust mites: Immunology, allergic disease, and environmental control. J Allergy Clin Immunol 1987;80:755–775.

Prof. G. Schultze-Werninghaus Berufsgenossenschaftliche Kliniken Bergmannsheil, Medizinische Klinik III Pneumologie, Allergologie, Schlaf- und Beatmungsmedizin Klinikum der Ruhr-Universität Bochum Bürkle-de-la-Camp-Platz 1, DE–44789 Bochum (Germany) Tel. ⫹49 0234 302 6444, Fax ⫹49 0234 302 6420 E-Mail [email protected]

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The Environment Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 30–48

The Immunological Basis of the Hygiene Hypothesis Harald Renz, Nicole Blümer, Simona Virna, Serdar Sel, Holger Garn Department of Clinical Chemistry and Molecular Diagnostics, Philipps-University Marburg, Germany

Abstract The dramatic increase of allergic disorders in the last decades made their study an imperious demand. The increasing incidence of the development of allergic disorders seems to be associated with the modern westernized lifestyle, but causal reasons and the underlying mechanisms are far from being completely understood. Evidences suggest that priming of the immune responses against allergens happens already in utero. In addition, early life events are essential in shaping the immune answer towards the Th1- or Th2-profile, associated with a nonallergic or allergic phenotype, respectively. The hygiene hypothesis suggests that an early life environment rich in normal microbial flora primes the immune system in the Th1 direction towards clinical balance while a ‘sterile’ environment rather promotes the development of pathological immune phenotypes. In this review we collect epidemiological evidence for this concept. The data suggest an association between environment, lifestyle and the development of allergic diseases. This is the basis for the development of new hypotheses regarding the underlying pathomechanisms. The current view of cellular and molecular mechanisms underlying these phenomena includes fine-balancing between innate immune mechanisms and Th1, Th2 and regulatory T cells. These novel immunoregulatory events may explain the hygiene hypothesis by an interaction of environmental factors with innate immune mechanisms and various subtypes of T-cell responses. Copyright © 2006 S. Karger AG, Basel

Epidemiological and Human Exposure Studies

The prevalence of atopic diseases has increased dramatically in the last decades, but reasons for this phenomenon are far from being completely understood. Areas with high-standard living conditions are particularly concerned from this development. One proposition developed to explain this increase is termed the ‘hygiene hypothesis’. This hypothesis was initially proposed by

Table 1. Summary of studies showing a strong association of farming environment or high endotoxin levels with low asthma prevalence The protective effect of farming environment Farmer

Nonfarmer

307 1,094 319 319

1,308 8,519 493 493

1,181

8,466

hay fever, specific IgE, asthma, rhinitis, asthma, hay fever, specific IgE asthma, hay fever, specific, IgE, Wheeze hay fever, asthma

Braun-Fahrländer et al. [9] Kilpeläinen et al. [11] Riedler et al. [70] Braun-Fahrländer et al. [12] Von Ehrenstein et al. [10]

High endotoxin concentration protects from allergic disease Farmer and Nonfarmer 61 1,884 219

Type 1 T-cell development, specific IgE atopic eczema atopic disease, specific IgE

Gereda et al. [14] Gehring et al. [13] Böttcher et al. [71]

Strachan [1] in 1989. It suggests that modern public health practices have led to a relative sterilization of the developed world. As a consequence a diminished exposure to bacterial antigens, viral infections or the commensal saprophyte microflora occurs, which reduces the inhibitory potential of these factors on the development of allergen-specific reactions. As a result, there is an imbalance of the immune system, which predisposes individuals to the development of allergic diseases. Meanwhile, there are several epidemiological studies which support the concept of the hygiene hypothesis (table 1). One of these epidemiological associations refers to the family size. In this context it could be demonstrated that the number of particularly older siblings is inversely related to occurrence of hay fever, atopic eczema and inhalant allergy. This ‘sibling effect’ is attributed to a higher infection rate of children with several siblings and is therefore in line with the hygiene hypothesis [2]. However, there are also other possibilities to explain the phenomena of the birth order. Another epidemiological observation indicates an association between the ownership of a pet and the development of allergic diseases. Pet exposure during the first years of life has been associated with a lower prevalence of allergic rhinitis and asthma. In a study performed among school children in Sweden the relationship between exposure to pets early in life, allergic sensitization and manifestation was assessed [3]. The corresponding results indicate that exposure

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to pets during infancy might have a protective effect later on in life as, for example, shown by less sensitization to cat in cat-allergen exposed children. In this case components mediating the protective ‘pet effect’ could be either the petspecific allergens or the exposure to the microbial flora associated with the pet. In accordance with these findings, various studies indicate that the lifestyle plays a key role in shaping the immune system. As an example, the prevalence of atopy was found to be lower in children from families which adopted an anthroposophic lifestyle. It seems that lifestyle factors associated with the anthroposophy (e.g. less antibiotic consumption, rich diet in fermented vegetables containing live bacteria) may reduce the risk of atopy in childhood [4]. In addition, day care attendance in early life may be another factor associated with a reduced risk of asthma and recurrent wheezing among children with no maternal history of asthma [5]. Moreover, a series of studies concerning the differences in the epidemiology of respiratory symptoms and allergies between former East and West Germany reported a significantly lower prevalence of asthma, wheezing and allergic rhinitis among the East German population compared to those of West Germany [6]. Studies conducted after German reunification in 1990 reported an increased prevalence of asthma, hay fever and atopic eczema among East German children born after 1990 [7]. These observations would suggest that the adopted ‘western lifestyle’ might be responsible for increases of bronchial asthma and atopic diseases. However, the hygiene hypothesis cannot explain the high asthma prevalence among the inner-city children in the USA. It is known that living conditions of Afro-American and Hispanic-American inner city populations having a low income, are characterized by a poor hygiene, a high level of allergen triggers, cigarette smoke and viral infections. Contrary to the expectations these populations have the highest rate of asthma [8]. Leading insights into the hygiene hypothesis have been gained through epidemiological studies of the traditional farming environment and its relation to the development of allergic disorders. Growing up on a farm with an environment of high microbial exposure has been shown in several studies to be associated with a decreased risk of developing allergic diseases. In this context the Swiss study SCARPOL showed that children born in a farming environment have got about 50% reduced risk to develop an allergic disorder compared to control groups [9]. Another study performed with children of two rural Bavarian regions could show that, in addition to the farm environment, an increased exposure to the livestock in the farm-stables is related with a reduced risk to develop atopic diseases [10]. This observation was confirmed by a Finish study [11]. A cross-sectional survey (Study of Allergy and Endotoxin – ALEX) with more than 800 children (6–13 years) from rural areas of Germany,

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Teichoic acid

Surface protein

Lipoteichoic acid

Peptidoglycan Gram-positive bacteria

Phospholipid (inner membrane)

Lipopolysaccharide

Surface protein

Outer membrane

Lipoprotein Gram-negative bacteria Peptidoglycan

Phospholipid (inner membrane)

Fig. 1. Structure of the cell wall of Gram-positive (top) and Gram-negative bacteria (bottom).

Austria and Switzerland further analyzed the effect of the traditional farming environment on the development of allergies. In this study investigators were able to find for the first time an association between a very early (first year of life) or even prenatal exposure to the farming environment and a reduced risk for wheeze, asthma and general atopy in the children. The prevalence of asthma was significantly lower among children exposed to stables and farm milk (unpasteurized) compared to children not exposed to these influences. When the mother in this traditional environment continue to work daily in the stable during pregnancy, the development of bronchial asthma is prevented to a large extent. Therefore, it is likely that the farm environment is able to shape the immune system very early in life, probably already in utero. The farming effect has been hypothesized to result from elevated exposures to microbial compounds. One microbial factor among others proposed to mediate this effect is endotoxin (lipopolysaccharide, LPS), a cell wall component of the Gram-negative bacteria (fig. 1 and 2). In the course of the ALEX study it

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O -specific polysaccharide chain

Core glycolipid

Outer

Inner

Lipid A

Core polysaccharide

Fig. 2. Structure of lipopolysaccharide from Gram-negative bacteria.

was demonstrated that endotoxin concentrations were highest in stables from farmer families, but farmer households also contain much more endotoxin than nonfarmer households [10]. A strong inverse association between endotoxin concentrations (measured in the mattress of the child) and the development of allergic diseases in the child was observed [12]. Another longitudinal prospective birth cohort study on endotoxin exposure suggests that endotoxin exposure is associated with less atopic dermatitis [13]. Furthermore, high endotoxin levels in the house dust were associated with higher levels of interferon (IFN)-
after mitogenic stimulation of peripheral blood mononuclear cells in 9- to 24month-old children [14]. IFN-
as a Th1 associated cytokine is thought to directly counteract Th2-driven allergic diseases. Endotoxin, however, represents only a small part of the total microbial burden, because it occurs only in Gramnegative bacteria. Peptidoglycan, in turn, is a major component of the cell wall of all bacterial species (fig. 1). A major component of peptidoglycan is N-acetylmuramic acid {2-acetamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose}. It was shown that children with higher muramic acid concentrations in mattress dust had a significantly lower prevalence of wheezing and asthma regardless of farming status and endotoxin exposure [15]. Another association between the exposure to Gram-positive bacteria and an antiallergic effect is described by Lauener et al. [16] showing that in farmer children Toll-like receptor-2 (TLR-2), a receptor of the innate immune system, is expressed at higher levels in blood samples of farmer children in comparison to nonfarmer children. TLR-2 is a receptor responsible for the recognition of components mainly from Gram-positive bacteria. Another substance which was found in very high concentrations in the dust of the farmhouses is bacterial DNA rich in hypomethylated CpG motifs (CpG-DNA) [17]. This opens the possibility that the immune system of farmers may be triggered by bacterial DNA that thereby exerts an antiallergic effect. Signaling of bacterial CpG-DNA is mediated via TLR-9. In addition, there are several studies indicating that the TLR signal cascade mediating the

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Table 2. Segments of population associated with protection from asthma and allergies Segments of population

Findings

Author

Farm environment

reduced development of allergic disorders in children from farm environment lower prevalence of asthma, positive skin-prick test among East as compared to West German children; low prevalence of atopic disease in Estonian children than in Swedish children the greatest reduction in atopy was observed in children who had been vaccinated during the first week of life a protective sibling effect on the presence and severity of atopy; day care attendance in early life was associated with a decreased risk of asthma lower risk of atopy in childhood from families with an anthroposophic lifestyle

Von Ehrenstein et al. [10]

Eastern countries

Early childhood microbial exposure

Sibling effect/day care attendance

Anthroposophic lifestyle

Nowak et al. [6]

Böttcher et al. [71]

Aaby et al. [21]

Koppelman et al. [72]

Celedon et al. [5]

Alm et al. [4]

immune response of bacterial components might be involved in the development of allergic diseases. Polymorphisms in the LPS recognizing TLR-4 gene could be associated with an increased severity of atopy [18]. Furthermore, a polymorphism in the TLR-2 gene was associated with an increased risk to develop asthma and allergies in children of farmers (table 2) [19, 20]. Evidence supporting the principle of the hygiene hypothesis is not only based on the influence of environmental factors, but also on situations were humans have been infected. Thus, a mycobacterial infection, or vaccination with Bacillus Calmette Guerin (BCG, containing attenuated mycobacteria) of

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humans has been associated with a reduced risk to develop allergic disorders. A putative link between exposure to mycobacteria and decreased atopy was illustrated in a study of Japanese children. In this study strongly positive tuberculin responses in early life were significantly associated with less asthma, rhinoconjunctivitis and eczema in later childhood. Positive tuberculin responses in early life were also associated with lower immunoglobulin E (IgE) levels and Th1-dominated cytokine profiles in the peripheral blood at 12 years of age. A part of this inverse relationship might be attributed to genetic determinants of Th1/Th2 skewing in these children who were immunized with BCG as neonates. However, other observations within the study pointed to environmental influences, including the likelihood of varying exposures to mycobacteria. The scale of the strong tuberculin responses suggested natural early life exposure to M. tuberculosis in addition to BCG in many of the children. The most prominent reduction in atopy was observed in children who had been vaccinated during the first week of life [21]. In addition, in an international study, tuberculosis notification rates were matched to the prevalence of atopic symptoms in nearly a quarter of a million children within the International Study of Asthma and Allergies in Childhood study population. Investigators found that an increase in the tuberculosis notification rate was associated with an absolute decrease in wheeze [22]. In this context the mycobacterial notification rate might be regarded as a surrogate marker for the mycobacterial exposure and is therefore in line with the hygiene hypothesis. However, there are also studies showing no association between BCG vaccination and atopy, but in this case children with atopic hereditary were chosen [23]. Not only influences by pathogenic bacteria but also by components of the commensal saprophyte microflora and/or probiotics are described to be associated with the prevalence of allergies. The two countries Estonian and Sweden show a different incidence of allergic disorders. Whereas the Estonian population had a relatively low prevalence of allergy, the Swedish population suffers from allergic diseases to a much greater extent. Since the intestinal microflora differed in children from these countries, it was suggested that changes in the microbial gut flora may mediate the changes in the prevalence of atopy [24]. Dietary studies have shown that long-term exposure to yoghurt could reduce serum levels of IgE and some of the clinical symptoms of allergy in patients with atopic rhinitis or nasal allergies [25]. Kalliomaki et al. [26] reported that a perinatal administration of the probiotic strain Lactobacillus rhamnosus GG reduces the incidence of atopic eczema in at risk children during the first 4 years of life. All of these studies and others indicate that microbial environmental influences as well as the nutrition (probiotica) could mediate allergy-preventing effects. These studies furthermore demonstrate that the timing of exposure is

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very important for this protection. Early life and prenatal exposure are reported to result in the most effective protection. Various in vitro studies support these epidemiological associations. For example, it was shown that a specific response to allergen stimulation might be achieved in mononuclear cells obtained from cord blood indicating that allergen priming must have occurred prenatally [27]. In fact, it appears that the development of the fetal immune response is the result of a complete interaction at maternal and placental interface. The presence of allergen-specific T-cell proliferation in cord blood cells has already been convincingly demonstrated for different allergens [27]. The immunological mechanisms involved in prenatal cell priming are not yet fully understood. Using an ex vivo placenta perfusion model, the ability of different allergens to cross placenta from mother to fetus has been demonstrated (e.g. Bet v1, the major allergen of Betula verrucosa and -lactoglobulin). However, some other allergens were not able to cross directly the placental barrier [28]. This does not exclude the possibility that some antigens are introduced into the fetus directly by the antigen-presenting cells or through the amniotic fluid swallowed by the fetus [29]. Moreover, the influence of the maternal adaptive immune system in shaping the allergic immune response in the offspring has been documented previously. To conclude, certain groups could be identified in Western population that show some degree of protection for allergies and asthma, and this is associated with specific lifestyle factors e.g. farm environment, early exposure to infections, older siblings, pets (table 2).

Animal Models: A Proof-of-Concept Approach

Since epidemiological studies may only provide information on possible associations between environmental factors and the development of a certain pathological outcome, for example development of allergic disease, studies using laboratory animals are helpful to assess a cause-effect relationship and to delineate the underlying mechanisms between environmental conditions and allergy development. In addition, natural environmental influences are always very complex and animal models open the possibility to explore the influence of single, well-defined compounds of the respective environment on the onset and/or perpetuation of the disease. From epidemiological studies it became clear, that pre- and postnatal exposure to stable environment and consumption of nonpasteurized milk protects children from respiratory allergies to a large extent. These environmental factors contain multiple antigenic molecules and microbial components. Therefore, the investigation of the role of prenatal antigen exposure and the influence

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of certain microbial components on the development of allergic responses has been in the focus of recent animal studies. Actually, several studies have shown that prenatal antigen exposure is able to induce long-lasting, antigen-specific tolerance in the offspring. The first observation within that line was already described in 1969, when Lewert and Mandlowitz [30] observed that infection of mothers with Schistosoma mansonii induced tolerance towards parasite antigens in the offspring. Later on, using the model antigen ovalbumin (OVA) our group was able to show that induction of an OVA-specific Th2 response in mother mice protects the littermates from the development of an IgE-response towards this allergen. It was demonstrated that antigen itself as well as antigen-specific IgG1 and IgG2a were transferred from the mother by diaplacentar uptake and breast milk transmission, and the protective effects were shown to be mediated by the transfer of allergen-specific IgG1 antibodies. These effects were antigen specific; in contrast, the allergic sensitization towards unrelated antigens was even accelerated due to a generalized suppression of Th1 responses as shown by a decreased frequency of IFN-
producing T cells in these offspring [31, 32]. Similar results were reported by Victor et al. [33] who could additionally demonstrate that offspring from mothers immunized towards house dust mite did not develop an antibody response towards that antigen following immunization in early life and secondary allergenic challenge. Taken together these observations imply that prenatal and early postnatal allergen contact leads rather to allergen-specific tolerance than to induction of allergic immunization. The environment is not only full of allergens but also of high amounts of microbes and microbial products. Therefore, exposure to these components has been hypothesized to prevent the induction of an allergic immune response. Indeed, there are several reports showing that exposure of mice to whole bacteria may prevent from the development of an allergic phenotype. Initial evidence came from experiments using mycobacterial preparations. Tükenmez et al. [34] observed that the total IgE response after an OVA sensitization was reduced in animals that have been preimmunized to heat-killed Mycobacterium bovis immediately after birth. This is accompanied by attenuated histopathological changes following intratracheal allergen challenge [35]. However, application of mycobacteria may not only influence sensitization towards allergens but exerts beneficial effects even when applied in sensitized mice and in animals that have an already established allergic inflammation. As shown by Hopfenspirger and Agrawal [36], intranasal application of BCG after a primary allergen challenge successfully diminished airway hyperreactivity to methacholine, peribronchial eosinophilia and interleukin (IL)-5 levels in the bronchoalveolar fluid following a secondary provocation. In addition to mycobacteria, other bacterial species as well may also provide protection from

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the development of allergic disorders, as it has been shown in different models with heat-killed preparations of Listeria monocytogenes [37] and Propionibacterium acnes [38], whole cell vaccines of Bordetella pertussis [39] and infection with live Chlamydia [40]. More recently, mucosal application of nonpathogenic bacteria like certain Lactococcus species was demonstrated to counter-regulate immune responses, and thereby preventing the development of an allergic phenotype [41, 42]. Finally, naturally occurring intestinal microbes are also involved in the regulation of allergic immune responses, since the application of antibiotics that changes number and composition of the intestinal microflora increases the susceptibility of mice to the induction of an allergic immune response as demonstrated by augmented IgE levels and enhanced Th2cell reactivities [43]. The observation that not only pathogenic bacterial species are involved in the protection from the development of certain atopic disorders and the fact that killed bacterial preparations may also be protective as well, clearly indicate that these effects are not related to the pathogenic nature of microbes as proposed by the original hygiene hypothesis. Thus, the advanced hygiene hypothesis proceeds from the assumption that such microbes or components and products of them act as effective immunomodulators. The by far best characterized microbial product in this regard is LPS, a structural component of the cell wall of Gram-negative bacteria (fig. 2). Indeed, it has recently been shown that local as well as systemic administration of LPS prior to allergen sensitization suppresses IgE production, development of airway eosinophilia and Th2 immune responses even though airway hyperreactivity was not influenced [44]. Interestingly, these effects were dose dependent with high-dose exposure exerting prevention, whereas low LPS doses led to even augmented allergic inflammatory responses [45]. When applied to neonates, LPS was able to induce an increased production of IgG2a following sensitization but failed to prevent Th2-mediated immune responses. However, when LPS was administered in the presence of the specific antigen, a complete suppression of allergic sensitization, airway inflammation and hyperreactivity was obtained [46]. In order to mimic the human situation, where the most efficient protection from the development of allergic diseases was found in children whose mothers have been exposed to stable environment during pregnancy and lactation period, we recently developed a murine model where LPS was applied intranasally to pregnant mice [47]. At birth, T cells from offsprings of those LPS-treated mothers showed an elevated IFN-
production. Following sensitization to OVA these mice developed reduced levels of antigen-specific IgE and IgG1, whereas anti-OVA IgG2a levels remained unaltered. In addition, a selective suppression of Th2 reactivities was observed since in vitro re-stimulated

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Mother

Offspring

Birth

LPS

IFN-


Wk 4 Wk 5

Wk 10

OVA sensitization

OVA aerosol

IgG1 IgE

Airway inflammation Airway hyperreactivity

Fig. 3. Effects of prenatal LPS exposure of mothers on postnatal immune parameters in the offspring. Female BALB/c mice received three intraperitoneal (i.p.) injections of 10 g of LPS on days 5, 3 and 1 prior to mating. During the gestation period mice were exposed intranasally to 10 g of LPS every third day. IFN-
production was assessed on day 2 postpartum. Neonates were sensitized with OVA at days 28 and 42 of age. Serum anti-OVA IgG1 and -IgE expression was assessed at day 70 of age. A subgroup of neonates were exposed to an additional OVA i.p. injection (day 63 of age) and to OVA aerosol (day 67, 68, 69, 70 of age). Airway reactivity of these animals was assessed 24 h after the last allergenaerosol exposure.

T cells produced significantly less IL-5 and IL-13 while the IFN-
response was not affected. Following airway provocation by OVA aerosol, the inflammatory reaction within the airways was less pronounced when compared to offsprings of untreated mothers; however, airway responsiveness to increased doses of methacholine was comparable in these two groups (fig. 3). Taken together, these data clearly show that LPS, on the one hand, may effectively influence the development of allergen-specific immune responses in adult animals. On the other hand, prenatal exposure to microbial components, such as LPS, potently reduced the risk of offspring to develop allergic immune responses later in life. It should be pointed out that LPS is not the only microbial component that has been found to exert allergy-protective properties. A number of components from Gram-positive and Gram-negative bacteria, yeasts, mycoplasma and even viruses were also shown to have similar effects, and the number of these agents is still growing. Among them are CpG oligonucleotides [48], lipoproteins and -glycans [49], zymosan, peptidoglycans, including muramic acid and macrophage-activating lipopeptide 2, a peptide derived from mycoplasma [50].

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In conclusion, animal models have much contributed to investigate components of the environment that might be responsible for the effects that have been described in epidemiological studies. Moreover, all of the above-mentioned compounds derived from different microbial organisms belong to the so-called ‘pathogen-associated microbial patterns (PAMPs)’. It is now well established that these structures are recognized by the hosts’ innate immune system via pattern-recognition receptors. Thus, animal models may also provide insights into cellular and molecular mechanisms that are responsible for the beneficial effects of microbial exposure on the development of allergic immune responses.

Cellular and Molecular Mechanisms of Allergy Protection

Several mechanisms have been proposed to explain the effect of the hygiene hypothesis during recent years. Initially, an early lack in Th1-responses was proposed as one important underlying mechanism. Naïve CD4 T cells differentiate into Th1 or Th2 cells to orchestrate an effective immune response. Thereby, Th2 cells are important for the initiation and maintenance of an allergic phenotype. They secrete mediators including IL-4, IL-5, IL-9 and IL-13 which regulate effector cells by triggering (1) B-cell IgE production, (2) the growth, differentiation and recruitment of eosinophils and (3) mucus production in the airways, respectively [51, 52]. Th1 cells produce the cytokine IFN-
that promotes the development of cell-mediated immune responses, particularly against intracellular microorganisms and also antagonizes the development of Th2 cells. Conversely, IL-4 inhibits Th1 cell development. The critical Th2inducing cytokine is IL-4, whereas IL-12 produced by cells of the innate immune system, such as macrophages and dendritic cells, drives CD4 T-cell differentiation towards the Th1 phenotype [53]. However, decreased numbers of microbial infections in early childhood caused by improved hygienic conditions in the industrialized countries result in reduced production of IL-12. Less frequent stimulation of Th1-biased immune responses facilitate the development of Th2-dominated allergic diseases [54]. In the ‘original hygiene hypothesis’ the microbes are considered as ‘dangerous’ pathogens. But, certain patterns of microbial load play an important role in immunoprotection depending on the type of microbe, colonization site (skin and mucosal surface), chronicity and occurrence in early life. We have termed this model the ‘advanced hygiene hypothesis’, where microbes are considered as immunomodulators. The first response of an organism to microbes is build up by the cells of the innate immune system enhancing the control of the adaptive immune system. Microbial organisms or components of them activate

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Table 3. Toll-like receptors (TLRs) and their most important ligands Receptor

Microbial and synthetic ligands

TLR-1 TLR-2

Triacyl lipopeptides (e.g. Pam3Cys) Lipoproteins, lipopeptides, peptidoglycan, zymosan, lipoteichoic acid, lipoarabinomannan Double-stranded RNA (e.g. poly I:C) Lipopolysaccharide, taxol, viral F protein Flagellin Lipoteichoic acid, zymosan Imidazoquinoline, single-stranded RNA, loxoribine Imidazoquinoline, single-stranded RNA CpG DNA and oligonucleotides Not yet determined Uropathogenic bacteria ligands Not yet determined Not yet determined

TLR-3 TLR-4 TLR-5 TLR-6 TLR-7 TLR-8 TLR-9 TLR-10 TLR-11 TLR-12 TLR-13

the innate immune system in a pathogen-unspecific fashion depending on ligand binding to germline-encoded receptors that recognize PAMPs shared by large classes of microbes. These molecular patterns are detected by patternrecognition receptors and within pattern-recognition receptors the TLR family today represents the best-characterized class [55]. So far, 10 human (TLR-1 – TLR-10) and 12 mouse (TLR-1 – TLR-13, except TLR-10) distinct TLRs and several corresponding PAMPs have been identified. Important examples are LPS (TLR-4 ligand), bacterial lipoproteins and lipoteichoic acids (TLR-2 ligand), CpG DNA (TLR-9 ligand) and double-stranded RNA (TLR-3 ligand) [56]. An overview of TLRs and their most important ligands is given in table 3. Binding of PAMPs to their specific TLR induces the expression of costimulatory molecules and also the production of several proinflammatory cytokines, such as tumor necrosis factor-, type-I IFNs, IL-1, IL-6, IL-10 and IL-12 in antigen-presenting cells [57]. Perhaps, the most convincing aspect of TLR ligands with regard to their immunomodulatory features is their potential usage as adjuvants in allergic inflammation. This is best demonstrated using the TLR-9 ligand CpG DNA and the TLR-4 ligand LPS. In murine models of experimental asthma, it has been clearly demonstrated that the application of CpG DNA or LPS prevents Th2 inflammatory responses and effectively interferes with the development of atopic airways disease [58, 59]. Moreover, when administered in conjunction with an experimental allergen, CpG DNA promotes the reversal of established eosinophilic inflammation. Furthermore, as already mentioned

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above, association studies between TLR polymorphisms and the risk of atopic disease development produced evidence that TLR activation is involved in the shaping of the immune system. Indeed, some studies could show associations between TLR-4 polymorphisms and higher prevalence of asthma with decreased IL-12 production by antigen-presenting cells after LPS stimulation [60]. In contrast to the initially widely accepted Th1-Th2 model is the observation that no clear beneficial effects were observed in clinical trials on asthma patients and patient with atopic dermatitis treated with subcutaneously applied IFN-
[61]. Also the administration of recombinant human IL-12 had no clear beneficial effects either on the early or late response to inhaled allergen challenge and on airway hyperreactivity in asthma patients [62]. This suggests that IL-12 production and subsequent Th1 activation by TLR stimulation seems not to be the main mechanism of the immunomodulatory effects of TLR ligands. Moreover, the incidence of Th1-mediated organ-specific autoimmune diseases such as Type-1 diabetes and multiple sclerosis is increasing as well [63]. These data emphasize that a deregulated Th1 and Th2 balance is not the major factor for the development of allergic and autoimmune diseases. The fact that both forms of diseases are caused by poorly regulated, and therefore pathologically exaggerated, immune responses implies that a failure in the regulation of overall T-helper cell responses might be responsible for allergy development. Recently it has been demonstrated that various regulatory T-cell (TReg) populations control the reactivity to self-antigens and alloimmune responses. Within several TReg populations which have been described so far (for review, see Robinson et al. 2004), the CD4 CD25 T-cell subset is currently attracting the most interest. It has been proposed that they are generated in the thymus as well as in the periphery, where they comprise about 5–10% of all peripheral CD4 T lymphocytes [64]. The development and function of CD4 CD25 TRegs is mainly dependent on the expression of the specific transcription factor Foxp3. Gene deletion of Foxp3 resulted in absence of suppressor activity of CD4 CD25 T cells, whereas ectopic Foxp3 expression in peripheral CD4 CD25 T cells conferred suppressor function [65]. TReg exert their immunosuppressive activities on Th1 and Th2 cells by production of inhibitory cytokines, such as IL-10 and transforming growth factor-, and the expression of negative costimulatory molecules such as CTLA4 and glucocorticoid-induced tumor necrosis factor receptor (GITR) on the surface [66]. It has been suggested that a deficiency or failure of TReg-mediated suppression of Th2 activation is one possible reason for development of allergic diseases. Indeed, a more recent study showed that the ability of CD4 CD25 T cells from atopic donors to suppress proliferation and Th2 cytokine production by autologous allergen-stimulated CD4 CD25 T cells was significantly reduced as compared to CD4 CD25 T cells from nonatopic donors [67].

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Physiological role

Role in pathology

lgG2a

• Control of bacterial and viral infections • Tumor immunity

Th1 IL-12

IFN-


APC

• Delayed type hypersensitivity • Some autoimmune diseases • Psoriasis

N

B

TGF-, IL-10

IL-4

Treg

APC

TGF-, IL-10 Th2 IL-3, IL-4

IL-5 IL-4 IL-13

Eo • Defense of parasite infections • Maintenance of pregnancy

B lgE

MC • Allergy • Certain autoimmune diseases

Fig. 4. Interaction of different immune cell populations in the regulation of allergic immune responses.

With regard to the hygiene hypothesis, Moseman et al. [68] demonstrated that CpG DNA induced the development of TRegs by the activation of human plasmacytoid dendritic cells. At least, in the mouse, TRegs may also respond to TLR ligands directly in an antigen-presenting cell-independent manner. TLR-4, TLR-5, TLR-7 and TLR-8 were found to be expressed selectively by TRegs. LPS stimulation induced several activation markers and supported cell survival and proliferation together with the enhancement of the suppressor functions of TReg [69]. Moreover, the application of a Mycobacterium vaccae suspension induced TRegs, which suppressed airway eosinophilia and bronchial hyperresponsiveness. These effects could be completely reversed by antagonizing antibodies against IL-10 and transforming growth factor- (TGF-). We conclude that the basis of the hygiene hypothesis is beyond the Th1 and Th2 balance and is based on the interaction between environment, innate immunity and all types of T-helper cells (fig. 4). Further work is required to determine when each type of TReg develops, and what their exact mechanisms of

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suppression are. Controversy still remains about the interaction between different TReg populations and between TReg and Th2 cells. Clinical and epidemiological studies are required to evaluate the impact of TRegs in different periods of life. A better knowledge about TRegs will help to understand the pathogenesis of several diseases which are caused by the disregulation of the immune response and this will provide new possibilities to modulate TReg cell function for therapeutic purposes. In conclusion, because of their high rate of prevalence in the developed countries, allergies meanwhile became a major health problem. Therefore, the study of factors that favor the development of allergies and of the underlying mechanisms is an imperious demand. Due to an extensive series of epidemiological studies under certain lifestyle circumstances, nowadays we have a good insight into the prevalence of allergic disorders. In addition, experimental animal models provide essential progress to understand molecular and cellular mechanisms leading to the development of these diseases. It became clear that multiple levels of interactions between environmental factors and the genetic background of the host define whether immunological tolerance or allergy will develop. With this respect, deeper insights into the mechanisms that may explain the hygiene hypothesis will not only offer novel information on the cause of increases in the prevalence of allergic disorders but may also lead to the development of new preventive and therapeutic strategies.

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8 9

10

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15

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Rodriguez D, Keller AC, Faquim-Mauro EL, et al: Bacterial lipopolysaccharide signaling through Toll-like receptor 4 suppresses asthma-like responses via nitric oxide synthase 2 activity. J Immunol 2003;171:1001–1008. Kitagaki K, Jain VV, Businga TR, Hussain I, Kline JN: Immunomodulatory effects of CpG oligodeoxynucleotides on established Th2 responses. Clin Diagn Lab Immunol 2002;9: 1260–1269. Fageras BM, Hmani-Aifa M, Lindstrom A, et al: A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children. J Allergy Clin Immunol 2004;114:561–567. Boguniewicz M, Martin RJ, Martin D, et al: The effects of nebulized recombinant interferongamma in asthmatic airways. J Allergy Clin Immunol 1995;95:133–135. Barnes PJ: Cytokine modulators as novel therapies for asthma. Annu Rev Pharmacol Toxicol 2002;42:81–98. Bach JF: The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 2002;347:911–920. Holm TL, Nielsen J, Claesson MH: CD4CD25 regulatory T cells. I. Phenotype and physiology. APMIS 2004;112:629–641. Fontenot JD, Gavin MA, Rudensky AY: Foxp3 programs the development and function of CD4CD25 regulatory T cells. Nat Immunol 2003;4:330–336. O’Garra A, Vieira P: Regulatory T cells and mechanisms of immune system control. Nat Med 2004;10:801–805. Ling EM, Smith T, Nguyen XD, et al: Relation of CD4CD25 regulatory T-cell suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease. Lancet 2004;363:608–615. Moseman EA, Liang X, Dawson AJ, et al: Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4CD25 regulatory T cells. J Immunol 2004;173:4433–4442. Caramalho I, Lopes-Carvalho T, Ostler D, Zelenay S, Haury M, Demengeot J: Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med 2003; 197:403–411. Riedler J, Braun-Fahrlander C, Eder W, Schrauer M, Waser M, Maisch S, Carr D, Schierl R, Nowak D, Von Mutius E: ALEX Study Team: Exposure to farming in early life and development of asthma and allergy: A cross-sectional survey. Lancet 2001;358:1129–1133. Böttcher MF, Bjorksten B, Gustafson S, Voor T, Jenmalm MC: Endotoxin levels in Estonian and Swedish house dust and atopy in infancy. Clin Exp Allergy 2003;33:295–300. Koppelman GH, Jansen DF, Schouten JP, van der Heide S, Bleecker ER, Meyers DA, Postma DS: Sibling effect on atopy in children of patients with asthma. Clin Exp Allergy 2003;33:170–175.

Prof. Harald Renz, MD Department of Clinical Chemistry and Molecular Diagnostics Philipps-University Marburg, Baldingerstrasse DE–35033 Marburg (Germany) Tel. 49 6421 2866234, Fax 49 6421 2865594, E-Mail [email protected]

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The Lung, Eosinophils and Asthma Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 49–58

Molecules Involved in the Regulation of Eosinophil Apoptosis Hans-Uwe Simon Department of Pharmacology, University of Bern, Switzerland

Abstract Apoptosis is the most common form of physiological cell death and a necessary process to maintain cell numbers in multicellular organisms. Eosinophils are constantly produced in the bone marrow and the same numbers die, under normal circumstances, within a relatively short time period. In many eosinophilic inflammatory diseases, reduced eosinophil apoptosis has been described. This mechanism may contribute to increased eosinophil numbers, a phenomenon called eosinophilia. Overexpression of interleukin-5 appears to be crucial for delaying eosinophil apoptosis in many allergic disorders. Survival factor withdrawal leads to the induction of apoptosis. Besides survival cytokines, eosinophil apoptosis is also regulated by death factors. Recent observations suggest a role for mitochondria in conducting eosinophil apoptosis, although the mechanisms that trigger mitochondria to release proapoptotic factors remain less clear. Drugs that specifically induce eosinophil apoptosis might be useful for triggering the resolution of unwanted eosinophilic inflammatory responses. Copyright © 2006 S. Karger AG, Basel

Eosinophils are prominent effector cells that are believed to play important roles in many chronic allergic and parasitic inflammatory responses [1, 2]. Under physiological conditions, they are constantly generated in the bone marrow. To keep cellular homeostasis, aged eosinophils undergo a programmed cell death, which is apoptosis. Eosinophil apoptosis also plays an important role in the resolution of an inflammatory response. Apoptotic eosinophils are phagocytosed by other cells, a process that is not associated with the induction of inflammatory responses [3] (fig. 1). Since eosinophils contribute to inflammatory responses by releasing toxic mediators and cytokine production [4–6], controlling their numbers and activation is a critical issue. In this context, studying regulatory mechanisms of eosinophil apoptosis appears to be important. Although apoptosis is physiologic, it might be deregulated and then it may contribute to pathophysiological mechanisms leading, or at least contributing,

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Fig. 1. Bronchoalveolar lavage fluid cells from a patient with bronchial asthma were stained with propidium iodide (PI) and anti-eosinophil cationic protein (ECP) antibody and analyzed by confocal microscopy. Some alveolar macrophages demonstrated evidence for ECP expression (arrow heads), indicating uptake of apoptotic eosinophils. The upper panel shows a physical association between an eosinophil (arrow) and an ECP-positive macrophage. The lower panel demonstrates an eosinophil (arrow) inside of a macrophage. Bars ⫽ 10 ␮M.

to diseases. For instance, disregulation of apoptosis can lead to pathophysiological changes, which result in either loss of cells (e.g., acquired immunodeficiency syndrome, degenerative diseases, etc.) or accumulation of cells (e.g., cancer, autoimmune and inflammatory diseases, etc.). In eosinophilic diseases, delayed eosinophil apoptosis has been proposed as one important mechanism contributing to eosinophilia [7]. However, although the phenomenon of delayed eosinophil apoptosis was shown in explanted nasal polyp tissues [8], most of the studies performed until now were performed in vitro. Delayed apoptosis of purified blood eosinophils has been observed in atopic dermatitis [9], asthma [10] and hypereosinophilic syndrome [11]. Like in many other cells, eosinophil apoptosis can be induced in response to specific ligands of the so-called ‘death receptors’ of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily [12]. For instance eosinophil express functional Fas receptors, at least under in vitro conditions [13, 14]. However, eosinophils also undergo apoptosis in the absence of death signaling. This phenomenon is called spontaneous apoptosis. Studying spontaneous

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apoptosis in vitro seems to be a good model for the induction of apoptosis during the resolution of an eosinophilic inflammatory response. Moreover, inflammation can be mimicked in this system by stimulating eosinophils with inflammatory mediators present at the inflammatory site. Such in vitro studies greatly helped to understand the molecular events that control eosinophil apoptosis under physiological and pathological conditions. In this article, I discuss survival and death pathways in eosinophils that might be relevant under both physiological and inflammatory conditions.

Proapoptotic Extracellular Stimuli

Death Receptor Ligands As mentioned above, eosinophils express functional Fas receptors (CD95, APO-1) [13, 14]. Autocrine and paracrine Fas ligand/Fas receptor interactions have been proposed to be crucial for activation-induced T-cell death, an important mechanism for downregulating immune responses. Based on this model, it has been suggested that the same mechanism might drive granulocyte apoptosis and be responsible for the short life span of these cells [15, 16]. However, studies using antagonistic anti-Fas receptor antibodies and blocking soluble Fas receptor molecules in neutrophils do not support the idea that spontaneous granulocyte apoptosis is the consequence of autocrine or paracrine Fas ligand/Fas receptor interactions in purified cell populations in vitro [17, 18]. Therefore, there is little evidence that production and/or release of Fas ligand can drive constitutive apoptosis in eosinophils. Studies on the role of TNF-␣ in the induction of eosinophil apoptosis yielded inconsistent results. Currently available data suggest that induction of apoptosis occurs only under conditions in which nuclear factor-␬B is suppressed [19]. If this is not the case, TNF-␣ may even be antiapoptotic for eosinophils. For instance, TNF-␣ was shown to activate the p38 mitogen-activated protein kinase pathway resulting in delayed eosinophil apoptosis [20]. Moreover, the survival effect of TNF-␣ under in vitro conditions was suggested to be mediated via granulocyte macrophage colony-stimulating factor (GMCSF) induction in eosinophils [21]. Another death ligand represents TNF-related apoptosis-inducing ligand (TRAIL). TRAIL was reported as promoting eosinophil survival [22]. Experiments in our own laboratory suggested that TRAIL has antiapoptotic effects on eosinophils, but only in approximately 50% of the donors [23]. Nevertheless, it appears to be clear that both TNF-␣ and TRAIL, although death ligands, do not induce eosinophil apoptosis under normal conditions, suggesting that they mediate alternative functions in these cells.

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CD137 represents another member of the TNF/nerve growth factor family, which is expressed on eosinophils from allergic donors. In contrast, normal eosinophils do not express CD137 [24]. The expression of CD137 is induced by a T-cell-derived factor, but its identity remains unclear. CD137 stimulation alone had no effect on eosinophil death and apoptosis. However, when CD137 expressing eosinophils were simultaneously stimulated with anti-CD137 monoclonal antibody and interleukin (IL)-5 (or GM-CSF), the inhibitory effect of the survival cytokine on eosinophil apoptosis was blocked [24]. Therefore, CD137 stimulation of eosinophils may help to prevent unlimited expansion of eosinophils in tissues with high levels of eosinophil-survival cytokines. Other Death Triggers: Siglec-8, CD30, CD45 and CD69 Apoptosis in eosinophils can be induced via several additional surface molecules. For instance, cross-linking of Siglec-8 [25], CD30 [26, 27], CD45 [28] and CD69 [29] was shown to induce eosinophil apoptosis. These studies were performed using agonistic antibodies and were performed under in vitro conditions. The physiological relevance of these findings remains unclear.

Antiapoptotic Extracellular Stimuli

Classical Survival Cytokines The short life of granulocytes can be prolonged by stimulation with cytokines that have antiapoptotic properties. It has been demonstrated that eosinophil apoptosis is delayed by IL-3, IL-5 and GM-CSF in vitro [30–32], and by IL-5 in nasal polyp tissues ex vivo [8]. The signaling events important for cytokine-mediated antiapoptosis include activation of tyrosine kinases [31, 32] and STAT proteins [33], resulting in the transcriptional activation of members of the Bcl-2 and inhibitor of apoptosis protein (IAP) families (see below). Since IL-5 is crucial for differentiation, activation and survival of eosinophils, there have been attempts to block this cytokine as a therapeutic approach in allergic diseases (anti-IL-5 antibody treatment) [34, 35]. Recently, CCR3-reactive chemokines, such as eotaxins, have also been demonstrated to prolong eosinophil survival [36]. TNF-Like Molecules As mentioned above, members of the TNF superfamily, such as TNF-␣ [20, 21] and TRAIL [22, 23], delay eosinophil apoptosis in vitro. Another member of this family, CD40L, has also been reported to promote eosinophil survival [37]. Taken together, multiple cytokines have been shown to mediate antiapoptotic effects on eosinophils.

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Intracellular Regulators

Caspases Despite a great variety of available apoptotic stimuli, final changes to the cell are similar and many of the signaling events appear to converge into common mechanisms involving activation of cysteine-containing proteases that cleave their target proteins at specific aspartic acids (caspases). Caspases are part of a family comprising 14 members up to now [38]. They are present in the cells as inactive zymogens that must be cleaved to generate free catalytic subunits able to associate and form active heterotetramers. The family of caspases can be divided into two functional subgroups, the initiator and the executioner caspases. Many apoptotic responses are initiated by activation of the initiator caspases-8 or -9. Initiator caspases necessitate special mechanisms of activation of zymogens. For instance, caspase-8 can be activated following recruitment and clustering at multicomponent apoptosis-signaling complexes, resulting from ligation of cell surface molecules of the TNF/nerve growth factor receptor family, presumably by autoprocessing of the zymogens according to the inducedproximity model [39]. Caspase-9 is activated by recruitment to Apaf-1 in the presence of ATP following release of cytochrome c from mitochondria [40]. Activation of either of these caspases can result in activation of executioner caspases such as caspase-3, leading eventually to apoptosis. Caspase-3 has been shown to be an important effector caspase in eosinophils following mitochondrial activation involving the Bcl-2 family protein Bax [41]. A caspase-9 inhibitor blocked eosinophil apoptosis, confirming the view that mitochondria are involved in proapoptotic signaling in eosinophils [42]. In addition, broadrange caspase inhibitors also blocked eosinophil apoptosis, suggesting that caspases are indeed critical elements of the death machinery in eosinophils. In these earlier studies, however, it appeared that their proteolytic activity is diminished in eosinophils, perhaps due to inadequate processing of the procaspase. For instance, the majority of the fragments generated by processing of caspase 3 did not correspond to the expected size of the active caspase (17 kDa). Instead, the major fragment is 20 kDa in size and might correspond to a fragment constituted of the 17 kDa attached to the 3-kDa prodomain [18]. Recent data provide a potential explanation for this phenomenon: using cell-free systems, we observed that activation of caspase-3 in eosinophils requires much more cytochrome c compared to Jurkat cells or neutrophils [unpubl. observations]. IAP Family Members The proteolytic activity of caspases must be tightly controlled. Active caspases can be specifically suppressed by the IAP family, a group of negative apoptosis regulators that has evolved to protect cells from unwanted self-execution

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Fig. 2. Bronchoalveolar lavage fluid cells from a patient with bronchial asthma were stained with propidium iodide (PI) and anti-survivin (upper panel) and anti-cIAP-2 (lower panel) antibodies, respectively, and analyzed by confocal microscopy. Eosinophils (arrows) demonstrate evidence for both survivin and cIAP-2 expression. Macrophages are indicated by arrow heads. Bars ⫽ 10 ␮M.

by fortuitous activation of the caspase cascade. IAPs were first identified as baculoviral proteins capable of inhibiting apoptosis in infected insect cells [43]. This class of cell death inhibitors is characterized by one to three zinc-binding motifs of approximately 70 residues, termed baculovirus IAP repeat, which enables binding to activated caspases. Several of these structurally related proteins also contain a highly conserved carboxy-terminal RING domain involved in the targeted degradation of proteins by ubiquitinylation [44]. In humans, eight IAPs have been identified so far, that include cIAP-1, cIAP-2, XIAP, ILP2, NAIP, ML-IAP, apollon and survivin. All can block the caspase cascade, but only for five of them direct physical interaction with caspases has been unequivocally demonstrated [44]. Sputum, but not blood eosinophils, from asthmatic patients expressed cIAP2. Under in vitro conditions, cIAP-2 was induced by cross-linking of CD40 on eosinophils. CD40 was not expressed on freshly blood isolated eosinophils, but was seen following culturing the cells in medium [37]. Recent work carried out in my laboratory confirmed the expression of cIAP-2 in bronchial eosinophils from asthmatic patients. As shown in figure 2, we performed immunofluorescence

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Growth factor withdrawal

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Fig. 3. A simplified scheme showing apoptosis pathways and their counter-regulation by survival factor signaling in eosinophils. FADD ⫽ Fas-associated death domain; FasR ⫽ Fas receptor.

analysis using bronchoalveolar lavage eosinophils to demonstrate cIAP-2 expression. Using these cells, we also obtained evidence for survivin expression (fig. 2). Survivin was also inducible and expressed in normal blood eosinophils following stimulation with IL-5 in vitro (fig. 3) [unpubl. observations]. Survivin has received great attention due to its expression in many tumors and its potential as a therapeutic target in cancer. Moreover, it was believed that it selectively blocks apoptosis in proliferating cells. Therefore, the finding that mature terminally differentiated neutrophils express survivin under inflammatory conditions was surprising [45]. That eosinophils are also able to express survivin supports the view that the antiapoptotic effect of this molecule is not restricted to the G2-M phase of the cell cycle [46]. Bcl-2 Family Members There have been several reports suggesting the involvement of members of the Bcl-2 family in the regulation of apoptosis that usually regulate the proapoptotic activity of mitochondria. This has been somewhat surprising since granulocytes have been described as cells with limited numbers of mitochondria [47]. Recently published work, however, suggests that eosinophils contain

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small numbers of mitochondria, which are essentially used for the induction of apoptosis only [42]. The proapoptotic Bax molecule was found to be expressed at high levels in eosinophils [48]. Moreover, eosinophil apoptosis was associated with translocation of cytosolic Bax into the outer membrane of mitochondria where it forms pores, allowing the release of proapoptotic factors such as cytochrome c [41]. Clearly, high levels of Bax may contribute but may not be sufficient to the short life span of eosinophils. The more proximal mechanisms that are responsible for Bax translocation to mitochondria in the absence of sufficient stimulation with survival cytokines remain to be determined. Recent results obtained in our own laboratory point to a role for calpain-1 in this process [49; unpubl. observations]. Besides the proapoptotic Bax, eosinophils also express antiapoptotic members of the Bcl-2 family. For instance, Bcl-xL was shown to play an antiapoptotic role and was inducible and expressed by IL-5 in eosinophils [48] (fig. 3). In contrast, Bcl-2 appears not to be expressed in eosinophils [48], although there are some contrasting reports in the literature [50, 51]. In our view, it is possible that eosinophils express Bcl-2 under hypereosinophilic conditions [11]. Conclusions

Delayed eosinophil apoptosis has been observed in association with many eosinophilic diseases and may be important in the pathogenesis of eosinophilia. Eosinophil apoptosis is regulated by several survival and death factors, which initiate signaling pathways leading either to inhibition or activation of caspases. Members of the Bcl-2 and IAP families are important intracellular regulators of eosinophil apoptosis. Their expression is largely controlled by IL-5 and other eosinophil hematopoietins. The initial events triggering eosinophil apoptosis under in vivo conditions remain to be identified. References 1 2 3 4

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Straumann A, Simon HU: The physiological and pathophysiological roles of eosinophils in the gastrointestinal tract. Allergy 2004;59:15–25. Simon D, Braathen LR, Simon HU: Eosinophils and atopic dermatitis. Allergy 2004;59:561–570. Sexton DW, Al-Rabia M, Blaylock MG, Walsh GM: Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells. Clin Exp Allergy 2004;34:1514–1524. Simon HU, Weber M, Becker E, Zilberman Y, Blaser K, Levi-Schaffer F: Eosinophils maintain their capacity to signal and release eosinophil cationic protein upon repetitive stimulation with the same agonist. J Immunol 2000;165:4069–4075. Yousefi S, Hemmann S, Weber M, et al: IL-8 is expressed by human peripheral blood eosinophils: Evidence for increased secretion in asthma. J Immunol 1995;154:5481–5490. Schmid-Grendelmeier P, Altznauer F, Fischer B, et al: Eosinophils express functional IL-13 in eosinophilic inflammatory diseases. J Immunol 2002;169:1021–1027.

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Simon HU, Blaser K: Inhibition of programmed eosinophil death: A key pathogenic event for eosinophilia? Immunol Today 1995;16:53–55. Simon HU, Yousefi S, Schranz C, Schapowal A, Bachert C, Blaser K: Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia. J Immunol 1997;158:3902–3908. Wedi B, Raap U, Lewrick H, Kapp A: Delayed eosinophil programmed cell death in vitro: A common feature in inhalant allergy and extrinsic and intrinsic dermatitis. J Allergy Clin Immunol 1997;100:536–543. Kankaanranta H, Lindsay MA, Giembycz MA, Zhang X, Moilanen E, Barnes PJ: Delayed eosinophil apoptosis in asthma. J Allergy Clin Immunol 2000;106:77–83. Plötz SG, Dibbert B, Abeck D, Ring J, Simon HU: Bcl-2 expression by eosinophils in a patient with hypereosinophilia. J Allergy Clin Immunol 1998;102:1037–1040. Smith CA, Farrah T, Goodwin RG: The TNF receptor superfamily of cellular and viral proteins: Activation, costimulation, and death. Cell 1994;76:959–962. Hebestreit H, Yousefi S, Balatti I, et al: Expression and function of the Fas receptor on human blood and tissue eosinophils. Eur J Immunol 1996;26:1775–1780. Hebestreit H, Dibbert B, Balatti I, et al: Disruption of Fas receptor signaling by nitric oxide in eosinophils. J Exp Med 1998;187:415–425. Liles WC, Kiener PA, Ledbetter JA, Aruffo A, Klebanoff SJ: Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: Implications for the regulation of apoptosis in neutrophils. J Exp Med 1996;184:429–440. Liles WC, Klebanoff SJ: Regulation of apoptosis in neutrophils – Fas track to death. J Immunol 1995;155:3289–3291. Brown SB, Savill J: Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes. J Immunol 1999;162:480–485. Daigle I, Simon HU: Critical role for caspase 3 and 8 in neutrophil but not eosinophil apoptosis. Int Arch Allergy Immunol 2001;126:147–156. Fujihara S, Ward C, Dransfield I, et al: Inhibition of nuclear factor-␬B activation un-masks the ability of TNF-␣ to induce human eosinophil apoptosis. Eur J Immunol 2002;32:457–466. Kankaanranta H, De Souza PM, Barnes PJ, Salmon M, Giembycz MA, Lindsay M: SB 203580, an inhibitor of p38 mitogen-activated protein kinase, enhances constitutive apoptosis of cytokinedeprived human eosinophils. J Pharmacol Exp Ther 1999;290:621–628. Levi-Schaffer F, Temkin V, Malamud V, Feld S, Zilberman Y: Mast cells enhance eosinophil survival in vitro: Role of TNF-alpha and granulocyte-macrophage colony-stimulating factor. J Immunol 1998;160:5554–5562. Robertson NM, Zangrilli JG, Steplewski A, et al: Differential expression of TRAIL and TRAIL receptors in allergic asthmatics following segmental antigen challenge: Evidence for a role of TRAIL in eosinophil survival. J Immunol 2002;169:5986–5996. Daigle I, Simon HU: Alternative functions for TRAIL receptors in eosinophils and neutrophils. Swiss Med Wkly 2001;131:231–237. Heinisch IV, Bizer C, Volgger W, Simon HU: Functional CD137 receptors are expressed by eosinophils from patients with IgE-mediated allergic responses but not by eosinophils from patients with non-IgE-mediated eosinophilic disorders. J Allergy Clin Immunol 2001;108:21–28. Nutku E, Aizawa H, Hudson SA, Bochner BS: Ligation of Siglec-8: A selective mechanism for induction of human eosinophil apoptosis. Blood 2003;101:5014–5020. Matsumoto K, Terakawa M, Miura K, Fukuda S, Nakajima, Saito H: Extremely rapid and intense induction of apoptosis in human eosinophils by anti-CD30 antibody treatment in vitro. J Immunol 2004;172:2186–2193. Berro AI, Perry GA, Agrawal DK: Increased expression and activation of CD30 induce apoptosis in human blood eosinophils. J Immunol 2004;173:2174–2183. Al-Rabia MW, Blaylock MG, Sexton DW, Walsh GM: Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line. J Leukoc Biol 2004;75: 1045–1055. Foerster M, Haefner D, Kroegel C: Bcl-2-mediated regulation of CD69-induced apoptosis of human eosinophils: Identification and characterization of a novel receptor-induced mechanism and relationship to CD95-transduced signalling. Scand J Immunol 2002;56:417–428.

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Rothenberg ME, Stevens RL, Silberstein DS, Soberman R, Austen KF, Owen WF: IL-5 promotes long-term culture and enhances functional properties of human eosinophils. J Immunol 1989;143: 2311–2316. Yousefi S, Hoessli DC, Blaser K, Mills GB, Simon HU: Requirement of Lyn and Syk tyrosine kinases for the prevention of apoptosis by cytokines in human eosinophils. J Exp Med 1996;183: 1407–1414. Simon HU, Yousefi S, Dibbert B, Levi-Schaffer F, Blaser K: Anti-apoptotic signals of granulocytemacrophage colony-stimulating factor are transduced via Jak2 tyrosine kinase in eosinophils. Eur J Immunol 1997;27:3536–3539. Pazdrak K, Stafford S, Alam R: The activation of the Jak-STAT1 signaling pathway by IL-5 in eosinophils. J Immunol 1995;155:397–402. Leckie MJ, ten Brinke A, Khan J, et al: Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyperresponsiveness, and the late asthmatic response. Lancet 2000;356: 2144–2148. Plötz SG, Simon HU, Darsow U, et al: Use of an anti-interleukin-5 antibody in the hypereosinophilic syndrome with eosinophilic dermatitis. N Engl J Med 2003;349:2334–2339. Shinagawa K, Trifilieff A, Anderson GP: Involvement of CCR3-reactive chemokines in eosinophil survival. Int Arch Allergy Immunol 2003;130:150–157. Bureau F, Seumois G, Jaspar F, et al: CD40 engagement enhances eosinophil survival through induction of the cellular inhibitor of apoptosis protein 2 expression: Possible involvement in allergic inflammation. J Allergy Clin Immunol 2002;110:443–449. Thornberry NA, Lazebnik Y: Caspases: Enemies within. Science 1998;281:1312–1316. Salvesen GS, Dixit VM: Caspase activation: The induced-proximity model. Proc Natl Acad Sci USA 1999;96:10964–10967. Green DR, Reed JC: Mitochondria and apoptosis. Science 1998;281:1309–1312. Dewson G, Cohen GM, Wardlaw AJ: Interleukin-5 inhibits translocation of Bax to the mitochondria, cytochrome c release, and activation of caspases in human eosinophils. Blood 2001;98:2239–2247. Peachman KK, Lyles DS, Bass DA: Mitochondria in eosinophils: Functional role in apoptosis but not respiration. Proc Natl Acad Sci USA 2001;98:1717–1722. Crook NE, Clem RJ, Miller LK: An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J Virol 1993;67:2168–2174. Salvesen GS, Duckett CS: IAP proteins: Blocking the road to death’s door. Nat Rev Mol Cell Biol 2002;3:401–410. Altznauer F, Martinelli S, Yousefi S, et al: Inflammation-associated cell cycle-independent block of apoptosis by survivin in terminally differentiated neutrophils. J Exp Med 2004;199:1343–1354. Zangemeister-Wittke U, Simon HU: An IAP in action: The multiple roles of survivin in differentiation, immunity and malignancy. Cell Cycle 2004;3:1121–1123. Bainton DF, Ullyot JL, Farquhar MG: The development of neutrophilic polymorphonuclear leukocytes in human bone marrow: Origin and content of azurophil and specific granules. J Exp Med 1971;134:907–934. Dibbert B, Daigle I, Braun D, et al: Role for Bcl-xL in delayed eosinophil apoptosis mediated by granulocyte-macrophage colony-stimulating factor and interleukin-5. Blood 1998;92:778–783. Altznauer F, Conus S, Cavalli A, Folkers G, Simon HU: Calpain-1 regulates Bax and subsequent Smac-dependent caspase-3 activation in neutrophil apoptosis. J Biol Chem 2004;279:5947–5957. Jang AS, Choi IS, Lee S, Seo JP, Yang SW, Park CS: Bcl-2 expression in sputum eosinophils in patients with acute asthma. Thorax 2000;55:370–374. El-Gamal Y, Heshmat N, Mahran M, El-Gabbas Z: Expression of the apoptosis inhibitor Bcl-2 in sputum eosinophils from children with acute asthma. Clin Exp Allergy 2004;34:1701–1706.

Prof. Dr. Hans-Uwe Simon Department of Pharmacology, University of Bern Friedbühlstrasse 49 CH–3010 Bern (Switzerland) Tel. ⫹41 31 632 3281, Fax ⫹41 31 632 4992, E-Mail [email protected]

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The Lung, Eosinophils and Asthma Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 59–75

The Role of T Lymphocytes in Asthma A. Barry Kay National Heart & Lung Institute, and Leukocyte Biology Section, Imperial College, London, UK

Abstract There is now overwhelming evidence to support a major role for T cells in asthma, in particular the involvement of T helper type 2 (Th2) cells in atopic allergic asthma as well as nonatopic and occupational asthma. There may also be a minor contribution from T cytotoxic type 2 CD8⫹ T cells. Several Th2 cytokines have potential to modulate airway inflammation, in particular interleukin-13 which induces airway hyperresponsiveness independently of IgE and eosinophilia in animal models. The identification of transcription factors controlling Th1, Th2 and T-regulatory cell (TReg) development further support the Th2 hypothesis since GATA3 is overexpressed and T-bet underexpressed in the asthmatic airway and Foxp3 is induced in asthma by corticosteroid treatment. Specific T-cell-directed immunotherapy may allow induction/modulation of T-cell responses, and elucidation of the mechanisms of TRegs may allow further optimization of immunotherapy. Recent advances in the understanding of dendritic cell function in directing T-cell responses may uncover further therapeutic targets. Efficacy of cyclosporin and anti-CD4 treatment in chronic severe asthma argues for continued T-cell involvement, but whether remodeling contributes to pathology inaccessible to antiinflammatory treatment or T-cell immunotherapy remains an important question. Copyright © 2006 S. Karger AG, Basel

The group in Davos, under the direction of Professor Kurt Blaser, were amongst the first to recognize the association between T cells, their products and asthma severity [1] and over the years they have published a succession of highly original papers which have significantly increased our understanding of T-cell function in allergy and asthma. A critical role for T helper type 2 (Th2) cells in asthma is now widely accepted [2]. The T-cell hypothesis of asthma developed from studies of late asthmatic reactions (LARs) [3] and acute severe asthma (‘status asthmaticus’) [4] and was supported by the observation that there was a Th2-type T-cell cytokine profile in this disease [5]. Asthma is a heterogeneous disease with several clinical subtypes and a wide spectrum ranging from mild, episodic, wheezy breathlessness to chronic, intractable, corticosteroid-dependent chronic airway narrowing. The classical

IgE-associated allergic asthma phenotype starting in childhood is the most widely studied, largely because this form of the disease can be provoked in the clinical laboratory under controlled conditions by inhalation of allergen, or allergen-derived T-cell peptides. In these patients airway cells have a predominant Th2-cytokine profile [i.e. interleukin (IL)-4⫹, IL-5⫹, IL-9⫹ and IL-13⫹ mucosal cells]. Some asthmatics have late-onset, nonallergic (so called ‘intrinsic’) asthma in which allergens cannot be identified but in which airway eosinophilia and Th2 cells are also prominent. The characteristic features of most asthma phenotypes, including allergic asthma, are airway inflammation, airway hyperresponsiveness (AHR; increased sensitivity or ‘irritability’ of the bronchi), 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. It is well known that allergen-specific IgE synthesis is T-cell dependent, through cognate activation of B lymphocytes and T-cell-derived cytokines such as IL-4 and IL-13. Thus, in atopic asthma and allergic rhinitis, allergen processing and presentation to allergen-specific T cells via antigen-presenting cells is a key initiation step. Growing interest in the role of the T-cell in asthma arose from the concept that, in addition to participating in IgE synthesis, T-cell products may also have direct effects on the airways through the recruitment of inflammatory cells, particularly eosinophils. A number of studies showed evidence for CD4⫹ T-cell activation in peripheral blood of asthmatics during exacerbations. Sampling of the airways either with bronchial biopsies and bronchoalveolar lavage (BAL) revealed T cells with features of activation [reviewed in 2]. In some studies T-cell activation could be related to measures of asthma severity, such as the degree of airway narrowing or AHR, as well as the bronchial eosinophil response. Similarly, after the description of the Th2/Th1 dichotomy mRNA⫹ cells for the signature Th2 cytokines, IL-4 and IL-5 were detected in airway samples from atopic asthmatics. This attractively linked IgE synthesis through IL-4, and eosinophilic airway inflammation through IL-5, together with IL-3 and granulocyte/macrophage colony stimulating factor [6, 7]. In addition, a number of investigators have isolated allergen-specific T-cell lines and clones from the BAL of asthmatics [2].

The Asthma Phenotypes

The contribution of allergy across the spectrum of asthma has always been vigorously debated, especially as only around half of asthmatics are atopic. An

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important question is: what is the role of T cells and more particularly Th2 cells, in the nonatopic (intrinsic) form of the disease, as well as occupational asthma and acute exacerbations where viruses often appear to be the triggering factor? Bronchial biopsies from nonatopic and occupational asthmatics have revealed remarkable similarities to atopic asthma, at least at the level of immunopathology [6, 8]. Thus, eosinophil infiltration and cells bearing markers of T-cell activation, such as CD25, were present in increased numbers in bronchial biopsies from all three forms of disease. Similarly, the cytokine profile in the airway of nonatopic asthma also showed prominence of IL-4, IL-5 and IL-13, with no increase in interferon-␥ (IFN-␥) compared with nonasthmatic control volunteers. More recent data have demonstrated evidence for local IgE synthesis in the bronchial mucosa of nonatopic asthmatics, supporting a Th2/IgE-mediated immunopathological process [9], despite the absence of specific serum IgE or positive skin prick test. Interestingly, it has been suggested that self-antigens may drive the Th2 response in nonatopic asthma. Further work will be required to understand the role of IgE in this disease, including how it might be triggered. Similarly, some occupational asthma studies involving low-molecular-weight agents, such as toluene diisocyanate, suggest Th2-type cytokine production in the airway, although it is of note that the IgE dependence of occupational asthma is not always demonstrated [2]. Initial studies of airway biopsies were restricted to mild asthmatics with well-preserved lung function, although there was also evidence of T-cell infiltration and activation in postmortem airway tissue from asthmatics dying both from asthma and from other causes, as well as BAL and transbronchial biopsy studies in more severe asthma [10]. In some patients there is marked eosinophilic inflammation of the small airways together with T-cell activation, but others show sparse inflammatory changes or neutrophilic inflammation. Whether such findings reflect different pathological mechanisms or the effect of treatment (often with high-dose inhaled or oral corticosteroids) remains to be established. Using an ‘immunoepidemiological’ approach Heaton et al. [11] found that in children the blood cytokine profiles differed according to the wheezing phenotype with asthma symptoms being associated with Th2 cytokines, and AHR with eosinophilia and IFN-␥ production.

Effector Mechanisms: How Do T Cells Cause Asthma?

Over the past few years a working hypothesis has been that Th2 cytokines contribute to asthma pathology through IgE synthesis, and maturation and activation of mast cell and basophil (and thus acute asthma), and through

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IL-5-mediated eosinophil infiltration leading to epithelial damage and AHR. Some studies also showed Th1 cytokines in serum and BAL from asthmatics, particularly during exacerbations, although most studies confirm Th2 predominance in stable disease. It also soon became apparent that mast cells, basophils and eosinophils were themselves potential sources of Th2-type cytokines [12]. Indeed, immunohistochemical staining for cytokines in bronchial biopsies suggested that these cytokines localized mainly to non-T cells [13]. However, these findings likely reflect storage of cytokines in mast cell eosinophils, and basophils, as mRNA for IL-4 and IL-5 localized predominantly to T cells with minor contributions from mast cells basophils and eosinophils. Data from animal studies suggested AHR could be initiated by T cells through mechanisms that were not dependent on either IgE or eosinophils. This may also be the case for human asthma, since direct interaction of T cells and airway smooth muscle is suggested and both IL-5 and IL-13 have been shown to have the ability to increase smooth muscle contractility to acetylcholine in vitro [2].

Are T Cells Required to Perpetuate Asthma?

Much attention has been focused on airway remodeling in asthma [14, 15]. This encompasses changes in the epithelium, subepithelial basement membrane deposition of collagen and other extracellular matrix proteins, increased vascularity and smooth muscle hypertrophy and hyperplasia. It has been suggested that the smooth muscle changes alone are sufficient to sustain AHR. Bronchial mucosal biopsies of asthmatics compared with those from patients with eosinophilic bronchitis (which is characterized by cough without reversible airway narrowing or AHR) showed no difference in mucosal eosinophil or T-cell infiltration. However, mast cell infiltration of smooth muscle was seen in the asthmatics but not the eosinophilic bronchitis patients, and it was therefore suggested that AHR and airway narrowing in asthma were more related to smooth muscle and mast cell interaction than the eosinophil-T cell axis [16]. These findings raise the question of whether T cells are important in sustaining chronic asthma or in initiating the pathological process. Data discussed below showing that T-cell-directed therapy is effective in chronic asthma argues for a continued role, although equally the persistent AHR and incomplete abolition of symptoms after broad anti-inflammatory treatment, such as corticosteroids, may result from structural airway changes. The development of specific T-celldirected therapy and better animal models of chronic airway inflammation might give further information in this important area.

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T-Cell-Derived Cytokines and Cytokines That Act on T Cells

Many cytokines with potential relevance to asthma have been described. IL-9, IL-11, IL-13, IL-16, IL-17 and IL-19 [17] have been linked to asthma, IL25 acts in Th2 differentiation, whilst IL-12, IL-18, IL-23 and IL-27 are involved in Th1 development and IFN-␥ production, which may be deficient in asthma. IL-9 was implicated in AHR initially from studies linking strain differences in baseline AHR with IL-9 expression and increased AHR and eosinophilia in IL-9 transgenics, although response to airway challenge was not reduced in sensitized IL-9 knockout animals. Like IL-9, transgenic expression of IL-11 and IL-13 in the airway of mice was sufficient to induce eosinophilia, AHR, mucus hypersecretion and variable changes similar to remodeling [18, 19]. Transgenic expression of IL-11 in the airway lead to lymphocytic infiltration and remodeling, yet inhibited the Th2 response to inhaled antigen [20]. IL11 has also been reported to favor Th2 polarization of naïve T cells [21]. Thus IL-11 may be associated with Th2 responses and chronic repair and remodeling. Several animal models have shown that IL-13 plays a key role in the development of AHR and mucus production [22] and Mattes et al. [23] provided evidence that eosinophils could modulate IL-13 production from CD4⫹ cells, possibly via IL-18. IL-16 is a CD4⫹ lymphocyte chemoattractant, whilst IL-17 is a T-cellderived cytokine that can induce fibroblast production of proinflammatory cytokines, including granulocyte/macrophage colony-stimulating factor [24]. IL-25 is a more recently characterized cytokine with homology to IL-17 and drives Th2 IL-4, IL-5 and IL-13 production in murine T cells [2]. Adenoviral transfer of IL-25 into the lung of mice causes eosinophilic inflammation, epithelial changes, mucus hypersecretion and AHR [25]. All of these cytokines (except IL-25) have been reported to be over-represented in the airways of asthmatic subjects when compared to nonasthmatic control volunteers, and IL-11 was detected in severe asthma, where it may act in remodeling [2]. Since the array of T-cell cytokines with potential to contribute to asthma pathology has expanded, this may, in turn, explain the disappointing results of single cytokine-directed therapy for asthma. Cytokines involved in Th1 development and phenotype expression have also been studied in animal models and human asthma. Thus IL-12 and IL-18 (with IL-12) have the potential to reduce airway inflammation to inhaled challenge after Th2 sensitization, and AHR and eosinophilia was increased in IL-18-deficient animals compared to wild-type controls [2]. Similarly it has been suggested that IL-12 and IL-18 are deficient in human airway samples in asthma, and this may relate to similar relative underexpression of T-bet [26].

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Controls of T-Cell Cytokine Production A number of transcription factor and signaling molecules have been shown to have potential roles in animal models, including c-MaF, NF-␬B, NF-AT and STAT6. At least two transcription factors with potential to control Th1 or Th2 development have been described. GATA3 is implicated in Th2 development in mouse and human T cells, and in particular is an important controller of the IL-5 gene locus. In mouse models, blocking GATA3 with a dominant negative construct or antisense DNA could prevent Th2 cytokine activation, eosinophilia and AHR in challenge models. Further knockout mice with a p50 NF-␬B defect failed to mount a GATA3 or Th2 response in an antigen sensitization and airway challenge model, suggesting that NF-␬B may control GATA3 expression in developing Th2 cells [2]. Numbers of GATA3-expressing cells were increased in bronchial biopsies from asthmatic patients, supporting this as a target for control of Th2 cells in asthma. Christodoulopoulos et al. [27] have examined other transcription factors and showed increased expression of cells bearing GATA3, c-MaF (a transcription factor for IL-4) and STAT6 (which transduces signals for IL-4 and IL-13) in bronchial biopsies in both atopic and nonatopic asthmatics compared to control volunteers. However STAT6 expression was less prominent in nonatopic asthma suggesting IL-4 signaling may play a less important role. T-bet is also a controller of Th1 development, and was shown to direct IFN-␥ production and IL-12R␤2 expression. Finotto et al. [28] described reduced numbers of cells staining for T-bet in bronchial biopsies from asthmatics, and spontaneous AHR in mice deficient in T-bet. These findings again suggest that asthma is associated with reduced airway Th1 cells, and the authors speculate that a defect in Th1 development through deficiency in T-bet might predispose to Th2 responses. It is of note that Th2 cytokines in human T cells appear to be coordinately regulated [29] through the activity of a number of these transcription factors, and it may thus be possible to target a range of cytokines through therapy directed on interfering with this process. However, this remains hypothetical at present.

TReg Cells and Asthma

There is much current interest in regulatory T cells (TRegs). A number of such suppressive T-cell subsets have been described in mouse and humans, including naturally occurring CD4⫹CD25⫹ T cells, IL-10-producing T cells and Tr1 cells [30]. Whether such cells can be induced therapeutically in asthma remains to be established, although, this is one possible mechanism of immunotherapy (both using peptides and whole allergen extracts). One type of

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TReg was induced by a combination of corticosteroids and vitamin D3 [31], and corticosteroids increase IL-10 both in vitro and in vivo in asthma [32, 33]. Thus current evidence suggests that human CD4⫹ CD25⫹ T cells and IL-10producing TRegs have the capacity to suppress Th2 responses to allergen, and that this may be defective in those who develop allergic sensitization [34]. Allergen immunotherapy modifies T-cell responses to allergen, and may do so through induction of IL-10-producing TRegs: although other mechanisms including Th1 immune deviation are likely involved. Controversy remains about the nature and interrelation of different TReg-populations: it is likely that CD4⫹ CD25⫹ T cells isolated from human adult blood represent a mixed population of ‘naturally occurring’ TRegs analogous to those studied in mice, and ‘adaptive’ TRegs induced by low- or high-dose allergen exposure. The exact mechanism of suppression also remains controversial and may differ for different regulatory populations. An important advance in the understanding of the biology of regulation by CD4⫹ CD25⫹ T cells was the demonstration of the dependence of their suppressive phenotype on expression of the transcription factor Foxp3. Interestingly, glucocorticosteroids upregulate Foxp3 and IL-10 mRNA in asthma [35]. Further work is required to determine the phenotype and derivation of T-regulatory cells in asthma and allergy, and whether modification of existing immunotherapy can make such treatment more widely applicable and more effective.

Provoked Asthma under Controlled Clinical Conditions

Inhaled allergen challenge of sensitized atopic asthmatics has been used for many years as a model of asthma, to study both the early and late reaction. Whilst the early reaction is thought be IgE- and mast-cell-dependent, the cause and significance of the LAR and associated increased AHR is less certain. Although cutaneous late responses could be induced by passive transfer of IgE and re-challenge, the skin and lung response are associated with eosinophil and T-cell infiltration and studies at 24 h after challenge show increases in T-cell activation and Th2 cytokine expression which can be correlated with the preceding airway narrowing during the LAR. This model is essentially similar to that used in mouse models, except that there is background chronic inflammation in the airway in human asthmatics. Quite how it relates to chronic asthma is uncertain, but like the mouse models human allergen challenge has proved instructive. T-cell dependence in mouse models could be directly demonstrated by anti-CD4 antibody depletion of T cells. Data to support a role of T cells in human LAR came from inhibition of the LAR with cyclosporin A (CsA), a drug that targets T-cell activation via NFAT-driven IL-2 production, and also inhibits T-cell IL-5 production [36]. In a further study, bronchoscopy revealed that

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inhibition of the LAR by CsA was associated with accelerated apoptosis of BAL CD3⫹ cells as well as decreases in eosinophils and IL-5 [37]. This provided indirect evidence that CsA is effective in this human model of asthmatic allergic inflammation principally through its effects on the T cells and associated eosinophil events, rather than actions on mast cells and basophils. Studies from our own group have shown that direct T-cell activation via intradermal injection of short peptides derived from the cat allergen Fel d 1 could induce an isolated LAR in cat-allergic asthmatics [38]. Peptides were shown to be capable of binding to 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 major histocompatibility class II able to bind the injected peptides, further supporting a role for T cells in the observed response. The mechanism of LARs induced by intradermal injection of peptide (without reaction in the skin) is uncertain. Unlike LAR induced after inhalation of whole allergen, bronchoscopic analysis of peptide-induced LAR showed no evidence of eosinophil infiltration, or T-cell activation in the airway, with no change in IL-5, IL-13, histamine or leukotrienes detected in paired BAL samples from peptide- and diluent-challenged subjects. This may be because the LAR was elicited via the circulation rather than the airway, or that there is direct T-cell-mediated smooth muscle contraction. We have also shown that inhaled peptide can induce LAR in cat-allergic asthmatics [39]. Reactions induced by inhaled peptides were accompanied by sputum eosinophilia, suggesting that changes in inflammatory mediators and mucosal infiltration of effector cells are only detectable by bronchoscopy after topical challenge with peptides. The potential for such peptides in therapy is discussed below.

Activation of T Cells in Asthma:Antigen-Presenting Cells

In addition to presentation of antigen peptide in the context of MHC, T-cell activation requires costimulation and additional signals. Recent evidence suggests a number of factors may influence antigen-presenting cells, and that in particular dendritic cells are plastic in their ability to drive T-cell responses to Th1- or Th2-type responses. Dendritic cell function is partially controlled by signals from the innate immune system, in part via Toll-like receptors, through control of IL-12 and IL-10 production. Different costimulation molecules and other factors may also influence DC function. For example inducible costimulator (ICOS) costimulation in the mouse lung favors an IL-10-producing T cell with potential suppressive activity [40]. Other mouse data suggests that ICOS favors Th2 responses and blocking ICOS can reduce AHR and eosinophilia in an animal model of allergen challenge [41]. The role of ICOS in human asthma

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remains to be defined. We have shown that human BAL T cells can be activated with allergen to proliferate and secrete IL-5 and that this was CD86 dependent. Furthermore, adherent cells from asthmatic BAL fluid (predominantly alveolar macrophages) were as effective at presenting antigen to T cells as blood monocytes [42]. Histamine has also been shown to affect dendritic cell function, driving DCs to favor Th2-cell expansion via H1 and H2 receptors. This may clearly be relevant to asthma. Whether targeting of costimulation or dendritic cell function can be sufficiently specific to be useful for asthma therapy remains to be established. There is also evidence that T-regs may influence DCs by maintaining them in an immature state [43]. CD8ⴙ T Cells and ␥␦ Cells

There are several reports that suggest that CD8⫹ cells may participate in the asthma process. An early study involving BAL after allergen inhalational challenge suggested that CD8⫹ cells may be involved in the regulation of the expression of the LAR since there was a relative increase in OKT8⫹ lavage cells in early-phase, as compared to late-phase responders [3]. On the other hand, several studies have indicated that CD8⫹ cells may be proinflammatory in the airways. In both atopic and nonatopic asthmatics mRNA for IL-4 and IL-5 colocalized, predominantly to CD4⫹, but also to CD8⫹ T cells. Others found that CD8⫹ as well as CD4⫹ T-cell lines from BAL from asthmatics elaborated the IL-5 protein. Cho et al. [44] demonstrated elevated numbers of IL-4 protein/CD8⫹ cells in blood from mild atopic asthmatics. All these observations are compatible with the concept of a population of CD8⫹ T cytotoxic type 2 lymphocytes. Some studies have suggested increases in CD8⫹ cells in occupational asthma, and in a mouse model it was shown that virus-specific CD8⫹ cells switch to IL-5 production and induce airway eosinophilia. This raises the possibility that in asthma certain viruses or chemical haptens may modify intrinsic antigens, which in turn are targeted by CD8⫹ cytotoxic cells. Although some animal data suggests that ␥␦ T cells may be important in models of asthma or allergic sensitization [45], there is conflicting evidence to support a role in human asthma [2, 46].

Homing of T Cells to the Airway in Asthma

It has been established for some time that T cells utilize adhesion molecules, such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, during trafficking to the airway, but there is increasing interest in

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the role of specific chemokines in either tissue-directed T-cell homing or in recruitment of different T-cell subtypes. Most of the current data comes from mouse models or human allergen challenge studies. In vitro, mouse and human Th2 cells are polarized to preferentially express CCR3, CCR4 and CCR8 and interact with their ligands: eotaxin, monocyte-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC) and I-309/TCA-3. In experiments involving adoptive transfer of polarized T-cell receptor transgenic Th2 cells to recipient mice followed by inhaled challenge, early infiltrating T cells (first challenge) were predominantly CCR3⫹ and influx was reduced by blocking antibodies to eotaxin, whereas later T-cell infiltration was of CCR4⫹ T cells and was blocked by antibody to MDC [47]. However, effects of CCR3, CCR4 and CCR8 knockout in mice are complex, and lack of these receptors does not totally prevent Th2 cell infiltration and AHR after antigen sensitization and inhaled challenge. In the CCR3 knockout mouse there was a reduction in eosinophil trafficking to the lung after allergen challenge, but an increase in intraepithelial mast cells, and an overall increase in AHR to challenge [48]. In the CCR4 knockout there was no effect on eosinophils or AHR in an ovalbumin antigen challenge model [49], but inflammatory response and AHR were reduced in an Aspergillus model [50]. For CCR8 the knockout mouse showed diminished eosinophil recruitment in response to ovalbumin or cockroach allergen sensitization and airway challenge, but AHR was not measured and there was no change in T cells [51]. Some of these changes may relate to the finding that CCR8 is expressed by murine eosinophils [52]. In human, PaninaBordignon et al. [53] observed that virtually all T cells from endobronchial biopsies from asthmatics, taken 24 h after allergen challenge, expressed IL-4 and CCR4. CCR8 was coexpressed with CCR4 on 28% of the T cells, while CCR3 was associated with eosinophils but not on T cells. Interestingly expression of the CCR4-specific ligands MDC and TARC was strongly upregulated on airway epithelial cells upon allergen challenge, suggesting an involvement of this receptor/ligand axis in the regulation of lymphocyte recruitment into the asthmatic bronchi. In contrast to asthma, T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis produce IFN-␥ and express high levels of CXCR3, while lacking CCR4 and CCR8 expression. These data supported a role of CCR4, its ligands MDC and TARC, and CCR8 in the pathogenesis of allergen-induced late asthma and a role for T cells in LARs. However, Campbell et al. [54] examined BAL T cells from asthmatics compared to nonasthmatic control volunteers and showed no differences (albeit numbers were small) in expression of CCR5 and CXCR3 with only low expression of CCR4. Furthermore Liu et al. [55] found that dual asthmatic responders (early- and late-phase reactions) had both Th2 (TARC, MDC) and Th1 (IP10, MIG) chemokines in the airways after allergen

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challenge. Such differences may result from analysis of allergen challenge versus baseline asthma, and further studies are required. Small molecule antagonists of CCR3 and other chemokine receptors should be available for study in humans in the near future.

T Cells and Treatment of Asthma

Corticosteroids Inhaled corticosteroids are the mainstay of asthma management, and these agents are extremely effective in vitro as inhibitors of T-cell activation and cytokine production. There are many studies showing reduction in T-cell activation and cytokine expression in the airway of asthmatics after steroid treatment [2]. It is of interest that corticosteroid-resistant asthmatics failed to suppress IL-5 in airway biopsies after treatment with oral prednisolone [56]. The mechanism is unclear, but T cells can be rendered steroid-resistant in vitro by IL-2 and IL-4. Such patients fail to suppress T-cell activation markers in vivo after corticosteroid treatment, and show abnormalities in T-cell transcription factor expression (AP-1, STAT5 and NF-␬B), all of which suggests that this may be a T-cell defect [57]. Cyclosporin A Further support for the T-cell hypothesis of asthma was obtained from controlled clinical trials of CsA in chronic asthma. In a group of severe steroiddependent asthmatics treatment over a 12-week period with CsA at low dosage (5 mg/kg/day) was associated with improvement in lung function and a reduction in the number of disease exacerbations requiring an increase in corticosteroid dosage [58]. Reductions in the concentrations of serum IL-2R after treatment with CsA were also observed. Both showed improvements in lung function despite the reduction in corticosteroid dose, although in one this improvement was small. More direct evidence of a role for T cells in the pathogenesis of chronic asthma was described by Kon et al. [59] who treated severe corticosteroiddependent asthmatics with a single infusion of a chimeric (primatized) monoclonal antibody to human CD4. Subjects were treated in three separate dose groups in addition to a placebo arm. Patients receiving a single infusion of 3.0 mg/kg displayed a significant change in the morning and evening peak expiratory flow rate with a trend towards an improvement in symptom scores. Taken together, these studies in which T lymphocytes and in particular CD4⫹ T lymphocytes were targeted in allergic asthma provide support for a significant role of these cells in the pathogenesis of asthma.

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Studies using monoclonal antibodies or receptor fusion proteins to block individual T-cell-derived cytokines have been disappointing. Although our recent data suggest that anti-IL-5 does not completely deplete airway eosinophils [60], the lack of clinical efficacy of this treatment argues against a major direct effect of IL-5 on airway smooth muscle as an important contributor to AHR. Some effects of a soluble IL-4 receptor have been reported in a protocol of steroid withdrawal in mild asthma [61]. Human studies on IL-13 may be of interest, but, as stated, targeting single T-cell cytokines seems unlikely to be very effective.

Antigen-Directed Targeting of T Cells and TRegs: The Future for Asthma Therapy?

Allergen injection immunotherapy has long been used for allergic asthma in many countries (though not the UK). Current evidence suggests that immunotherapy modulates the T-cell response to allergen, either through immune deviation to a Th1 response, or through induction of a ‘modified Th2’ or TReg response with high IL-10 secretion. IL-10 inhibits T-cell activation and cytokine secretion, and switches B cells from IgE to IgG4 production. Whole allergen immunotherapy has modest effects in asthma compared to corticosteroids, possibly because of dose limitation due to the potential for anaphylaxis due to IgE cross-linking. Several studies in mice have shown that the development of T-cell tolerance in vivo is preceded by transient T-cell activation. Webb et al. [62] reported that the clonal elimination of V␤6⫹ cells responding to a superantigen was preceded by marked expansion of these cells, whereas Vidard et al. [63] found that prior to the establishment of specific T-cell tolerance to ovalbumin, T cells displayed transient responsiveness and synthesized IL-2 upon antigenic stimulation in vitro. Similar findings were reported by Hoyne et al. [64] using an immunodominant peptide derived from house dust mite. In this model a strong, transient, T-cell CD4⫹ response in vitro preceded the development of tolerance in vivo. Tsitoura et al. [65] observed transient T-cell activation and production of Th1 and Th2 cytokines following tolerogenic intranasal administration of ovalbumin (OVA). Re-challenge of asthmatic subjects with Fel d 1 peptides via the intradermal route resulted in reduction or abrogation of the isolated LAR in those individuals in whom such reactions occurred following initial challenge [38, 66, 67]. Additionally, the magnitude of both early- and late-phase skin reactions to whole allergen challenge were also reduced, together with a reduction of proinflammatory immunological parameters and an increase in the immunomodulatory

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cytokine IL-10 [67]. Müller et al. [68] observed similar changes in immunological parameters after treating bee-venom-allergic individuals with peptides from phospholipase A2. Analysis of the extent of reduction in the magnitude of peptide-induced LAR and the interval between the first and second challenge with peptides demonstrated a ‘window’ of optimal tolerance induction that appeared when injections were administered between approximately 2 weeks and 2 months apart. One implication of this observation is that the ability of peptides to activate a memory T-cell response that manifests itself as an isolated LAR in certain individuals, and the ability to induce hyporesponsiveness in the allergen-specific T-cell compartment are unlikely to be linked temporally. Furthermore, reductions in early- and late-phase skin reactions to whole allergen challenge were observed with equal frequency and magnitude in those subjects who experienced an LAR and those who did not. Thus, it appears likely that the induction of transient T-cell activation and induction of hyporesponsiveness in this clinical model are separate events. Whether these can be separated, and whether this treatment can reduce AHR and symptoms in asthma is the subject of ongoing work. Also of relevance for the future of immunotherapy is whether specific immunotherapy can be used to prevent asthma: certainly initial data is promising [69].

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Christodoulopoulos P, Cameron L, Nakamura Y, Lemiere C, Muro S, Dugas M, Boulet LP, Laviolette M, Olivenstein R, Hamid Q: TH2 cytokine-associated transcription factors in atopic and nonatopic asthma: Evidence for differential signal transducer and activator of transcription 6 expression. J Allergy Clin Immunol 2001;107:586–591. Finotto S, Neurath MF, Glickman JN, Qin S, Lehr HA, Green FH, Ackerman K, Haley K, Galle PR, Szabo SC, Drazen JM, De Sanctis GT, Glimcher LH: Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 2002;295:336–338. Cousins DJ, Lee TH, Staynov DZ: Cytokine coexpression during human Th1/Th2 cell differentiation: Direct evidence for coordinated expression of Th2 cytokines. J Immunol 2002;169: 2498–2506. McHugh RS, Shevach EM: The role of suppressor T cells in regulation of immune responses. J Allergy Clin Immunol 2002;110:693–702. Barrat FJ, Cua DJ, Boonstra A, Richards DF, Crain C, Savelkoul HF, de Waal-Malefyt R, Coffman RL, Hawrylowicz CM, O’Garra A: In vitro generation of interleukin 10-producing regulatory CD4(⫹) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)and Th2-inducing cytokines. J Exp Med 2002;195:603–616. Richards DF, Fernandez M, Caulfield J, Hawrylowicz CM: Glucocorticoids drive human CD8(⫹) T cell differentiation towards a phenotype with high IL-10 and reduced IL-4, IL-5 and IL-13 production. Eur J Immunol 2000;30:2344–2354. Hawrylowicz C, Richards D, Loke TK, Corrigan C, Lee T: A defect in corticosteroid-induced IL10 production in T lymphocytes from corticosteroid-resistant asthmatic patients. J Allergy Clin Immunol 2002;109:369–370. Robinson DS, Larché M, Durham SR: Tregs and allergic disease. J Clin Invest 2004;114: 1389–1397. Karagiannidis C, Akdis M, Holopainen P, Woolley NJ, Hense G, Ruckert B, Mantel PY, Menz G, Akdis CA, Blaser K, Schmidt-Weber CB: Glucocorticoids upregulate FOXP3 expression and regulatory T cells in asthma. J Allergy Clin Immunol 2004;114:1425–1433. Sihra BS, Kon OM, Durham SR, Walker S, Barnes NC, Kay AB: Effect of cyclosporin A on the allergen-induced late asthmatic reaction. Thorax 1997;52:447–452. Khan LN, Kon OM, Macfarlane AJ, Meng Q, Ying S, Barnes NC, Kay AB: Attenuation of the allergen-induced late asthmatic reaction by cyclosporin A is associated with inhibition of bronchial eosinophils, interleukin-5, granulocyte macrophage colony-stimulating factor, and eotaxin. Am J Respir Crit Care Med 2000;162:1377–1382. Haselden BM, Kay AB, Larché M: Immunoglobulin E-independent major histocompatibility complex-restricted T cell peptide epitope-induced late asthmatic reactions. J Exp Med 1999;189: 1885–1894. Ali FR, Oldfield WLG, Higashi N, Larché M, Kay AB: Late asthmatic reactions induced by inhalation of allergen-derived T-cell peptides. Am J Respir Crit Care Med 2004;169:20–26. Akbari O, Freeman GJ, Meyer EH, Greenfield EA, Chang TT, Sharpe AH, Berry G, DeKruyff RH, Umetsu DT: Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med 2002;8:1024–1032. Gonzalo JA, Tian J, Delaney T, Corcoran J, Rottman JB, Lora J, Al-garawi A, Kroczek R, Gutierrez-Ramos JC, Coyle AJ: ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses. Nat Immunol 2001;2:597–604. Larché M, Till SJ, Haselden BM, North J, Barkans J, Corrigan CJ, Kay AB, Robinson DS: Costimulation through CD86 is involved in airway antigen-presenting cell and T cell responses to allergen in atopic asthmatics. J Immunol 1998;161:6375–6382. Kuipers H, Lambrecht BN: The interplay of dendritic cells, Th2 cells and regulatory T cells in asthma. Curr Opin Immunol 2004;16:702–708. Cho SH, Stanciu LA, Begishivili T, Bates PJ, Holgate ST, Johnston SL: Peripheral blood CD4⫹ and CD8⫹ T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects. Clin Exp Allergy 2002;32:427–433. Miyahara N, Swanson BJ, Takeda K, Taube C, Miyahara S, Kodama T, Dakhama A, Ott VL, Gelfand EW: Effector CD8⫹ T cells mediate inflammation and airway hyperresponsiveness. Nat Med 2004;10:865–869.

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Krug N, Erpenbeck VJ, Balke K, Petschallies J, Tschernig T, Hohlfeld JM, Fabel H: Cytokine profile of bronchoalveolar lavage-derived CD4(⫹), CD8(⫹), and gammadelta T cells in people with asthma after segmental allergen challenge. Am J Respir Cell Mol Biol 2001;25: 125–131. Lloyd CM, Delaney T, Nguyen T, Tian J, Martinez-A C, Coyle AJ, Gutierrez-Ramos JC: CC chemokine receptor (CCR)3/eotaxin is followed by CCR4/monocyte-derived chemokine in mediating pulmonary T helper lymphocyte type 2 recruitment after serial antigen challenge in vivo. J Exp Med 2000;191:265–274. Humbles AA, Lu B, Friend DS, Okinaga S, Lora J, Al-Garawi A, Martin TR, Gerard NP, Gerard C: The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergeninduced airway inflammation and hyperresponsiveness. Proc Natl Acad Sci USA 2002;99: 1479–1484. Chvatchko Y, Hoogewerf AJ, Meyer A, Alouani S, Juillard P, Buser R, Conquet F, Proudfoot AE, Wells TN, Power CA: A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock. J Exp Med 2000;191:1755–1764. Schuh JM, Power CA, Proudfoot AE, Kunkel SL, Lukacs NW, Hogaboam CM: Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4⫺/⫺ mice. FASEB J 2002;16:1313–1315. Chensue SW, Lukacs NW, Yang TY, Shang X, Frait KA, Kunkel SL, Kung T, Wiekowski MT, Hedrick JA, Cook DN, Zingoni A, Narula SK, Zlotnik A, Barrat FJ, O’Garra A, Napolitano M, Lira SA: Aberrant in vivo T helper type 2 cell response and impaired eosinophil recruitment in CC chemokine receptor 8 knockout mice. J Exp Med 2001;193:573–584. Oliveira SH, Lira S, Martinez-A C, Wiekowski M, Sullivan L, Lukacs NW: Increased responsiveness of murine eosinophils to MIP-1␤ (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors, CCR5 and CCR8. J Leukoc Biol 2002;71:1019–1025. Panina-Bordignon P, Papi A, Mariani M, Di Lucia P, Casoni G, Bellettato C, Buonsanti C, Miotto D, Mapp C, Villa A, Arrigoni G, Fabbri LM, Sinigaglia F: The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics. J Clin Invest 2001; 107:1357–1364. Campbell JJ, Brightling CE, Symon FA, Qin S, Murphy KE, Hodge M, Andrew DP, Wu L, Butcher EC, Wardlaw AJ: Expression of chemokine receptors by lung T cells from normal and asthmatic subjects. J Immunol 2001;166:2842–2848. Liu L, Jarjour NN, Busse WW, Kelly EA: Enhanced generation of helper T type 1 and 2 chemokines in allergen-induced asthma. Am J Respir Crit Care Med 2004;169:1118–1124. Leung DY, Martin RJ, Szefler SJ, Sher ER, Ying S, Kay AB, Hamid Q: Dysregulation of interleukin 4, interleukin 5, and interferon gamma gene expression in steroid-resistant asthma. J Exp Med 1995;181:33–40. Adcock IM, Lane SJ, Brown CR, Lee TH, Barnes PJ: Abnormal glucocorticoid receptor-activator protein 1 interaction in steroid-resistant asthma. J Exp Med 1995;182:1951–1958. Alexander AG, Barnes NC, Kay AB, Corrigan CJ: Can clinical response to cyclosporin in chronic severe asthma be predicted by an in vitro T-lymphocyte proliferation assay? Eur Respir J 1996;9:1421–1426. Kon OM, Sihra BS, Compton CH, Leonard TB, Kay AB, Barnes NC: Randomised, dose-ranging, placebo-controlled study of chimeric antibody to CD4 (keliximab) in chronic severe asthma. Lancet 1998;352:1109–1113. Flood-Page PT, Menzies-Gow AN, Kay B, Robinson DS: Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003;167:199–204. Borish LC, Nelson HS, Lanz MJ, Claussen L, Whitmore JB, Agosti JM, Garrison L: Interleukin-4 receptor in moderate atopic asthma: A phase I/II randomized, placebo-controlled trial. Am J Respir Crit Care Med 1999;160:1816–1823. Webb S, Morris C, Sprent J: Extrathymic tolerance of mature T cells: Clonal elimination as a consequence of immunity. Cell 1990;63:1249–1256. Vidard L, Colarusso LJ, Benacerraf B: Specific T-cell tolerance may be preceded by a primary response. Proc Natl Acad Sci USA 1994;91:5627–5631.

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Hoyne GF, Askonas BA, Hetzel C, Thomas WR, Lamb JR: Regulation of house dust mite responses by intranasally administered peptide: Transient activation of CD4⫹ T cells precedes the development of tolerance in vivo. Int Immunol 1996;8:335–342. Tsitoura DC, DeKruyff RH, Lamb JR, Umetsu DT: Intranasal exposure to protein antigen induces immunological tolerance mediated by functionally disabled CD4⫹ T cells. J Immunol 1999;163: 2592–2600. Oldfield WLG, Kay AB, Larché M: Allergen-derived T cell peptide-induced late asthmatic reactions precede the induction of antigen-specific hyporesponsiveness in atopic allergic asthmatic subjects. J Immunol 2001;167:1734–1739. Oldfield WLG, Larché M, Kay AB: Effect of T-cell peptides derived from Fel d 1 on allergic reactions and cytokine production in patients sensitive to cats: A randomised controlled trial. Lancet 2002;360:47–53. Müller U, Akdis CA, Fricker M, Akdis M, Blesken T, Bettens F, Blaser K: Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol 1998;101:747–754. Moller C, Dreborg S, Ferdousi HA, Halken S, Host A, Jacobsen L, Koivikko A, Koller DY, Niggemann B, Norberg LA, Urbanek R, Valovirta E, Wahn U: Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study). J Allergy Clin Immunol 2002;109:251–256.

Prof. A.B. Kay, MD PhD Room 374, Sir Alexander Fleming Building, Leukocyte Biology Section Imperial College, South Kensington London, SW7 2AZ (UK) Tel. ⫹44 20 7594 3174, Fax ⫹44 20 7594 1478, E-Mail [email protected]

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The Skin Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 76–86

Allergic Manifestations of Skin Diseases – Atopic Dermatitis Kristine Breuer a,b, Thomas Werfel b, Alexander Kapp b a

Nordseeklinik Norderney, Norderney, bDepartment of Dermatology and Allergology, Hannover Medical University, Hannover, Germany

Abstract Atopic dermatitis (AD) is a chronic inflammatory skin disease which often becomes manifest in early infancy and is characterized by itchy eczematous lesions with characteristic localization. The cellular infiltrate of allergic eczematous skin diseases (i.e. AD, allergic contact dermatitis) is mainly composed of mononuclear cells. Whereas allergic contact dermatitis is always triggered by allergen-specific T cells, a number of allergic and nonallergic trigger factors appear to be relevant in AD. This article discusses data coming from immunological studies focusing on T-cell responses in AD. The concept of a switch from a T helper type 1 (Th1) to a Th2 cytokine profile in lesional skin of AD is well accepted. Besides CD4
T lymphocytes, CD8
cells are likely to play an important role in the pathogenesis of AD. Recent studies point to the induction of apoptosis in keratinocytes by interferon- derived from skin-homing T cells as a further important mechanism for the induction and maintenance of the eczema. Recent clinical studies have confirmed the major role of food allergy and infectious microorganisms as trigger factors of AD. New therapeutic strategies for AD include topical calcineurin inhibitors which were introduced as a new therapeutic principle at the beginning of this decade. Copyright © 2006 S. Karger AG, Basel

Atopic Dermatitis – A Chronic Inflammatory Skin Disease

Eczematous skin diseases are extremely common with a lifetime prevalence of more than 15% in Western industrialized countries. Atopic dermatitis (AD) may begin in early infancy and is then not as easily diagnosed as the flexural eczema or atopic hand eczema of later childhood. In a recent study, we confirmed in a large cross-sectional study performed in Hannover that the incidence of AD in school entrants is associated with individual lifestyle factors, but not with local environmental factors [1]. The frequency of AD was

significantly increased with more privileged socioeconomic status. Independent factors that were associated with a higher frequency of AD were German nationality (12.4% AD compared with 2.1% in non-German), higher paternal socioeconomic status (i.e. father’s profession), higher daily duration of the fathers’ professional work and the lack of paternal shift work. The cellular infiltrate of allergic eczematous skin diseases (i.e. AD, allergic contact dermatitis) is mainly composed of mononuclear cells. Whereas allergic contact dermatitis is always triggered by allergen-specific T cells, a number of allergic and nonallergic trigger factors appear to be relevant in AD [2]. Allergen-specific T cells and specific immunoglobulin E (IgE) are involved in the clinical responses to food and inhalant allergens in the extrinsic variant of AD. The number of CD4
cells is increased and CD8
suppressor/cytotoxic lymphocytes are decreased in peripheral blood of patients with AD [3]. However, in addition to the CD4
subset, the CD8
CLA
memory/effector T cells are capable of responding to superantigenic stimulation and play an important role in the pathogenesis of AD [4]. Psychological stress which is a possible trigger factor of AD has recently been shown to lead to immunological changes in AD patients compared to healthy controls. We detected a higher increase of CD8
T lymphocytes on mental stress, including the subpopulations of CD8
/CD11b
T lymphocytes and eosinophils in patients with AD compared with healthy volunteers [5]. Interestingly, CLA
and interleukin (IL)-5expressing T cells were also increased upon mental stress. Allergen-specific T cells isolated from the blood of patients with AD show a T helper type 2 (Th2)-like cytokine profile. Akdis et al. [6] could show that the Th1 compartment of activated memory/effector T cells in the peripheral blood selectively undergoes activation-induced cell death, skewing the immune response toward surviving Th2 cells. This mechanism was confined to atopic individuals, whereas nonatopic patients such as psoriasis, intrinsic-type asthma, contact dermatitis, intrinsic type of AD, bee-venom-allergic patients and healthy controls showed no evidence for enhanced T-cell apoptosis in vivo. Studies focusing on intralesional cytokine expression showed that acute patch test lesions and spontaneous eczematous lesions are dominated by a Th2like cytokine milieu, whereas Th1 cytokines are predominantly found in chronic patch test lesions [3]. Th1 cytokines derived from activated T cells present in inflamed skin of AD are likely to be involved into the induction of keratinocyte (KC) apoptosis. Interferon- secreted by activated T cells was shown to upregulate Fas receptor on KCs. Subsequent KC apoptosis could be induced by antiFasR monoclonal antibodies, soluble Fas ligand, supernatants from activated T cells or direct contact between T cells and KCs [7]. This mechanism may play an important role in the exacerbation and maintenance of eczematous lesions in AD.

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The intrinsic variant of AD is characterized by the typical clinical features of AD and the lack of specific IgE [8]. Akdis et al. [9] could show that skin T cells derived from intrinsic AD patients expressed lower IL-5 and IL-13 levels compared with extrinsic AD patients. In addition, B-cell activation with high CD23 expression was observed in the peripheral blood of extrinsic AD, but not intrinsic AD patients. These data suggest a lack of IL-13-induced B-cell activation and consequent IgE production in intrinsic AD. Allergen-specific T cells may be involved in the pathophysiology of this variant but experimental data are still lacking. Histamine is a well-known inflammatory mediator, in particular in immediate-type hypersensitivity reactions. In AD, histamine is released into inflamed skin by activated mast cells, basophils and also by mononuclear cells. Histamine affects the maturation of immune system cells and alters their activation, polarization, chemotaxis and effector functions. Histamine also regulates antigen-specific Th1 and Th2 cells, as well as related antibody isotype responses. Histamine binds to four different G protein-coupled receptors that transduce signals to cells through distinct pathways. The expression of these receptors on different cells and cell subsets is regulated, and apparently, the diverse effects of histamine on immune regulation are due to differential expression of four histamine receptors and their distinct intracellular signals [10]. As a possible link between histamine and cell-mediated reactions, the expression and function of histamine receptors on dendritic cells were investigated by our group. Monocyte-derived dendritic cells were shown to express histamine H1-, H2-, H3- and H4-receptors and respond to histamine stimulation with immunological and chemotactic effects via at least the H2R and H4R. Therefore, histamine might represent a link between immediate-type hypersensitivity reactions and cellular inflammation in allergic disease, for example in AD [11].

Food Allergy in AD

Food allergens can elicit systemic immunological responses and subsequently cutaneous reactions in AD. Many patients with AD (or their parents) suspect certain foods to trigger skin lesions. In general, oral food challenges represent the ‘gold standard’ for the diagnosis of food allergy. Immediate reactions can quite easily be related to the suspected food in most cases but the cause of late eczematous reactions are difficult to identify. This is probably the reason why food allergens are not generally accepted as provocation factors for late eczematous reactions in children with AD, and this topic is poorly discussed in the literature. Therefore, we have investigated the role of cow’s milk,

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hen’s egg, wheat and soy as a provocation factor of eczema in children with AD [12]. The results of 106 double-blind placebo-controlled food challenges in 64 children with AD aged 1–10 years were analyzed retrospectively. The severity of AD was determined by means of the Scoring Atopic Dermatitis Index (SCORAD). Total IgE and specific IgE to food were determined by CAP RAST. Forty-six percent of the double-blind placebo-controlled food challenges resulted in immediate and/or late-phase allergic symptoms upon ingestion of foodstuff. A worsening of AD occurred 24–48 h upon oral challenge with a significant difference in SCORAD before and after challenge and was seen in 57% of all positive oral provocations, with 12% isolated late-type reactions and 45% combined immediate/late-type reactions. Sensitivity and specificity of food-specific IgE were 75.5 and 63.2%, respectively, whereas the atopy patch test showed worse results. Our results confirm the important role of food allergens as trigger factors of AD. Due to the poor reliability of the history and in vitro tests, the double-blind placebo-controlled food challenge is still to be regarded as the gold standard for the diagnosis of AD. Since eczematous lesions are probably triggered by T lymphocytes, new diagnostic approaches may come from the characterization of allergen-specific T-cell parameters. In earlier studies we had found significant differences in the proliferative response of blood lymphocytes between patients who reacted to milk with worsening of AD and control groups and were able to generate casein-specific T-cell clones from the blood of these patients. No correlation between specific lymphocyte proliferation and specific IgE was found [13]. Interestingly, this indicates that IgE-independent mechanisms may be involved in the eczematous reaction to food in some patients stressing the potential pathophysiological role of allergen-specific T lymphocytes in AD. Respiratory atopic diseases are often associated with pollen-related food allergy. The clinical significance of pollen-related food antigens was also evaluated in AD. We identified a subgroup of patients suffering from AD who reacted exclusively, with worsening of eczema on oral provocation with pollenassociated foods. The clinical response was associated with an allergen-specific T-lymphocyte response which could be detected in the blood and in lesional skin upon oral provocation [14]. Since pollen allergy increases later in life this observation may be important for many adolescent and adult patients. The role of birch-pollen-related food for the induction of allergic symptoms in children with AD has largely been unclear. Recently, we could show that a subgroup of children with a sensitization to birch pollen react to birch-pollen-related food with a worsening of their eczema [15]. Twelve children (median age 5 years) with AD who were sensitized to birch pollen underwent an oral challenge with those birch-pollen-related foods which were reported to induce no immediate symptoms, but were ingested on a regular basis. Seven of twelve children

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showed immediate and/or late eczematous reactions upon ingestion of birchpollen-related foodstuff. Four children showed a worsening of eczema after 24 h following oral challenge with a significant difference in SCORAD before and after challenge. There were no differences in terms of total IgE or birch-pollenspecific IgE between children with a late eczematous response and nonreacting children. These results show that challenge tests should be performed in those children who suffer from severe AD and who are highly sensitized to birch pollen allergens even in the absence of a history suggestive of food allergy.

Inhalant Allergens and AD

During the last few years it has become clear that protein allergens can also trigger eczematous skin responses upon external skin contact, or possibly upon inhalation, in AD. An important observation pointing to the significance of inhalant allergens in AD was that positive eczematous patch test reactions to house dust mite, pollen or animal dander are observable in sensitized patients. The results of positive patch tests are associated with circulating IgE directed to the corresponding inhalant allergen in the majority of patients [16]. Proliferative responses can be elicited in blood lymphocytes with inhalant allergens, including house dust mite and pollen, which indicates the presence of specific T-cell-mediated hypersensitivity in AD. The clinical relevance of these findings was confirmed by the observation that proliferative responses to house dust mite, grass pollen or cat antigens were significantly higher in patients with positive patch test reactions to the corresponding allergens than in nonreactive patients with AD [17]. This observation points to an immunological mechanism which targets allergen-specific T cells to the skin in AD.

The Importance of Staphylococcus aureus as a Trigger Factor of AD

In many patients with AD, the disease is further complicated by the concurrent skin colonization with Staphylococcus aureus. Many, but not all, S. aureus strains carry the capacity to produce exotoxins with superantigenic properties able to activate T cells in an unspecific manner [18]. In addition to their function as superantigens, the staphylococcal exotoxins may function as ‘conventional’ allergens. We found an inter-relationship between a sensitization to staphylococcal enterotoxin B (SEB) and the severity of AD in adult patients. Patients, who had specific IgE to SEB had a significantly higher SCORAD than patients who had not [19]. This could be shown not only for the severity of

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eczema, but also for the in vitro parameters serum eosinophil cationic protein and urine eosinophil protein X. Furthermore, staphylococcal enterotoxins exert proinflammatory effects also on eosinophils. The enterotoxins SEA, SEB, SEC and toxic shock syndrome toxin 1 significantly inhibited eosinophil apoptosis in a manner comparable to that of higher concentrations of IL-3. Moreover, the expression of CD11b, CD45, CD54 and CD69 was increased by staphylococcal enterotoxins [20]. In order to prove the clinical relevance of S. aureus as trigger factor for the eczema, we treated 10 adult patients with AD who were colonized with S. aureus, but showed no signs of bacterial superinfection. In addition, colonized contact persons, i.e. partners, of these patients received a topical antimicrobial treatment. S. aureus was eradicated in 9 of 10 patients. The skin condition was significantly improved following this treatment. Specifically patients with severe AD had a benefit which points to the clinical relevance of S. aureus colonization for the pathogenesis of eczema. However, the therapeutic effect was also seen in patients who were colonized with staphylococci producing no enterotoxins, which points to the fact that pathogenic factors other than enterotoxins may be involved in the pathogenesis of AD [21]. Since modification of host immune responses is apparently not limited to S. aureus enterotoxins, we investigated whether S. aureus -toxin is also involved in the pathogenesis of AD [22]. -Toxin is known to be a potent cytolysin. Twenty-two of sixty-four subjects who did not suffer from cutaneous infections harbored -toxin-producing strains and had a significantly higher SCORAD than patients who did not carry this kind of strain. By immunohistochemistry of skin sections obtained from lesional skin of these patients, toxin reactivity was seen in the epidermis and the upper dermis. Since CD4
T cells are the most abundant cells present in lesional skin of AD, we investigated the effects of -toxin on peripheral blood mononuclear cells (PBMC) and isolated T cells: incubation of PBMC with 1–10 g/ml -toxin for 30 min and longer resulted in increasing numbers of necrotic cells. Up to 25% of these cells were in early stages of apoptosis during an incubation period of 0.5–24 h, while lower concentrations did not induce apoptosis. Concentrations of 10 pg/ml–100 ng/ml induced proliferation of isolated CD4
T cells. Proliferation of PBMC-induced by 10–100 ng/ml -toxin was comparable to that induced by 100 ng/ml SEB. Stimulation of PBMC and isolated CD4
T cells with sublytic concentrations of -toxin resulted in an upregulation of interferon- both on the protein and the mRNA level. In contrast, incubation of PBMC with -toxin did not lead to significant secretion of IL-4 as measured in the cell culture supernatants. Furthermore, as shown in the electrophoretic mobility and shift assay, -toxin induced DNA binding of the Th1-associated transcription factor T-bet. These data suggest that the staphylococcal exotoxin

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-toxin, well known as a pore-forming toxin and cytolysin, represents a further virulence factor of S. aureus in being able to activate T cells. The induction of interferon- by sublytic concentrations of -toxin indicates that this toxin might play a critical role in the chronification of eczema. Whether this activation is the result of membrane damage caused by formation of pores and subsequent ion flux or a direct effect of -toxin on the transcriptional processes is not yet clear. Since enhanced staphylococcal colonization is a prominent feature of AD as compared to other inflammatory skin diseases, we investigated potential defects of acquired and innate immune responses to S. aureus. To detect a potential defect of innate immunity we have genotyped serum DNA derived from 78 patients with AD for Toll-like receptor (TLR) single nucleotide polymorphisms. In our control population, the heterozygous TLR-2 R753Q single nucleotide polymorphism had a prevalence of 2.5%. In contrast, 9 of 78 AD patients (11.5%) were heterozygous carriers of the mutant R753Q allele. Heterozygous patients with the TLR-2 R753Q allele variant exhibited a unique phenotype characterized by severe to moderate AD (median SCORAD score 55.8 pts ranging from 30.3–90.5 pts). None of them had a SCORAD score 30 pts, whereas the median SCORAD score of the nonpolymorphic AD group was 44.8 pts (14.9–89 pts; min–max values). Furthermore, patients with the TLR-2 R753Q missense mutation showed highest levels of total serum IgE, superantigen as well as Der p-specific IgE titers. Thus we provided evidence for a disease-promoting effect of the TLR-2 R753Q single nucleotide polymorphism [23].

New Treatment Options for AD

The course of AD is influenced by multiple triggers which are relevant for the treatment. The mainstays of therapy include regular use of emollients coupled with antimicrobial substances, corticosteroids and immune modulators as required. Ultraviolet radiation and immunosuppressive regimens represent further options for the treatment of severe exacerbations and may lead to long-term improvement. Data from experimental studies provide insight into possible future treatment methods [24]. Topical calcineurin inhibitors were introduced as a new therapeutic principle at the beginning of this decade [25, 26]. Tacrolimus and pimecrolimus interact with macrophilin-12 (FKBP12), a cyclophilin-like cytoplasmic protein, and this complex inhibits the ability of calcineurin to dephosphorylate the transcription factor NF-AT [27]. Since only dephosphorylated NF-AT is able to

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translocate into the nucleus, the transcription of various proinflammatory cytokines and other mediators of the allergic inflammatory reaction is inhibited. Pimecrolimus and tacrolimus are able to inhibit the early activation and proliferation of human T cells by downregulation of IL-2 at nanomolecular concentrations. Furthermore, the production of Th1- and Th2-type cytokines are downregulated. In a previous study, we compared early intervention with pimecrolimus cream to a vehicle preparation. The purpose of this investigation was to assess whether early treatment in infants of AD signs/symptoms with pimecrolimus may influence long-term outcome by preventing disease flares. In this one-year, double-blind controlled study, 251 infants aged 3–23 months with AD were randomized 4:1 to a pimecrolimus-based regimen (n  204) or a conventional treatment regimen (n  47). Both groups used emollients for dry skin. Early AD signs and symptoms were treated either with pimecrolimus cream to prevent flares or, in the control group, with vehicle. Vehicle was used to maintain blinding conditions. In the event of flares, moderately potent corticosteroid was permitted in both groups. The primary efficacy endpoint was the incidence of flares at 6 months. Pimecrolimus significantly reduced the incidence of flares compared with control treatment with 67.6 versus 30.4% of patients completing 6 months with no flare and 56.9 versus 28.3% completing 12 months with no flare. Overall corticosteroid use was substantially lower in the pimecrolimus group: 63.7 versus 34.8% of patients did not use corticosteroids at all during the study. Pimecrolimus was also more effective than control treatment in the longterm control of pruritus and the signs of AD. There were no clinically significant differences in the incidence of adverse events between the two treatment groups. Treatment of early signs and symptoms with pimecrolimus significantly modified the disease course in infants by reducing the incidence of flares and improving overall control of AD. Pimecrolimus was safe and well tolerated [28]. Recently, we also compared the influence of pimecrolimus cream 1% on different standard eczema scores in infants with AD and analyzed the impact of treatment on the individual morphological key signs of eczema. Pimecrolimus cream 1% (n  129) or double-blind vehicle control (n  66) were administered for 4 weeks. The Eczema Area and Severity Index (EASI), Investigators’ Global Assessment (IGA) and SCORAD were determined and were correlated with each other. Following treatment with pimecrolimus, the EASI, IGA and SCORAD were significantly reduced on day 29 as compared with the vehicle group (p  0.001, p  0.001, p  0.002, respectively). There was a close correlation between EASI, IGA and SCORAD. The single parameters of the EASI were already significantly decreased by day 4 in the pimecrolimus group as compared to vehicle (each p  0.001). Thus treatment with pimecrolimus 1% cream led to a rapid improvement of all morphological signs of eczema. The close correlation of different scores was shown for the first time [29].

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Eosinophils may play an important role in the pathogenesis of AD. IL-5 is essential for eosinophil growth, differentiation and migration. A monoclonal antibody to human IL-5 (mepolizumab) was developed for atopic diseases. We took part in a multicenter study which was designed to study the effect of mepolizumab in AD [30]. Two single doses of 750 mg mepolizumab given one week apart were administered to patients with moderate to severe AD using a randomized, placebo-controlled parallel group design. The primary endpoint of ‘success’ to treatment was defined as the percentage of patients with at least ‘marked improvement’ after 2 weeks as assessed by the Physician’s Global Assessment of Improvement (PGA). Furthermore SCORAD, pruritus scoring, number of blood eosinophils and serum thymus and activation-regulated chemokine values served as secondary endpoints. Fluticasone propionate cream 0.05%, once daily, could be used as rescue medication from day 16 if no improvement was recorded. Eighteen patients received mepolizumab and 22 received placebo treatment. Peripheral blood eosinophil numbers were significantly reduced in the treatment group compared to placebo (p  0.05). No clinical success was reached by PGA assessment (p  0.115), SCORAD (p  0.293), pruritus scoring and thymus and activation regulation chemokine values in the mepolizumab-treated group compared to placebo. However, modest improvement (50% improvement) assessed by PGA was scored significantly more in the mepolizumab-treated group compared to placebo (p  0.05). Since two single doses of 750 mg mepolizumab did not result in profound clinical success in patients with AD, prolonged application of anti-IL-5 should be studied in future trials.

Conclusions

Cutaneous inflammation in AD is the result of a complex interaction between various cell types, among them CD4
and CD8
T cells, dendritic cells and KCs. Although recent studies have brought light to the immunopathogenesis of AD, there is still no causative treatment for this disease. The factors that influence the development of different phenotypes of AD are poorly known but susceptibility genes, environmental and immunological factors are likely to be involved. Future studies have to address these questions.

References 1

Werner S, Buser K, Kapp A, Werfel T: The incidence of atopic dermatitis in school entrants is associated with individual life-style factors but not with local environmental factors in Hannover, Germany. Br J Dermatol 2002;147:95–104.

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Werfel T, Kapp A: Atopic dermatitis and allergic contact dermatitis; in Holgate ST, Church MK, Liechtenstein L (eds): Allergy, ed 2. Mosby, 2000. Leung DYM, Boguniewicz M, Howell MD, Nomura I, Hamid QA: New insights into atopic dermatitis. J Clin Invest 2004;113:651–657. Akdis M, Simon HU, Weigl L, Kreyden O, Blaser K, Akdis CA: Skin homing (cutaneous lymphocyte-associated antigen-positive) CD8
T cells respond to superantigen and contribute to eosinophilia and IgE production in atopic dermatitis. J Immunol 1999;163:466–475. Schmid-Ott G, Jaeger B, Adamek C, Koch H, Lamprecht F, Kapp A, et al: Levels of circulating CD8(
) T lymphocytes, natural killer cells, and eosinophils increase upon acute psychosocial stress in patients with atopic dermatitis. J Allergy Clin Immunol 2001;107:171–177. Akdis M, Trautmann A, Klunker S, Daigle I, Kucuksezer UC, Deglmann W, et al: T helper (Th) 2 predominance in atopic diseases is due to preferential apoptosis of circulating memory/effector Th1 cells. FASEB J 2003;17:1026–1035. Trautmann A, Akdis M, Kleemann D, Altznauer F, Simon HU, Graeve T, et al: T cell-mediated Fasinduced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis. J Clin Invest 2000;106:25–35. Heratizadeh A, Breuer K, Kapp A, Werfel T: Intrinsic atopic dermatitis – An update; submitted. Akdis CA, Akdis M, Simon D, Dibbert B, Weber M, Gratzl S, et al: T cells and T cell-derived cytokines as pathogenic factors in the non-allergic form of atopic dermatitis. J Invest Dermatol 1999;113:628–634. Akdis CA, Blaser K: Histamine in the immune regulation of allergic inflammation. J Allergy Clin Immunol 2003;112:15–22. Gutzmer R, Langer K, Lisewski M, Mommert S, Rieckborn D, Kapp A, et al: Expression and function of histamine receptors 1 and 2 on human monocyte-derived dendritic cells. J Allergy Clin Immunol 2002;109:524–531. Breuer K, Heratizadeh A, Wulf A, Baumann U, Constien A, Tetau D, et al: Late eczematous reactions to food in children with atopic dermatitis. Clin Exp Allergy 2004;34:817–824. Werfel T, Breuer K: Role of food allergy in atopic dermatitis. Curr Opin Allergy Clin Immunol 2004;4:379–385. Reekers R, Schmidt P, Kapp A, Werfel T: Evidence of a lymphocyte response to birch pollen related food antigens in atopic dermatitis. J Allergy Clin Immunol 1999;104:466–472. Breuer K, Wulf A, Constien A, Tetau D, Kapp A, Werfel T: Birch pollen-related food as a provocation factor of allergic symptoms in children with atopic eczema/dermatitis syndrome. Allergy 2004;59:988–994. Darsow U, Laifaoui J, Kerschenlohr K, Wollenberg A, Przybilla B, Wuthrich B, et al: The prevalence of positive reactions in the atopy patch test with aeroallergens and food allergens in subjects with atopic eczema: A European multicenter study. Allergy 2004;59:1318–1325. Wistokat-Wuelfing A, Schmidt P, Darsow U, Ring J, Kapp A, Werfel T: Atopy patch test reactions are associated with T lymphocyte-mediated allergen-specific immune responses in atopic dermatitis. Clin Exp Allergy 1999;29:513–521. Bunikowski R, Mielke M, Skarabis H, Worm M, Anagnostopoulos I, Kolde G, et al: Evidence for a disease-promoting effect of Staphylococcus aureus-derived exotoxins in atopic dermatitis. J Allergy Clin Immunol 2000;105:814–819. Breuer K, Wittmann M, Bosche B, Kapp A, Werfel T: Severe atopic dermatitis is associated with sensitization to staphylococcal enterotoxin B (SEB). Allergy 2000;55:551–555. Wedi B, Wieczorek D, Stunkel T, Breuer K, Kapp A: Staphylococcal exotoxins exert proinflammatory effects through inhibition of eosinophil apoptosis, increased surface antigen expression (CD11b, CD45, CD54, and CD69), and enhanced cytokine-activated oxidative burst, thereby triggering allergic inflammatory reactions. J Allergy Clin Immunol 2002;109:477–484. Breuer K, Häussler S, Kapp A, Werfel T: Staphylococcus aureus: Colonizing features and influence of an antibacterial treatment in adults with atopic dermatitis. Br J Dermatol 2002;147: 55–61. Breuer K, Kempe K, Kohlrautz V, Kapp A, Mai U, Dittrich-Breiholz O, Kracht M, Werfel T: Alpha-toxin induces IFN-gamma in skin and blood derived T cells of patients with atopic dermatitis. J Allergy Clin Immunol 2004;113:S96.

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Ahmad-Nejad P, Mrabet-Dahbi S, Breuer K, Klotz M, Werfel T, Herz U, et al: The toll-like receptor 2 R753Q polymorphism defines a subgroup of patients with atopic dermatitis having severe phenotype. J Allergy Clin Immunol 2004;113:565–567. Heratizadeh A, Breuer K, Kapp A, Werfel T: Systemic therapy of atopic dermatitis. Hautarzt 2003; 54:937–945. Breuer K, Werfel T, Kapp A: Safety and efficacy of topical immunomodulators in the treatment of childhood eczema. Am J Clin Dermatol 2005;6:65–77. Breuer K, Werfel T, Kapp A: Pimecrolimus for therapy of atopic dermatitis. Therapy 2005;2: 37–56. Liu J, Farmer JD, Lane WS, Friedman J, Weissman I, Schreiber SL: Calcineurin is a common target of cyclophiline-cyclosporin A and FKBP-FK506 complexes. Cell 1991;66:807–815. Kapp A, Papp K, Bingham A, Fölster-Holst R, Ortonne JP, Potter P, et al: Long-term management of atopic dermatitis in infants with topical pimecrolimus, a nonsteroid anti-inflammatory drug. J Allergy Clin Immunol 2002;110:277–284. Breuer K, Braeutigam M, Kapp A, Werfel T: Influence of pimecrolimus cream 1% on different morphological signs of eczema in infants with atopic dermatitis. Dermatology 2004;209:314–320. Oldhoff M, Darsow U, Werfel T, Katzer K, Wulf A, Laifaoui J, Hijnen D, Plötz S, Knol E, Kapp A, Bruijnzeel-Koomen C, Ring J, de Bruin-Weller M: Anti-IL-5 recombinant humanized monoclonal antibody (Mepolizumab) for the treatment of Atopic dermatitis. Allergy 2005;60:693–696.

Kristine Breuer, MD Nordseeklinik Norderney Bülowallee 6 DE–26548 Norderney (Germany) Tel.
49 4932 881500, Fax
49 4932 881702, E-Mail [email protected]

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The Skin Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 87–97

Skin-Homing T Cells in Cutaneous Allergic Inflammation Luis F. Santamaría-Babí Almirall Prodesfarma Research Center, Barcelona, Spain

Abstract The involvement of circulating cutaneous lymphocyte-associated (CLA)⫹ T cells in skin allergy was initially described in atopic and contact dermatitis in a series of experiments performed in Davos, at the Swiss Institute of Allergy and Asthma Research. Since then, numerous studies have confirmed and extended those initial findings. Both basic and clinical research data obtained on this subset of memory T cells support a relevant role of CLA⫹ T cells in the regional cutaneous immune system. The spectrum of cutaneous diseases where CLA⫹ T cells have been shown to play a relevant role has expanded far beyond the field of allergy, highlighting the relevance of this memory T-cell subpopulation in cutaneous diseases. The goal of this chapter is to review known features of those cells in atopic dermatitis, contact dermatitis and nonimmediate cutaneous allergic reactions to drugs. Copyright © 2006 S. Karger AG, Basel

Effector memory T cells constitute a subset of antigen-experienced T cells that express surface receptors involved in tissue tropism and produce cytokines with immediate effector functions. Circulating T cells with skin-seeking capacity are effector memory T cells that can be identified by the expression of the cutaneous lymphocyte-associated (CLA) antigen on their surface. CLA is expressed by more than 90% of infiltrating T cells present in inflamed skin, but by less than 20% of T cells in other anatomical regions like joints, lungs or the gastrointestinal tract under inflammatory conditions [1, 2]. In addition, CLA⫹ T cells are present in 15% of peripheral blood T cells [1]. The CLA antigen is a ligand for E-selectin, an adhesion molecule that is induced in endothelial cells under inflammatory conditions in response to interleukin (IL)-1 and tumor necrosis factor-␣ [3]. The molecular interaction between CLA and E-selectin mediates cell adhesion and was the initial information that allowed proposing the CLA antigen as a skin-homing receptor involved in the localization of circulating T cells to the skin [3]. The CLA antigen is a carbohydrate similar to the sialyl Lewis X antigen, which is expressed on effector memory CD45RO⫹

T cells as an epitope of a single cell-surface protein named PSGL-1 [4]. The induction of CLA antigen takes place during the naive (CD45RA⫹) to memory (CD45R0⫹) T-cell transition upon activation in lymph nodes that drain the skin. Glycosyltransferases are involved in the generation of sialyl Lewis X [4], fucosyltransferase VII (FucTVII) and FucTIV being the best-characterized glycosyltransferase enzymes involved in T-cell skin homing. FucTVII-deficient mice manifest diminished contact hypersensitivity capacity but not a reduced inflammation in other organs [5]. Interestingly, mice deficient for both FucTVII and FucTIV show a complete abrogation of contact hypersensitivity [6], suggesting that FucTVII and FucTIV participate in the synthesis of E-, L-, and P-selectin ligands. These ligands are relevant in lymphocyte recruitment to lymph nodes and the skin in the mouse. IL-12 may probably be the best-characterized mediator responsible for the induction of CLA in T cells. This cytokine induces the synthesis of FucTVII on T cells undergoing naive to memory transition, in contrast to IL-4 that has been shown to inhibit FucTVII expression. Activation of peripheral blood lymphocytes by the superantigen staphylococcal enterotoxin B has been shown to induce the generation of CLA⫹ T cells, a mechanism that depends on the production of IL-12 [7]. Interestingly, in a study where melanoma patients received recombinant human IL-12 s.c., a peripheral burst of CLA⫹ T cells was documented [8]. Altogether, this suggests that IL-12 may be involved in the induction of CLA in T cells. When we decided to study immunological features of circulating CLA⫹ T cells, we realized that a cell purification procedure from blood was required. It took time to develop such a method, but finally one was established by using three consecutive immunomagnetic separations [9]. Highly purified CLA⫹CD45R0⫹ T cells could then be purified from blood to perform both studies of in vitro transendothelial migration through human endothelium and T-cell activation. Since the number of purified memory CLA⫹ T cells from blood was usually low, we also generated a human T-cell line that constitutively expressed the CLA antigen and behaved similarly to nontransformed T cells. This cell line helped to characterize some basic mechanisms between CLA⫹ T cells and endothelium under static and nonstatic conditions [2]. The CLA/E-selectin adhesive interaction is part of the multistep process of lymphocyte migration to inflamed skin that, together with very late antigen (VLA)-4/vascular cell adhesion molecule (VCAM)-1 and lymphocyte functionassociated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1, facilitate human CLA⫹ T-cell transendothelial migration [9]. To define adhesion molecules and chemokines involved in CLA⫹ T-cell transendothelial migration we used an in vitro assay previously used to study eosinophil transendothelial migration. It consisted of a matrix of collagen where human endothelial cells were grown. Once those cells reached a monolayer, they were activated to

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express adhesion molecules such as E-selectin, VCAM-1, ICAM-1 and chemokines. When purified CLA⫹ memory T cells were added to those activated endothelial cell layers, it was possible to quantify transmigrated T cells. Altogether, this allowed us to establish an in vitro assay to measure CLA⫹ T-cell migration [9]. Using this assay, we identified a different usage of cell adhesion molecules in the CLA⫹- or CLA⫺-dependent T-cell migration through activated endothelial cell monolayers. VLA-4 was preferentially required in the transendothelial migration of CLA⫹ T cells, whereas the LFA-1/ICAM-1 interaction was crucial for transendothelial migration of memory T cells, either CLA⫹ or CLA⫺ [9]. Several in vivo studies in different species have highlighted the relevance of VLA-4-dependent migration of T cells to inflamed skin in different species [10]. VLA-4 appears to be a relevant adhesion pathway for migration of effector memory T cells to inflamed nonlymphoid tissues. On the other hand, the role of LFA-1 in T-cell mediated extravasation in cutaneous inflammation has been confirmed by the clinical efficacy of anti-LFA-1 (Efalizumab/Raptiva®) in the treatment of psoriasis, a T-cell-mediated skin inflammation. Chemokines are involved in leukocyte migration from blood to tissues by attracting cells and inducing activation of integrins among other activities. Up to now, several chemokine receptors present on the surface of CLA⫹ T cells has been described; however, most of them are shared with other leukocyte subsets. The chemokine receptor repertoire expressed on circulating CLA⫹ T cells include: CC4, CCR6, CCR10, CXCR1, CXCR2 and CXCR3 [2]. From all those receptors, CCR10 would be the most specific since it is preferentially expressed by CD4⫹CLA⫹ T cells, and their ligand, CCL27/cutaneous T-cell-attracting chemokine (CTACK) is only produced by basal keratinocytes [11]. The capacity of cutaneous migration of human circulating CLA⫹ T cells has been studied in severe combined immunodeficient mice grafted with human skin [12]. In those animal models, injected human T cells can home into grafted human skin and their migration can be reduced by an anti-E-selectin antibody and an E-selectin antagonist [12]. Besides all this information obtained from the basic mechanism of skinhoming physiology, other studies have focused on this memory T-cell subset in cutaneous allergic inflammatory diseases, such as atopic dermatitis, contact dermatitis and drug-induced allergic reactions. Recent information on the role of CLA⫹ T cells in these diseases is highlighted below.

Atopic Dermatitis

Circulating allergen-specific CLA⫹ T cells are thought to be involved in the initiation of atopic dermatitis lesions [13]. Patch testing with house dust

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mite extract in atopic dermatitis patients triggers a steady increase in infiltrating CLA⫹ T cells that rises parallel to the upregulation of expression of E-selectin, VCAM-1 and ICAM-1 in the lesions [14]. Chemokines attracting CLA⫹ T cells are also present in atopic dermatitis lesions. CCL17/thymus and activation-regulated chemokine (TARC) and CCL27/CTACK are produced by keratinocytes and CCL22/macrophage-derived chemokine (MDC) by antigen-presenting cells [15]. Interestingly, serum levels of CCL17/TARC, CCL22/MDC and CCL27/CTACK have been shown to relate to clinical severity of atopic dermatitis, indicating that chemokines are involved in the attraction of CLA⫹ T cells to the lesions. Also CRTH2, a new type of prostaglandin D2 receptor, is present in circulating CLA⫹ T cells in atopic dermatitis. Not much is known about the cytokine profile and allergen specificity of circulating CLA⫹ T cells expressing different chemokine receptors in atopic dermatitis. This is probably important since allergen specificity is present in a very low percentage of memory T cells and the expression of chemokine receptors has been studied in large populations. Therefore, specific information has not yet been obtained in order to clarify the relevance of each receptor in both effector T cells and bystander cells. Circulating CLA⫹ T cells from allergic atopic dermatitis patients preferentially respond to Dermatophagoides pteronyssinus. When autologous antigenpresenting cells from atopic dermatitis patients were incubated with D. pteronyssinus extract together with purified memory cells CLA⫹ or CLA⫺, higher allergen-induced proliferation was present in the CLA⫹ T-cell subset [16]. Such preferential proliferation of CLA⫹ T cells to allergen was not found either in healthy controls or asthmatic patients allergic to the same allergen. This was a relevant finding because it related skin-homing and allergen response with CLA⫹ T cells in atopic dermatitis. Circulating CLA⫹ T cells from atopic dermatitis patients spontaneously produce IL-4 [16] and IL-13 [17]. In addition, the presence of the activation markers HLA-DR [16] and CD25 [17] on the surface of those cells indicates that they have been activated in the skin and re-circulate. CLA⫹ T cells that spontaneously produce cytokines might be activated memory effector T cells present in the circulation. For this reason, studying the phenotype of circulating CLA⫹ T cells during atopic dermatitis might provide information about the immunoinflammatory processes that take place in the skin. In addition, in atopic dermatitis the frequency of circulating CLA⫹HLA-DR⫹ T cells parallels clinical symptoms, suggesting that memory T cells are released into the circulation during the evolution of the lesion [2]. Cutaneous infection by Staphylococcus aureus is a common event in atopic dermatitis lesions that exacerbates the disease [13]. Interestingly, combined treatment with antibiotics and corticosteroids reduces disease severity [18].

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Superantigens produced by S. aureus can activate resident T cells and have an impact on ongoing atopic dermatitis. In fact, S. aureus colonies can be found between keratinocyte layers of the skin [18]. Furthermore, superantigens are present in the dermis in close contact with inflammatory cells [18]. Due to the association between S. aureus infection and exacerbation of atopic dermatitis, we investigated whether there was a T-cell receptor V␤-skewing for superantigens produced by S. aureus in circulating T cells. We only found such T-cell receptor V␤-skewing when analyzing circulating CLA⫹ T cells. Concretely, patients with active atopic dermatitis presented a higher percentage of cells positive for the T-cell receptor V␤2 and V␤5.1 segments in the CLA⫹ but not in the CLA⫺ subset. Moreover we found an increased percentage of HLA-DR expression on CLA⫹V␤5.1 T cells in patients with active atopic dermatitis, but not in those patients whose eczema was not active [19]. Those results were confirmed by other groups and were also associated with disease severity [20]. In atopic dermatitis, most of the studies focusing in circulating CLA⫹ T cells have involved patients that have not been classified according to their acute or chronic stage. In addition, T-cell cytokines present in acute and chronic cutaneous lesions can be associated to Th2 and Th1 cytokine profiles, respectively [13]. Based on those results we wondered whether circulating CLA⫹ T cells could manifest a different phenotype in patients in different phases of disease. We have compared the phenotype of circulating CLA⫹ T cells in acute and chronic atopic dermatitis patients [21]. Circulating CLA⫹CD4⫹ cells in patients with acute atopic dermatitis produced significantly higher levels of IL-4, IL-13 and tumor necrosis factor-␣ than patients in the chronic stage, or controls [21]. In addition, chronic atopic dermatitis patients manifested significantly increased levels of circulating CD4⫹ HLA-DR⫹ cells and decreased production of IL-4 and IL-13 compared to acute patients. These results indicate that, when studying circulating CLA⫹ T cells, the stage of atopic dermatitis can be of relevance and that specific information about the chronic stage of atopic dermatitis can be obtained from those cells. During the atopic march, atopic dermatitis is considered to be an entry point for further development of asthma or rhinitis. The evolution from a cutaneous to a respiratory T-cell-mediated allergic inflammation for the same allergen may require a change in tissue tropism of allergen-experience memory T cells present in the blood. Interestingly, in atopic dermatitis and asthmatic patients allergic to house dust mite the subset of circulating memory T cells that recognize the allergen are different. In allergic asthmatic patients it is the CLA⫺CD45R0⫹CD3⫹ subset that recognizes house dust mites, suggesting that memory T cells with lung tropism use a homing receptor other than CLA [16]. In fact, conversely to cutaneous sites, in lung inflammation there is no enrichment of infiltrating CLA⫹ T cells, although after allergen provocation,

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circulating allergen-specific Th2 T cells are depleted from the circulation and are present in bronchial lumen [22]. Allergen-specific T lymphocytes are present in the blood of allergic patients. How this shift from skin to lung homing occurs in the peripheral Th2 allergen-specific memory T-cell pool is still unknown. For example, prostaglandin E2 is a mediator that is highly abundant in atopic dermatitis lesions and has been shown to suppress CCL27/CTACK production in human keratinocytes and superantigen-induced CLA expression on T cells [23]. It has been shown that CLA induction is independent of the T helper cytokine profile, and that homing receptor expression plasticity on T cells is subjected to dendritic-cell activation [24]. All this information suggests that tissue-homing specificity can be modified without affecting memory T-cell cytokine profile. Understanding this mechanism may be of use to understand how asthma develops in atopic dermatitis patients.

Allergic Contact Dermatitis

In allergic contact dermatitis (ACD), nickel patch testing also induces upregulation of the cell adhesion molecules responsible for CLA⫹ T-cell infiltration [14]. It has recently been published that contact dermatitis patients undergoing nickel oral provocation develop an acute flare of contact dermatitis paralleled by a reduction in circulating CLA⫹CD45R0⫹CD3⫹ cells, which suggests a sequestration of the circulating skin-homing pool in the skin. In addition, as in the case of atopic dermatitis, circulating CLA⫹ T cells from ACD patients preferentially respond to a triggering allergen [16]. CLA⫹ T-cell attracting chemokines such as CCL27/CTACK, CXCL10/IP-10, CCL17/TARC and CCL22/MDC have also been described in ACD lesions [25]. In addition, after patch testing in ACD an increase in CCL27 expression is associated with the infiltration of T cells. Interestingly, CLA⫹CD4⫹ memory T cells are preferentially attracted by CCL17/TARC and CCL22/MDC, whereas CLA⫹CD8⫹ T cells by CXCL10/IP-10. Peripheral blood- or skin-derived nickel-specific Tcell clones express the CLA antigen on their surface and produce interferon-␥ which is able to induce cytotoxicity in autologous keratinocytes pretreated with the same cytokine [26]. Those studies suggest that circulating allergen-specific CLA⫹ T cells home to the skin in ACD.

Nonimmediate Cutaneous Allergic Reactions to Drugs

The skin is the organ most frequently affected in nonimmediate cutaneous reactions to drugs. Since T cells were suspected to be implicated in such

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reactions, the involvement of CLA⫹ T cells was investigated. CLA⫹ T cells were originally described to be related to drug allergic reactions when it was found that patients manifested increased frequency of both circulating CLA⫹ T cells and CLA⫹ HLA-DR⫹ T cells in comparison to healthy controls [27]. Subsequent studies showed that patients’ re-exposure to the drug showed an increase in circulating CLA⫹HLA-DR⫹ T cells [27]. Interestingly, the percentage of circulating CLA⫹ T cells and CLA⫹HLA-DR⫹ T cells paralleled clinical signs [27]. Because similar features in terms of percentage and activation state of circulating CLA⫹ T cells have been found in atopic dermatitis and psoriasis, it appears that this could be a common property of those cells during skin inflammation in vivo. In toxic epidermal necrolysis induced by anticonvulsants it has been shown that within cutaneous lesions the presence of infiltrating CLA⫹ T cells is associated with increased expression of E-selectin and VCAM-1 in endothelium [28]. However, the question still remained open in order to fully involve CLA⫹ T cells in the pathology of cutaneous drug-induced allergic reactions: it was not known whether those cells preferentially recognized triggering allergens, as in the case of atopic and contact dermatitis. Recently, the memory T-cell response to betalactams in patients with nonimmediate reactions to drugs has been analyzed. Purified CLA⫹ and CLA⫺ memory T cells from blood were activated with autologous antigen-presenting cells and drugs. Data indicated a higher proliferative response to betalactams in the CLA⫹ T-memory subset [29]. In addition several other authors have also related CLA⫹ T cells with cutaneous drug allergy. Most of the drug-specific T-cell clones derived from lesions elicited by phenobarbital, lamotrigine or cotrimoxazole express the CLA antigen [30]. Thus, CLA⫹ T cells appear to participate in the induction of cutaneous lesions induced by drugs; however, the number of studies focused on this topic is still low and there are many interesting activities of those cells to be determined in such heterogeneous type of cutaneous allergic reactions.

Features of Circulating CLAⴙ T Cells in Cutaneous Allergic Inflammation

CLA⫹ T cells are probably the best-characterized subset of human memory T cells with a known tissular tropism in disease [2]. Their features in terms of allergen/antigen specificity, cytokine production, cell adhesion molecules and chemokines allow proposing them as early players in the initiation of atopic dermatitis, contact dermatitis or drug-induced cutaneous lesions. Based on the characteristics of circulating T cells it appears that, during cutaneous inflammation, some infiltrating CLA⫹ T cells may migrate back to circulation. The spontaneous production of T-cell cytokines, the expression of activation markers on

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Chemokines Superantigens Neuropeptides

T

T

T

T

T

HLA-DR

Antigens (Ni, DPT, drugs)

T T

Cellular interactions, cytokines

HLA-DR

T

T

T

T

Fig. 1. CLA⫹ T cells migrate to skin and re-circulate to blood during cutaneous inflammation. Circulating CLA⫹ T cells migrate to inflamed skin at the early stage and are involved in the elicitation of the lesion. Adhesion molecules and chemokines allow them to selectively traffic to cutaneous sites. Once into the skin, CLA⫹ T cells respond to cutaneous allergens and different stimuli present in the inflammatory milieu. Some skin-activated CLA⫹ T are present in the blood during the development of the cutaneous inflammation. Those cells spontaneously produce cytokines and express activation markers such as HLA-DR.

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those circulating cells, and the parallelism in their frequency and clinical symptoms support this hypothesis. Skin-activated memory effector T cells present in circulation during cutaneous inflammation can be used to explore cutaneous environmental signals to which those cells have been exposed, helping to understand immunoinflammatory processes in skin allergy (fig. 1). The information that the study of CLA⫹ T cells is providing in nonallergic diseases, such as psoriasis, vitiligo, herpes simplex, cutaneous T-cell lymphoma or alopecia areata, reinforces the relevance of this subset in different types of cutaneous T-cellmediated diseases [2]. In summary, circulating memory T cells constitute a heterogeneous subset of cells in terms of tissue tropism. As it has been shown during this last decade of research, studying circulating CLA⫹ T cells allows us to focus on a skin-homing population that is helping to understand cutaneous allergic disorders.

References 1

2 3 4 5

6

7

8

9

10 11

Picker LJ, Michie SA, Rott LS, Butcher EC: A unique phenotype of skin-associated lymphocytes in humans: Preferential expression of the HECA-452 epitope by benign and malignant T cells at cutaneous sites. Am J Pathol 1990;136:1053–1068. Santamaria-Babi LF: CLA(⫹) T cells in cutaneous diseases. Eur J Dermatol 2004;14:13–18. Picker LJ, Kishimoto TK, Smith CW, Warnock RA, Butcher EC: ELAM-1 is an adhesion molecule for skin-homing T cells. Nature 1991;349:796–799. Fuhlbrigge RC, Kieffer JD, Armerding D, Kupper TS: Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells. Nature 1997;389:978–981. Erdmann I, Scheidegger EP, Koch FK, Heinzerling L, Odermatt B, Burg G, Lowe JB, Kündig TM: Fucosyltransferase VII-deficient mice with defective E-, P-, and L-selectin ligands show impaired CD4⫹ and CD8⫹ T cell migration into the skin, but normal extravasation into visceral organs. J Immunol 2002;168:2139–2146. Smithson G, Rogers CE, Smith PL, Scheidegger EP, Petryniak B, Myers JT, Kim DS, Homeister JW, Lowe JB: Fuc-TVII is required for T helper 1 and T cytotoxic 1 lymphocyte selectin ligand expression and recruitment in inflammation, and together with Fuc-TIV regulates naive T cell trafficking to lymph nodes. J Exp Med 2001;194:601–614. Leung DY, Gately M, Trumble A, Ferguson-Darnell B, Schlievert PM, Picker LJ: Bacterial superantigens induce T cell expression of the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin 12 production. J Exp Med 1995;181: 747–753. Mortarini R, Borri A, Tragni G, Bersani I, Vegetti C, Bajetta E, Pilotti S, Cerundolo V, Anichini A: Peripheral burst of tumor-specific cytotoxic T lymphocytes and infiltration of metastatic lesions by memory CD8⫹ T cells in melanoma patients receiving interleukin 12. Cancer Res 2000;60: 3559–3568. Santamaria Babi LF, Moser R, Perez Soler MT, Picker LJ, Blaser K, Hauser C: Migration of skinhoming T cells across cytokine-activated human endothelial cell layers involves interaction of the cutaneous lymphocyte-associated antigen (CLA), the very late antigen-4 (VLA-4), and the lymphocyte function-associated antigen-1 (LFA-1). J Immunol 1995;154:1543–1550. Issekutz AC, Issekutz TB: The role of E-selectin, P-selectin, and very late activation antigen-4 in T lymphocyte migration to dermal inflammation. J Immunol 2002;168:1934–1939. Hudak S, Hagen M, Liu Y, Catron D, Oldham E, McEvoy LM, Bowman EP: Immune surveillance and effector functions of CCR10(⫹) skin homing T cells. J Immunol 2002;169:1189–1196.

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Biedermann T, Schwarzler C, Lametschwandtner G, Thoma G, Carballido-Perrig N, Kund J, de Vries JE, Rot A, Carballido JM: Targeting CLA/E-selectin interactions prevents CCR4-mediated recruitment of human Th2 memory cells to human skin in vivo. Eur J Immunol 2002;32: 3171–3180. Leung DY, Boguniewicz M, Howell MD, Nomura I, Hamid QA: New insights into atopic dermatitis. J Clin Invest 2004;113:651–657. Jung K, Linse F, Pals ST, Heller R, Moths C, Neumann C: Adhesion molecules in atopic dermatitis: Patch tests elicited by house dust mite. Contact Dermatitis 1997;37:163–172. Pastore S, Mascia F, Mariotti F, Dattilo C, Girolomoni G: Chemokines networks in inflammatory skin diseases. Eur J Dermatol 2004;14:203–208. Santamaria Babi LF, Picker LJ, Perez Soler MT, Drzimalla K, Flohr P, Blaser K, Hauser C: Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen. J Exp Med 1995;181:1935–1940. Akdis M, Akdis CA, Weigl L, Disch R, Blaser K: Skin-homing, CLA⫹ memory T cells are activated in atopic dermatitis and regulate IgE by an IL-13-dominated cytokine pattern: IgG4 counterregulation by CLA⫺ memory T cells. J Immunol 1997;159:4611–4619. Morishita Y, Tada J, Sato A, Toi Y, Kanzaki H, Akiyama H, Arata J: Possible influences of Staphylococcus aureus on atopic dermatitis – The colonizing features and the effects of staphylococcal enterotoxins. Clin Exp Allergy 1999;29:1110–1117. Torres MJ, Gonzalez FJ, Corzo JL, Giron MD, Carvajal MJ, Garcia V, Pinedo A, MartinezValverde A, Blanca M, Santamaria LF: Circulating CLA⫹ lymphocytes from children with atopic dermatitis contain an increased percentage of cells bearing staphylococcal-related T-cell receptor variable segments. Clin Exp Allergy 1998;28:1264–1272. Zollner TM, Wichelhaus TA, Hartung A, Von Mallinckrodt C, Wagner TO, Brade V, Kaufmann R: Colonization with superantigen-producing Staphylococcus aureus is associated with increased severity of atopic dermatitis. Clin Exp Allergy 2000;30:994–1000. Antunez C, Torres MJ, Mayorga C, Cornejo-Garcia JA, Santamaria-Babi LF, Blanca M: Different cytokine production and activation marker profiles in circulating cutaneous-lymphocyte-associated antigen T cells from patients with acute or chronic atopic dermatitis. Clin Exp Allergy 2004;34:559–566. Borgonovo B, Casorati G, Frittoli E, Gaffi D, Crimi E, Burastero SE: Recruitment of circulating allergen-specific T lymphocytes to the lung on allergen challenge in asthma. J Allergy Clin Immunol 1997;100:669–678. Kanda N, Mitsui H, Watanabe S: Prostaglandin E(2) suppresses CCL27 production through EP2 and EP3 receptors in human keratinocytes. J Allergy Clin Immunol 2004;114:1403–1409. Mora JR, Cheng G, Picarella D, Briskin M, Buchanan N, von Andrian UH: Reciprocal and dynamic control of CD8 T cell homing by dendritic cells from skin- and gut-associated lymphoid tissues. J Exp Med 2005;201:303–316. Sebastiani S, Albanesi C, De PO, Puddu P, Cavani A, Girolomoni G: The role of chemokines in allergic contact dermatitis. Arch Dermatol Res 2002;293:552–559. Traidl C, Sebastiani S, Albanesi C, Merk HF, Puddu P, Girolomoni G, Cavani A: Disparate cytotoxic activity of nickel-specific CD8⫹ and CD4⫹ T cell subsets against keratinocytes. J Immunol 2000;165:3058–3064. Blanca M, Posadas S, Torres MJ, Leyva L, Mayorga C, Gonzalez L, Juarez C, Fernandez J, Santamaria LF: Expression of the skin-homing receptor in peripheral blood lymphocytes from subjects with nonimmediate cutaneous allergic drug reactions. Allergy 2000;55:998–1004. Leyva L, Torres MJ, Posadas S, Blanca M, Besso G, O’Valle F, del Moral RG, Santamaría LF, Juarez C: Anticonvulsant-induced toxic epidermal necrolysis: Monitoring the immunologic response. J Allergy Clin Immunol 2000;105:157–165. Blanca M, Leyva L, Torres MJ, Mayorga C, Cornejo-Garcia J, Antunez-Rodriguez C, Santamaria LF, Juarez C: Memory to the hapten in non-immediate cutaneous allergic reactions to betalactam resides in a lymphocyte subpopulation expressing both CD45RO and CLA markers. Blood Cells Mol Dis 2003;31:75–79.

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Pichler WJ: Immune mechanism of drug hypersensitivity. Immunol Allergy Clin North Am 2004; 24:373–397.

Dr. Luis F. Santamaría-Babí Almirall Prodesfarma Research Center Cardener, 68–74 ES–08024 Barcelona (Spain) Tel. ⫹34 93 291 3466, Fax ⫹34 93 291 2827, E-Mail [email protected]

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The Skin Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 98–109

The Role of Sensitization to Malassezia sympodialis in Atopic Eczema Peter Schmid-Grendelmeiera, Annika Scheyniusb, Reto Crameric a

Allergy Unit, Department of Dermatology, University Hospital, Zurich, Switzerland; bDepartment of Medicine, Unit of Clinical Allergy Research, Karolinska Institute and Hospital, Stockholm, Sweden; cSwiss Institute for Allergy and Asthma Research (SIAF), Davos, Switzerland

Abstract Atopic eczema (AE) is a chronic relapsing, highly pruritic inflammation of the skin with a worldwide prevalence of 10–20% in children and of 1–3% in adults. Malassezia sympodialis has been reported as the most frequent skin-colonizing yeast in both AE patients and healthy individuals. Approximately 50% of the AE patients show immediate-type skin reactions or have specific serum IgE against M. sympodialis. Sensitization to the yeast occurs almost exclusively in AE patients. The main cause for this specific sensitization may be the disrupted skin barrier facilitating allergen uptake. So far thirteen allergens of Malassezia have been cloned produced, characterized and partly studied in vitro and in vivo. Phylogenetically conserved allergen structures, such as manganese superoxide dismutase, may play a role as cross-reactive allergens in a subset of AE patients as a result of molecular mimicry and cross-reactivity with structurally related human proteins and might contribute to the perpetuation of the inflammatory skin reactions. The use of recombinant Malassezia allergens will contribute to elucidate the pathways of sensitization occurring in AE, the underlying immunological mechanisms governing IgE- and T-cell-mediated responses and may provide new therapeutic options to alleviate Malassezia-related symptoms in AE. Copyright © 2006 S. Karger AG, Basel

Erythema, serous exudation and excoriation characterize the acute eczematous lesions in atopic eczema (AE), whereas lichenification and dry, fibrotic papules are a chronic manifestation of AE. Impetiginization, periauricular fissuration and superficial pustules are clinical signs of secondary infected AE. The worldwide prevalence of AE in children and adults is estimated to be in the range of 10–20% and of 1–3%, respectively [1]. During the past few years an increased prevalence of AE in highly industrialized countries has been reported following the general trend observed for all forms of atopic diseases [2].

The pathogenesis of AE is not yet fully understood; however, the manifestations of AE result from a complex interaction between environmental factors, pharmacological abnormalities, skin barrier dysfunction, susceptibility genes and immunological phenomena [3]. Among several contributing factors, cutaneous hyperreactivity and inappropriate immune response to various bacteria, fungi and other microorganisms as well as secondary infections appear to play important roles not only in the underlying pathology but also as factors responsible for sustained disease activity [3, 4]. At least two forms of AE have been distinguished: an ‘extrinsic’ form associated with IgE-mediated sensitization involving 70–80% of the patients, and an ‘intrinsic’ (nonatopic) form without detectable IgE-mediated sensitization involving 20–30% of the patients [5, 6]. Since, AE often becomes chronic and leads to asthma and allergic rhinitis (the so-called ‘atopy march’) [6] an anti-inflammatory or antimicrobial therapy against colonization is most likely of importance in management of AE.

Skin Barrier Dysfunction and Its Impact on Microbial Colonization

Patients with AE present an altered skin structure, which is characterized by the following features: deficient skin barrier due to scratching [7], changed, less optimal lipid layer [7], predominantly alkaline pH [8], decreased IgA secretion through sweat production [9], upregulated expression of molecular adhesins for Staphylococcus aureus (fibronectin and fibrinogen) [10] and decreased expression and synthesis of antimicrobial peptides [11]. Skin barrier dysfunction in patients with AE together with inappropriate immunological responses favors colonization both of lesional skin and unaffected skin by different microorganisms. A disruption of the primary skin defense system supports superinfection, increased antigen absorption and bacterial invasion in the skin.

Fungi as Contributing Factors to AE

Various fungal species can be identified in higher densities on atopic skin compared to healthy skin. Among these the yeasts Cryptococcus diffluens and Cryptococcus liquefaciens colonize the skin of AE patients more frequently than those of healthy subjects [12]; however, a direct involvement of specific IgE against these species has not yet been demonstrated. Coprinus comatus, a basidiomycete involved in the development of respiratory allergies [13] has been proposed as a potential aeroallergen that may provoke AE [14], although the number of patients enrolled in the study was quite low. Conflicting results

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have been reported on the role of intestinal or skin colonization with Candida albicans in AE. Candida species, especially C. albicans have been cultured more frequently from patients with AE, preferentially from the gastrointestinal tract, than from healthy donors, but reports about any correlation between disease severity and fungal colonization are rare and the role of C. albicans in the pathogenesis of AE may be questioned. In contrast, a number of studies have shown that Malassezia species play an important role in the pathogenesis of AE [4, 15]. The yeast Malassezia formerly known as Pityrosporum, belongs to our normal cutaneous microflora, but can cause skin diseases such as pityriasis versicolor, Pityrosporum folliculitis and seborrheic dermatitis and even systemic infections [15]. Malassezia species are lipophilic yeasts commonly found on the body surface in humans and warm-blooded animals, typically predominating seborrheic skin sites, such as head and neck, because of their defect in synthesis of fatty acids, and thus their need for nutritive requirement of exogenous fatty acids [16, 17]. Today, eleven different Malassezia species are known [4]. M. sympodialis has been reported in studies from Canada, Russia and Sweden as the most frequent species in both AE patients and healthy individuals, whereas in Japan M. furfur was the most common species cultured from the skin of patients with AE [4, 18]. Most likely, both methodological and climate factors can contribute to contradictory reports. Several studies have now demonstrated the presence of specific serum IgE and/or positive skin prick test (SPT) and atopy patch tests (APT) against Malassezia in patients suffering from AE [4]. The exclusivity of IgE-sensitization to M. sympodialis for AE patients has recently been shown in a large patient collective with 655 individuals suffering from different allergic diseases [Fischer et al., in preparation].

The Special Role of Malassezia Species in Allergic Reactions Related to AE

The mechanisms governing allergic response in AE are still not fully understood. The route of entry of the yeast Malassezia most likely occurs through the skin, especially bearing in mind that the skin barrier of AE patients is often disrupted [18]. Only recently, interactions between dendritic antigenpresenting cells and M. sympodialis have been investigated in detail. Monocytederived dendritic cells, when in an immature state reflecting the Langerhans cells in the skin, can internalize whole Malassezia yeast cells within one hour [19], which leads to monocyte-derived dendritic cell maturation, production of proinflammatory and immunoregulatory cytokines, favoring induction of a

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Th2-like immune response [20]. Therefore this yeast is considered to be one of the factors that can contribute to AE. In accordance, patients with AE show an altered gene expression profile of proinflammatory and costimulatory molecules in monocyte-derived dendritic cells exposed to Malassezia compared to healthy individuals [21]. Another important linking element between Malassezia colonization and AE might be the decreased expression of antimicrobial peptides in the skin [11]. The human ␤-defensin-2 can kill M. furfur [22]. The expression of human ␤-defensin-2 is upregulated in normal cultured human keratinocytes after internalization of M. furfur which thus limits the uptake of further Malassezia cells from the extracellular environment and reflects regulated innate host defence [22]. This immunomodulatory ability is impaired in AE which is why uncontrolled uptake of Malassezia spp. into keratinocytes can take place.

The Allergen Repertoire of M. sympodialis

Like other fungi, also M. sympodialis is able to produce a large amount of IgE-binding molecules ranging from 10 to 100 kDa in molecular mass detectable by immunoblotting of extracts [23]. However, different cultivation methods and time, different protocols used for the preparation of yeast extracts and the use of different species or strains of Malassezia contribute to varying results between studies [24]. These problems are not new and reflect the difficulties encountered during the standardization of fungal extracts in general. Therefore, efforts to clone, produce and characterize single allergens from the yeast have been undertaken. To date thirteen allergens from Malassezia species have been cloned, three from M. furfur strain TIMM 2782 and ten from M. sympodialis strain ATCC 42132 (table 1). Interestingly, four of the so-far-identified Malassezia allergens show no significant sequence similarity to known proteins, whereas others share similarities with each other or other yeast proteins or human structures with potential cross-reactivity to human homologs (table 1). However, the allergen repertoire of the yeast is much larger as demonstrated by high throughput screening of a cDNA library expressed on phage surface which yielded twenty-seven complete and partial sequences encoding IgE-binding molecules [25]. Since cDNA cloning can detect only genes expressed at the time where mRNA is prepared, the allergen repertoire of M. sympodialis might be even larger. At structural level, beside cDNAs encoding proteins with unknown biological function, the allergens cloned does not reveal any new structures in comparison to the allergen repertoires of other fungi [26]. Cyclophilin (Mala s 6), peroxisomal protein (Mala s 5), heat shock protein (Mala s 10) and manganese superoxide dismutase (MnSOD, Mala s 11) have

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Table 1. Allergen structures cloned as IgE-bining proteins from Malassezia species Allergen

Size (kDa)

Function/sequence similarity

Strain

Acc. no

Mala s 1 Mala f 2 Mala f 3 Mala f 4

36 20 21 35

ATCC 42132 TIMM 2782 TIMM 2782 TIMM 2782

X96486 AB011804 AB011805 AF084828

Mala s 5 Mala s 6 Mala s 7 Mala s 8 Mala s 9 Mala s 10 Mala s 11 Mala s 12

18 17 16 18 14 86 22 67

ATCC 42132 ATCC 42132 ATCC 42132 ATCC 42132 ATCC 42132 ATCC 42132 ATCC 42132 ATCC 42132

AJ011955 AJ011256 AJ011957 AJ011958 AJ011959 AJ428052 AJ548421 AJ871960

Mala s 13

12

Unknown Peroxisomal protein Peroxisomal protein Mitochondrial malate dehydrogenase Peroxisomal protein Cyclophilin Unknown Unknown Unknown Heat shock protein MnSOD Glucose-methanol-choline oxidoreductase family Thioredoxin

ATCC 42132

AJ9377431

1

Provisional submission, not yet accessible.

been identified as allergens also in other fungi and moulds including C. albicans, C. comatus and A. fumigatus and represent phylogenetically conserved proteins with a high degree of sequence homology expected to be crossreactive structures. In fact there is a good correlation between serum IgE to Asp f 6 and Mala s 11 in patients sensitized to MnSOD (fig. 1). Recently the threedimensional structures of Mala s 6 and Mala s 13 have been solved at 1.5 and 1.41 Å resolution, respectively [in preparation]. The crystal structure of Mala s 6 reveals the typical fold of cyclophilins, and the solvent-accessible surface of this allergen and human cyclophilin B were calculated and compared in order to identify conserved, putative IgE-binding amino acids. The analysis revealed three conserved, contiguous surface patches of identical amino acid residues likely to define conformational, cross-reactive IgE-binding epitopes. Similar results were obtained comparing the solved crystal structure of Mala s 13 with the structure of human thioredoxin. Not surprisingly, cyclophilin (Mala s 6 [27]), thioredoxin (Mala s 13) and MnSOD (Mala s 11 [28]) cross-react with the corresponding enzymes from Aspergillus fumigatus and Homo sapiens at least with respect to their IgE-binding capacity (Limacher and Glaser, pers. commun.]. The presence of conserved allergens in fungal extracts explains the cross-reactivity reported also between Malassezia and other fungal species [29]

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Serum lgE Asp f 6 (ImmunoCAP Rm222) (kU/l)

1,000 100 10 1 0.1 0 0.1

1

10

100

1,000

10,000

Serum lgE MnSOD (OD)

Fig. 1. Correlation between IgE against Asp f 6 and IgE against Mala s 11 in AE patients sensitized to M. sympodialis extract. The serology indicates strong cross-reactivity between the two MnSOD structures which has been confirmed also by Western blot analyzes and inhibition ELISA [Glaser, pers. commun.].

and may be a complication factor for a clear-cut diagnosis of fungal allergy [Crameri et al., this volume]. However, knowledge about the specific allergens and their cross-reactive structures is essential to understand the pathophysiology of allergic diseases, to predict clinically relevant sensitization to allergen sources, and for the development of new diagnostic and therapeutic concepts. Since the efficiency of detecting AE patients sensitized to M. sympodialis by ImmunoCAP measuring specific serum IgE, SPT and APT differs considerably, it has been suggested that there are different molecular structures of Malassezia responsible for either immediate hypersensitivity (SPT and IgE) and delayed-type hypersensitivity (APT). If this is the case, it remains to be demonstrated; however, the availability of pure recombinant allergens will allow clarifying the specific immune responses occurring in Malassezia-sensitized AE patients in detail. The availability of recombinant allergens will allow evaluating the diagnostic value of this panel of allergens in the subset of AE patients sensitized to M. sympodialis and might contribute to improve diagnostic sensitivity and specificity and to study the involvement of each single structure in the pathogenesis of AE (see below).

M. sympodialis in the Intrinsic Form (Nonatopic Type) of AE

Approximately 80% of the AE patients show immediate-type skin reactions to environmental allergens together with elevated serum IgE levels [30].

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However, approximately 20% of patients with clinical signs of AE have low serum IgE levels and lack detectable allergen-specific serum IgE and positive SPT reactions to environmental allergens. This subgroup is also defined as intrinsic, nonallergic or more recently nonatopic type of AE [5, 31, 32]. As the serum level of IgE to aeroallergens in the extrinsic type is directly associated with the severity of AE, it is assumed that allergens are involved in the onset and/or maintenance of the disease. Furthermore, the discovery of IgE antibodies on the epidermal Langerhans cells indicates an involvement of allergens in the pathogenesis of AE through IgE-mediated pathways [33]. Interestingly, in the intrinsic type of AE also we have found IgE- and Tcell-mediated sensitization against M. sympodialis: in 7 out of 15 patients (47%) by ImmunoCAP, in 5 of 15 patients by SPT and APT and in 6 out of 15 patients in peripheral blood mononuclear cell proliferation assays [Fischer et al., in preparation]. These patients, who all show a disrupted skin barrier and clinical signs of AE, have neither associated respiratory nor food allergy or detectable IgE-mediated sensitization against common inhalant or food allergens routinely tested in clinical practice. At least the patients showing specific IgE-mediated reactions to M. sympodialis extract have to be re-classified as belonging to the group of patients suffering from extrinsic AE. It is tempting to speculate that the remaining ‘intrinsic’ AE patients also – so far defined by the absence of sensitization to standard aero/food allergens – might suffer from an undetected IgE sensitization not revealed by the routine tests. As in fungal allergy, in the diagnosis of AE also the testable allergen panel is limited and detection of an unknown source of sensitization in absence of a clear clinical history is difficult.

Therapeutic Long-Term Options and Anti-Inflammatory Approaches

The golden standard for AE patients is an excellent skin care to reconstitute skin barrier, identification and elimination of trigger factors and antiinflammatory treatment. Long-term management with topical steroids alone should be avoided due to their numerous side effects. In combination with FK 506 or other anti-inflammatory treatment, steroids reduce colonization with S. aureus efficiently. The newer calcineurin inhibitors (tacrolimus and pimecrolimus) inhibit transcription of various Th1 and Th2 cytokine genes without significant side effects and are safe to use for facial and eyelid eczema [Brauer et al., this volume]. Antifungal therapy by oral itraconazole has been demonstrated in a randomized, double-blind, placebo-controlled trials in patients suffering from head

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and neck dermatitis [34]. This study shows that the antifungal systemic treatment has a significant Scoring Atopic Dermatitis Index reduction of AE, irrespective of the presence of detectable IgE sensitization [34]. The use of ketoconazole or itraconazole, both inhibiting IL-4 and IL-5 production and ergosterol synthesis, in AE patients may decrease Malassezia- and C. albicansspecific IgE and total IgE levels which correlate with the improvement of clinical symptoms. However, although this and other studies indicate that antifungal treatment of both Malassezia and C. albicans may be effective in the treatment of some patients with AE, we need further controlled studies to assess the real benefit of such therapies for generalized treatments.

Fungal Allergens Leading to Autoreactivity in AE

Autoreactivity to human proteins has been postulated as a pathogenetic factor for AE patients based on the detection of IgE directed against various proteins in vitro [35]. There is no doubt that a variety of human proteins, including MnSOD [36] and acidic ribosomal P2 protein [37] can induce strong B- and T-cell-mediated responses in peripheral blood mononuclear cell of patients suffering from chronic inflammatory allergic diseases. Moreover, skin test reactivity of patients sensitized to environmental MnSOD and P2 protein and to the corresponding homologous human self-antigens is basically indistinguishable [36, 37]. These responses to self-antigens in individuals sensitized to structurally related environmental allergens indicate an autoantigenic T-cell-mediated pathogenesis. In many infectious diseases, dominant antibody responses are directed towards microbial antigens with a high degree of homology to selfproteins indicating molecular mimicry at the T-cell level to homologous peptide sequences shared between environmental and self-antigens. Simple comparisons of the amino acid sequences by alignments show that short identical linear amino acid sequences potentially representing conserved T-cell epitopes are present in such homologous molecules [28]. On the other hand, as earlier mentioned, comparison of the crystal structures of the environmental allergens with the solved structures of the corresponding human proteins reveals patches of conserved solvent-exposed amino acids potentially defining conformational, cross-reactive IgE-binding epitopes [38, 39]. More work is needed to clarify if conserved T- or B-cell epitopes, or both, are responsible for the clinically observed cross-reactivity. Based on these results and on the fact that reactivity to self-antigens has so far been observed only in long-lasting chronic allergic diseases with inflammatory background [35–37], we have recently tested the ability of human MnSOD to induce eczematous reactions in patients sensitized to M. sympodialis and

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a

b Fig. 2. Immunohistochemistry staining of MnSOD expression in cryostat-embedded healthy skin (a), and lesional skin of a patient suffering from severe AE (b). Upregulation of the stress-inducible MnSOD is found in the skin of AE patients but not in patients suffering from other skin diseases [40].

suffering from AE [40]. This study showed that human MnSOD – a stressinducible enzyme – is able to induce eczematous reactions on unaffected skin areas of patients suffering from AE sensitized to M. sympodialis. In concordance with the presence of MnSOD-specific IgE antibodies determined by ELISA, the human enzyme also elicited positive SPT reactions in all ELISApositive patients. Interestingly, reactivity against human MnSOD strongly correlated with the severity of the disease further corroborating the postulated pathogenic role of self-reactivity in the exacerbation of AE. A strong indication that such reactions contribute to the pathogenesis of AE derive from the observation that human MnSOD is upregulated in eczematous areas as shown by immunohistochemistry [fig. 2, 40], most likely as a consequence of mechanical stress due to scratching. The fact that all patients reacting to human MnSOD were sensitized against the skin-colonizing yeast M. sympodialis indicates that sensitization most likely occurs primarily by exposure to the environmental fungal MnSOD. M. sympodialis MnSOD has now been cloned and expressed as a recombinant allergen [28]. The availability of a whole array of recombinant MnSODs of different origin [41] will allow further detailed studies to elucidate the role of cross-reactivity and self-reactivity in the pathogenesis of AE and other chronic allergic diseases with an inflammatory background.

Conclusions

There is increasing evidence that Malassezia represent a contributing factor in AE. Although it is still unknown how, where and why patients suffering from AE become sensitized to allergens of this lipophilic yeast, it becomes

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evident that sensitization to Malassezia, particularly M. sympodialis, is almost specific for AE. Substantial work aimed at cloning, characterizing and producing recombinant allergens from the yeast opens up new possibilities to study the pathogenic mechanisms underlying molecular interactions between the host and fungus. The use of these allergens will contribute to elucidate the pathways of sensitization occurring in AE, the underlying immunological mechanisms governing IgE- and T-cell-mediated responses and may provide new therapeutic options to alleviate Malassezia-related symptoms in AE.

Acknowledgements Supported by the Swiss National Science Foundation grants No. 3600–063381.00/2 and 3200B0–108139/1, by the OPO-Stiftung Zürich and by the EMDO-Stiftung, Zürich and by grants from the Swedish Research Council (project nos 74x-7924 and 74EF-15193). We are grateful to Prof. K. Blaser for his continuous support.

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7 8 9

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Schultz Larsen F, Diepgen T, Svensson A: The occurrence of atopic dermatitis in North Europe: An international questionnaire study. J Am Acad Dermatol 1996;34:760–764. ISAAC – The international Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee: Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivities, and atopic eczema: ISAAC. Lancet 1998;351:1225–1232. Leung DY, Bieber T: Atopic dermatitis. Lancet 2003;361:151–160. Scheynius A, Johansson C, Buentke E, Zagari A, Linder MT: Atopic eczema/dermatitis syndrome and Malassezia. Int Arch Allergy Immunol 2002;127:161–169. Novak N, Bieber T: Allergic and nonallergic forms of atopic disease. J Allergy Clin Immunol 2003;112:252–262. Wuthrich B, Schmid-Grendelmeier P: The atopic eczema/dermatitis syndrome: Epidemiology, natural course, and immunology of the IgE-associated (‘extrinsic’) and the nonallergic (‘intrinsic’) AEDS. J Investig Allergol Clin Immunol 2003;13:1–5. Fartach M: Epidermal barrier in disorders of the skin. Microsc Res Tech 1997;38:361–372. Gfesser M, Rakoski J, Ring J: The disturbance of epidermal barrier function in atopy patch test reactions in atopic eczema. Br J Dermatol 1996;135:560–565. Imayama S, Shimozono Y, Hoashi M, Yasumoto S, Ohta S, Yoneyama K, Hori Y: Reduced secretion of IgA to skin surface of patients with atopic dermatitis. J Allergy Clin Immunol 1994;94: 195–200. Cho SH, Strickland I, Boguniewicz M, Leung DY: Fibronectin and fibrinogen contribute to the enhanced binding of Staphylococcus aureus to atopic skin. J Allergy Clin Immunol 2001;108: 269–274. Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, Gallo RL, Leung DY: Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 2002;347: 1151–1160. Sugita T, Saito M, Ito T, Kato Y, Tsuboi R, Takeuchi S, Nishikawa A: The basidiomicetous yeast Cryptococcus diffluens and C. liquefaciens colonize the skin of patients with atopic dermatitis. Microbiol Immunol 2003;47:945–950.

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Brander KA, Borbléy P, Crameri R, Pichler WJ, Helbling A: IgE-binding, proliferative responses and skin test reactivity to Cop c 1, the first recombinant allergen from the basidiomycete Coprinus comatus. J Allergy Clin Immunol 1999;104:630–636. Fischer B, Yawalkar N, Brander KA, Pichler WJ, Helbling A: Coprinus comatus (shaggy cap) is a potential source of aeroallergen that may provoke atopic dermatitis. J Allergy Clin Immunol 1999;104:836–841. Faergemann J: Pityrosporum species as a cause of allergy and infection. Allergy 1999;54: 413–419. Midgley G: The lipophilic yeasts: State of the art and prospects. Med Mycol 2000;38(suppl 1): 9–16. Midgley G: The diversity of Pityrosporum (Malassezia) yeasts in vivo and in vitro: Mycopathologia 1989;106:143–153. Sandström Falk MH, Tengvall Linder M, Johansson C, Bartosik J, Bäck O, Särnhult T, Wahlgren CF, Scheynius A, Faergemann J: The prevalence of Malassezia yeasts in patients with atopic dermatitis, seborrhoeic dermatitis and healthy controls. Acta Derm Venereol 2005;85:17–23. Hirai A, Kano R, Makimura K, Duarte ER, Hamdan JS, Lachance MA, Yamaguchi H, Hasegawa A: Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals. Int J Syst Evol Microbiol 2004;54:623–627. Sugita T, Tajima M, Takashima M, Amaya M, Saito M, Tsuboi R, Nishikawa A: A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects. Microbiol Immunol 2004;48:579–583. Gabrielsson S, Buentke E, Liedén A, Schmidt M, D’Amato M, Tengvall-Linder M, Scheynius A: Malassezia sympodialis stimulation differently affects gene expression in dendritic cells from atopic dermatitis patients and healthy individuals. Acta Derm Venereol 2004;84:1–7. Donnarumma G, Paoletti I, Buommino E, Orlando M, Tufano MS, Baroni A: Malassezia furfur induces the expression of beta-defensin-2 in human keratinocytes in a protein kinase C-dependent manner. Arch Dermatol Res 2004;295:474–481. Johansson S, Karlström K: IgE-binding components in Pityrosporum orbiculare identified by an immunoblotting technique. Acta Derm Venereol 1991;71:11–16. Zagari A, Doekes G, van Ieperen-van Dijk AG, Landberg E, Härfast B, Scheynius A: Influence of culture period on the allergenic composition of Pityrosporum orbiculare extracts. Clin Exp Allergy 1995;25:1235–1245. Crameri R, Kodzius R, Konthur Z, Lehrach H, Blaser K, Walter G: Tapping allergen repertoires by advanced cloning technologies. Int Arch Allergy Immunol 2001;124:43–47. Weichel M, Flückiger S, Crameri R: Molecular characterisation of mould allergens involved in respiratory complications. Recent Res Dev Resp Crit Care Med 2002;2:29–45. Flückiger S, Fijten H, Whitley P, Blaser K, Crameri R: Cyclophilins, a new family of crossreactive allergens. Eur J Immunol 2002;32:10–17. Andersson N, Rasool O, Schmidt M, Kodzius R, Flückiger S, Zagari A, Crameri R, Scheynius A: Cloning, expression and characterization of two new IgE-binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase. Eur J Biochem 2004;271:1885–1894. Huang X, Johansson SGO, Zagari A, Nordwall SL: Allergen cross-reactivity between Pityrosporum orbiculare and Candida albicans. Allergy 1995;50:648–656. Werfel T, Kapp A: Environmental and other major provocation factors in atopic dermatitis. Allergy 1998;53:731–739. Johansson SGO, Bieber T, Dahl R, Friedmann, PS, Lanier BQ, Lockey RF, Motala C, Ortega Martell JA, Platts-Mills TA, Ring J, Thien F, Van Cauwenberge P, Williams HC: Revised nomenclature for allergy for global use. Report of the Nomenclature Review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol 2004;113:832–836. Schmid-Grendelmeier P, Simon D, Simon HU, Akdis CA, Wüthrich B: Epidemiology, clinical features, and immunology of the ‘intrinsic’ (non-IgE-mediated) type of atopic dermatitis (constitutional dermatitis). Allergy 2001;56:841–849.

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Bruynzeel-Koomen C, van Wichen DF, Toonstra J, Berrens L, Bruynzeel PL: The presence of IgE molecules on epidermal langerhans cells in patients with atopic dermatitis. Arch Dermatol Res 1986;278:199–205. Svejgaard E, Larsen PO, Deleuran M, Ternowitz T, Roed-Petersen J, Nilsson J: Treatment of head and neck dermatitis comparing itraconazole 200 mg and 400 mg daily for 1 week with placebo. J Eur Acad Dermatol Venereol 2004;18:445–449. Valenta R, Seiberler S, Natter S, Mahler V, Mossabeb R, Ring J, Stingl G: Autoallergy: A pathogenetic factor in atopic dermatitis? J Allergy Clin Immunol 2000;105:432–437. Crameri R, Faith A, Hemmann S, Jaussi R, Ismail C, Menz G, Blaser K: Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus. J Exp Med 1996;184:265–270. Mayer C, Appenzeller U, Seelbach H, Achatz G, Oberkofler H, Breitenbach M, Blaser K, Crameri R: Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus. J Exp Med 1999;189:1507–1512. Flückiger S, Limacher A, Glaser AG, Scapozza L, Crameri R: Structural aspects of cross-reactive allergens. Rec Res Dev Allergy Clin Immunol 2004;5:57–75. Flückiger S, Mittl PRE, Scapozza L, Fijten H, Folkers G, Grütter MG, Blaser K, Crameri R: Comparison of the crystal structures of the human manganese superoxide dismutase and the homologous Aspergillus fumigatus allergen at 2-Å resolution. J Immunol 2002;168:1267–1272. Schmid-Grendelmeier P, Flückiger S, Disch R, Trautmann A, Wüthrich B, Blaser K, Scheynius A, Crameri R: IgE-mediated and T-cell-mediated autoimmunity against manganese superoxide dismutase in atopic dermatitis. J Allergy Clin Immunol 2005;115:1068–1075. Mayer C, Hemmann S, Faith A, Blaser K, Crameri R: Cloning, production, characterization and IgE cross-reactivity of different manganese superoxide dismutases in individuals sensitized to Aspergillus fumigatus. Int Arch Allergy Immunol 1997;113:213–215.

PD Dr. med. Peter Schmid-Grendelmeier Allergy Unit, Department of Dermatology University Hospital, Gloriastrasse 31 CH–8091 Zurich (Switzerland) Tel. ⫹41 1 255 8955, Fax ⫹41 1 255 4403, E-Mail [email protected]

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The Skin Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 110–120

Allergic Conjunctivitis:The Forgotten Disease Sergio Bonini Chair Allergology and Clinical Immunology, Second University of Naples and IRCCS San Raffaele, Rome, Italy

Abstract The eye is largely exposed to foreign substances, and ocular tissues have a complete array of immune cells to interact with offending antigens. In particular, the external eye represents an ideal site for immediate hypersensitivity reactions because of the high number of mast cells in the eyelids and bulbar conjunctiva, as well as for the potential local synthesis of IgE. In fact, the involvement of the eye was part of the original description of hay fever by Charles Blackley in 1873. In a large epidemiological survey of allergic patients we reported since 1987, 40% had symptoms possibly related to allergic conjunctival disease. However, the participation of the conjunctiva to the multiorgan picture of allergy is largely underestimated. Allergic eye diseases have long been referred to the classical Type I hypersensitivity mechanism according to Coombs and Gell. Recently, however, the tremendous progress in understanding allergic mechanisms and inflammation has brought new insights into the pathophysiology of several allergic diseases, including those of the eye. Accordingly, several systematic descriptions of allergic eye diseases should be revisited. In particular, the classification of the various forms of a ‘red eye’ of allergic origin appears inadequate to answer the progress achieved in their multifactorial pathogenesis. Copyright © 2006 S. Karger AG, Basel

Definition

Allergic diseases of the eye should include all diseases in which immune mechanisms are responsible for pathological changes of ocular tissues – including conjunctiva, cornea, lens, sclera and retina – and eye symptoms [1]. However, this paper will be confined to hypersensitivity reactions of the external eye, in which both IgE-mediated and non-IgE-mediated mechanisms produce symptoms usually defined with the generic term of ‘a red eye’. The term allergic conjunctivitis does not appear satisfactory to encompass these clinical and pathophysiological entities, since in some forms cornea is affected too. Moreover, while allergic mechanisms can be clearly documented even in

forms not referable to a specific IgE response to common allergens (such as in some cases of vernal and atopic keratoconjunctivitis), the allergic component is far less documented in other forms and is usually classified under the umbrella term of ‘allergic conjunctivitis’, such as contact lens conjunctivitis (CLC) [2]. Classification

Allergic eye diseases are usually classified on the basis of their clinical presentation as acute, seasonal or hay fever, perennial, vernal and atopic. An immunological mechanism has also been postulated for conjunctival symptoms in contact lens wearers [3]. Acute allergic conjunctivitis is an acute hypersensitivity reaction with hyperemia and chemosis accompanied by intense tearing, itching and burning of the eye, caused by an accidental exposure to several substances such as gas and liquid ‘irritants’ or animal danders. Seasonal allergic conjunctivitis (SAC) is the typical conjunctival reaction in hay fever rhino-conjunctivitis or following exposure to seasonal pollen allergens in sensitized subjects. Perennial allergic conjunctivitis (PAC) is a less intense, but continuous, conjunctival reaction related to exposure to perennial allergens such as house dust mites. Vernal conjunctivitis (VKC) is a severe bilateral eye condition of children with frequent involvement of the cornea (vernal keratoconjunctivitis) characterized by conjunctival hypertrophy and mucus excess. Atopic conjunctivitis (AKC) is a keratoconjunctivitis associated with eczematous lesions of the lids and skin. Contact lens conjunctivitis (CLC) is a giant-papillary conjunctivitis observed in hard and soft contact lens wearers. Therefore, patients with a red eye are heterogeneous. Different clinical entities only answer eye examination criteria, while the age and distribution, the levels of IgE as well as the frequency and the type of sensitization might be different in various forms, as it will be shown below. On the other hand, overlapping and resemblances do occur between patients with different ‘clinical labels’. Accordingly, a new classification of allergic eye diseases might be desirable on the basis of pathophysiological rather than clinical criteria. Etiopathogenesis

The release of preformed and de novo synthesized mediators from mast cells (MCs) and basophils represents the main pathophysiological mechanism

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for explaining the effects and symptoms of many forms of red eye. However, additional mechanisms can be relevant for a better understanding of some cases of allergic eye diseases where the clinical and pathological picture is not explained by a Type I hypersensitivity reaction only. (1) The possibility that MC and basophil mediator release can be induced by several triggers other than IgE (such as anaphylotoxins C3a and C5a, interleukins, etc.) is now largely proven. This mechanism might explain how triggers other than allergens (such as food, drugs, bacterial and viral infections) might induce pseudo-allergic reactions which are very similar, at clinical level, to topical IgE-mediated immediate hypersensitivity reactions. This might occur in the eye too, as suggested by several in vitro and in vivo experiments. (2) We were able to produce original evidence [4] that allergen challenge of human conjunctiva causes persisting inflammatory changes of ocular tissues similar to those previously described in rats. Therefore, the concept of late-phase allergic reaction (LPR) can be applied to the eye too, as previously to the skin, nose and bronchi. In the eye, LPR is characterized, at the clinical level, by redness (with minor itching and tearing) persisting long after allergen provocation, accompanied by a sensation of foreign body [5]. At the cytological level, the analysis of conjunctival scrapings or tears shows an early accumulation of neutrophils (mainly) during the immediate phase, followed by a prevalent recruitment of eosinophils 6–10 h after provocation and of lymphocytes and monocytes later on, with persistent inflammatory changes up to 24 h after challenge [4, 5]. The recruitment of inflammatory cells during LPR is associated with the detection in tears of mediators released by primary and secondary effector cells. For instance, we were able to detect significant amounts of leukotriene C4, eosinophil peroxidase, eosinophil cationic protein (ECP) and histamine (but not tryptase) during ocular LPR. This last finding is in agreement with the hypothesis that basophils, but not MCs, have a role in LPR. Interestingly enough, mediators present in tears (6 h after allergen challenge) can passively transfer the ocular LPR. Clinical and cytological changes of conjunctival LPR appear to be a continuous dose-dependent process [5]. When low allergen doses are used for challenge, or when the sensitivity of the patient is low, only cytological changes are observed during the early phase of conjunctival reaction, not associated with clinical symptoms. Increasing the dose of allergen, results in a more intense cellular reaction and in typical clinical immediate response of the conjunctiva. When the dose of allergen is further increased, or when patients with higher sensitivity are challenged, a more intense and prolonged cell recruitment is induced, with a clinical LPR when consistent allergen doses are used. Therefore, from our studies in the eye it appears that LPR is a constant outcome of the immediate reaction, provided that large allergen doses are administered to highly sensitized patients.

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(3) Neural and nonimmune factors. Allergic inflammation is a complex phenomenon modulated by several factors. Several nonimmune factors, such as neural and endocrine factors, may have a role in influencing pathological changes and clinical expression of allergic eye diseases. Plasma levels of nerve growth factor (NGF) are significantly increased in patients with VKC and related to the number of MC infiltrating the tarsal and bulbar conjunctiva [7]. Patients with VKC also show increased plasma levels of substance P [8], a neuropeptide, the synthesis and release of which are modulated by several factors including NGF. In fact, VKC patients with higher levels of NGF also had high levels of substance P. Increased levels of NGF, however, are not confined to VKC, but also occur in other forms of allergic conjunctivitis as well as in other allergic diseases such as asthma [9]. In addition, increased expression of estrogen and progesterone receptors in conjunctival biopsies of patients with VKC has also been demonstrated [10]. (4) Extensive evidence in target organs other than the eye indicates that allergic patients are hyperreactive to various nonspecific substances in addition to the sensitizing allergen. Although close relationships exist between reactivity to allergens and to nonallergic substances, specific and nonspecific hyperreactivity represent distinct concurrent mechanisms in the pathogenesis of several allergic diseases such as bronchial asthma. We compared the conjunctival responsiveness to histamine diphosphate (0.1–1.0 mg/ml) in patients with VKC and in healthy controls [11]. Both VKC patients and controls reacted to histamine with a dose-dependent conjunctival redness 2–5 min after allergen challenge. However, at low histamine doses (0.01–0.05 mg/ml) VKC had a significantly (p ⬍ 0.05) more intense reaction than controls. Moreover, the concentration of histamine diphosphate causing a threshold conjunctival redness (provoking concentration of histamine) was significantly lower (p ⬎ 0.02) in patients with VKC than in controls. The existence of a conjunctival hyperresponsiveness to histamine in VKC might be relevant for a better understanding of the pathogenesis and some clinical features of this disease. For instance, nonspecific conjunctival hyperreactivity may play a role in causing the variable course of the disease not correlating with environmental changes of the sensitizing allergen. In fact, sun, wind, dust or other natural agents may represent triggers of a nonspecific abnormal reactivity of the conjunctiva. We can also speculate that nonspecific conjunctival hyperreactivity has a primary pathogenetic role in forms of ‘allergic’ conjunctivitis where no clinical sensitization is detectable, as in many cases of VKC and CLC. In summary, the above-reported data strongly suggest that the pathogenesis of ‘allergic conjunctivitis’ cannot be confined to the classic Type I hypersensitivity reaction. Several mechanisms other than the IgE-dependent MC mediator release can be operating in different forms of ‘allergic conjunctivitis’, including

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the participation of a complex network of inflammatory cells and mediators, the occurrence of late-phase persistent inflammatory changes, the role of neuroendocrine factors and the presence of an enhanced reactivity of the conjunctiva. For instance, a specific IgE response and the classical hypersensitivity reaction are the major pathophysiological abnormalities in both SAC and PAC. However, in this last form as well as in highly sensitive subjects with SAC, latephase inflammatory events are often associated with pathophysiological changes and symptoms. A Th2-type eosinophilic inflammation dominates the pathogenesis of VKC and AKC [12–18]. In fact, specific IgE antibodies – usually to multiple allergens – are not always detectable in these forms of allergic conjunctivitis and are possibly the consequence of a polyclonal IgE activation rather than the major pathogenetic factor for the disease. Finally, conjunctival hyperreactivity – associated with eosinophilic inflammation in VKC and AKC – might well represent the major pathophysiological abnormality in forms with no detectable IgE antibodies or signs of allergic inflammation.

Clinical Presentation

We studied the clinical presentation of different forms of allergic eye disease in a large epidemiological survey of allergic patients [1]. More recently we revisited the clinical features of VKC in a case series of 195 patients [16]. In the first survey, clinical data of 898 consecutive patients referred to our allergy clinic were gathered via questionnaires and correlated, by means of computerized analysis, to clinical ocular manifestations and to allergy tests (prick test, total and specific IgE measurement, functional and provocation tests). Three hundred sixty-three patients (40%) had symptoms possibly related to allergic conjunctival disease. In 64 of these (18%), eye symptoms were associated with other allergic diseases (rhinitis 50%, asthma 30%, atopic dermatitis 2%, urticaria-angioedema 1%, more than one allergic disease 26%). The number of males presenting with symptoms was approximately equal to the number of females (178 vs. 184). The mean age of subjects with ocular involvement was 27 ⫾ 13 years. Skin test sensitization to inhalant allergens could be shown in 68% of cases, 23% of whom had conjunctival symptoms only, and 77% of whom had other concomitant allergic diseases. Frequencies of sensitization to grass, house dust mites, Parietaria or more than one inhalant allergen were 28, 6, 3 and 30%; respectively. Food and drug allergy or intolerance was present in 6 and 7% of cases, respectively (in 6 and 14% of patients with ocular symptoms only; in 6 and 5% of subjects with involvement of more than one target organ). In the 363 patients with eye symptoms, the clinical diagnosis was SAC or PAC in 239 cases (66%), VKC in 29 cases (8%), AKC in 9 cases (3%) and CLC in 19

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cases (5%). In 67 cases (18%), it was not possible to make a definite diagnosis by history and clinical examination at time of study. These patients are often referred to as having ‘nonspecific conjunctivitis’. SAC and PAC were the forms of allergic conjunctivitis most frequently observed clinically (66%). Male: female ratio was 113:126. The mean age of patients at time of observation was 28.2 ⫾ 10.4 years, with a mean age at time of appearance of symptoms of 20.8 ⫾ 12.9 years. SAC or PAC occurred without other allergic manifestations in 6% of the patients, whereas it was associated with other allergic diseases in 95.2% (65% of patients had rhinitis, 1% asthma, 1.8% atopic eczema, 0.4% urticaria-angioedema, 27% more than one allergic disease). Although SAC should by definition occur in patients with hay fever and sensitization to seasonal pollen allergens, 0.4% of SAC patients were skin test- and radioallergosorbent test (RAST)-negative. Furthermore, of the 229 cases with positive findings after history, skin, RAST and provocation testing, only 38% were sensitive to grass, with 2% sensitive to a nonseasonal pollen allergen (Parietaria), 5% to house dust mites and 48% showing sensitization to more than one allergen. Food and drug allergy were present in 4 and 5% of cases, respectively. Total serum IgE was higher (0.05 ⬍ p ⬍ 0.1) in subjects with SAC or PAC than in controls. However, subjects with associated allergic diseases had significantly higher IgE values than patients with SAC or PAC only (284 ⫾ 296 kU/l vs. 21 ⫾ 15 kU/l; p ⬍ 0.001). It has been reported that total and specific IgE is present in tears of subjects with SAC or PAC and is possibly locally produced. However, in our study total IgE values in tears were often below the sensitivity level of the test. There was no patient who had positive RAST in tears but not in serum. As reported in previous studies in our survey [16] VKC was more common in males (M:F: 2.8) and during the first decade of life. The mean age of VKC patients was 11.0 ⫾ 5.8 years at time of observation, whereas the mean age at time of appearance of symptoms was 7.1 ⫾ 4.7 years. VKC was associated with other allergic diseases in 41.5% of cases (with asthma in 62.4% of patients; with rhinitis in 49.4%; with atopic eczema in 23.5%; and with more than one allergic disease in 81.5%). Data regarding the frequency of sensitization in VKC patients are conflicting. We found 57.8% of patients to be sensitive to one or more inhalant allergens at skin tests and 52.2% at RAST. In previous studies of VKC, pollen allergens were more frequently responsible for sensitization, although the frequencies of sensitization to house dust mites and animal dander were equally high. We found lower frequencies of sensitization to these allergens, although many patients showed more than one sensitization. The high frequency of food allergy reported by others was not observed in our series. Drug intolerance was present in 17% of cases. Total IgE has been shown to be elevated in serum and tears of patients with VKC. The mean value of serum IgE in VKC patients was

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129 ⫾ 545 kU/l. Patients with VKC and positive RAST had a significantly higher level of serum IgE than RAST-negative patients. However, total serum IgE was increased versus healthy control even in the absence of a clinical sensitization to common allergens. A local production of IgE (and IgG) antibodies in VKC patients has been postulated. However, we never observed a positive RAST in tears and a negative RAST in serum. AKC occurs in children older than 3 years of age with atopic eczema and in 25% of eczematous adults. A few patients with AKC were recorded in our series. All had eczema, often-associated allergic diseases and IgE antibodies to inhalant or food allergens [17]. We studied 19 patients with CLC, 14 of them females. Mean age was 22.9 ⫾ 5.8 years at time of observation, with a mean age at time of appearance of symptoms of 20.0 ⫾ 1.6 years. An association with other allergic diseases was present in only 2 cases. IgE levels have been reported as elevated in the tears, but not in the serum, of CLC patients. Total IgE serum levels measured in our study (101 ⫾ 133 kU/l) did not differ significantly from those of normal subjects. Evidence of sensitization to inhalant allergens was present in only 3 of 16 cases (18%). Two patients had food allergy and 3 patients had drug intolerance.

Diagnosis

The diagnosis of allergic eye diseases on the basis of the current classification is based on clinical criteria. Therefore, an accurate eye examination is essential for the differential diagnosis of different clinical entities and, mainly, for distinguishing forms confined to the conjunctiva – such as SAC and PAC – from forms with possible localization to the cornea, such as VKC and AKC. The diagnosis of specific allergy in allergic eye diseases does not differ from that of other allergic diseases. In fact, the clinical value of the measurement of IgE in tears as well as in specific conjunctival provocation tests is doubtful, and these additional diagnostic tools appear at present more useful for research purposes rather than for routine clinical practice. Since the assessment of the type and degree of allergic inflammation is important for distinguishing among different forms of allergic eye diseases and for adequate management of individual cases, ad hoc tests should be standardized for a wider use in current practice. Impression and tear cytology are easily performed techniques to evaluate the type and degree of inflammation in ocular tissues, showing a good correlation with conjunctival scrapings and the more invasive conjunctival biopsies. Several mediators are measurable in tears, such as tryptase [19], histamine, leukotrienes, ECPs, interleukins and other cytokines. They have been reported to document a Th2-type allergic inflammation, monitor changes after allergen challenge and better

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define different forms of allergic eye diseases. However, so far, these interesting research results cannot be extrapolated for current use in diagnosis of ocular allergy. The measurement of ECP has been extensively used as a marker of inflammation in allergic diseases. ECP serum levels are increased in SAC during pollen season, in PAC and – in parallel to eosinophil-derived neurotoxin/protein X – in VKC [20, 21]. However, the increased ECP serum levels in conjunctivitis – as well as in other allergic diseases – reflect the severity of atopic status and the releasibility of eosinophil, while their value in predicting the severity of inflammation at the level of target organs involved is less relevant.

Treatment

The treatment of allergic eye diseases is based on hygienic measures and on the use of oral and topical antihistamines, cromones, vasoconstrictors, antiinflammatory drugs and immunotherapy [22]. The first-choice drug depends not only on the type of allergic conjunctivitis but also, in individual cases, on the prevalent pathogenetic mechanism as well as on the coexisting clinical symptoms, if any. Frequent eye washing and cold compresses are simple measures which may benefit the majority of allergic conjunctivitis patients. In VKC ocular lubrication, artificial tears and mucolytic agents – such as acetylcysteine – may be helpful in softening and enabling the evacuation of the mucus discharge. Allergic conjunctivitis patients – and mainly those with AKC and VKC in which rubbing of the eyes is constant because of the severe itching – should be instructed to a frequent hand washing to avoid superimposed infections. If blepharitis occurs, lid hygiene and topical antibiotics should be added to antiallergic treatment. The use of contact lens should of course be discontinued in CLC. However, contact lenses are often not tolerated in SAC and PAC too, until symptoms and nonspecific conjunctival hyperreactivity are under control. Topical and oral antihistamines are usually effective in treating SAC and PAC. Topical antihistamines are usually preferred, since they lack systemic effects and because of the beneficial symptomatic effect of vasoconstrictors associated with topical antihistamines in several preparations. However, rebound effects and a clinical condition of the eye similar to rhinitis medicamentosa can be induced by the prolonged use of vasoconstrictor agents. Moreover, oral antihistamines might be preferable if coexisting rhinitis symptoms are present (as it is in the majority of cases of SAC and PAC). The potential usefulness of receptor antagonists of other allergy mediators – such as leukotrienes – is a promising perspective, not yet supported by enough clinical trials.

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Sodium cromoglycate and nedocromil sodium have also shown to be effective in SAC and PAC. They represent the prototype agents of a group of compounds commonly referred to as MC stabilizers, although their precise mechanism of action is still only partially known. A MC-stabilizing effect has also been documented for lodoxamide. All the above antiallergic agents have proven to be of some efficacy in VKC and AKC too. However, the severe allergic inflammation and the corneal involvement often present in these forms require the use of topical or oral corticosteroids. All corticosteroids are associated with increased risk of elevation in intraocular pressure and cataract formation. Therefore, their use should be restricted to short courses when the doctor – and not the patient – feels they are necessary. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been used in allergic eye diseases to treat inflammation, while avoiding the possible severe side effects of corticosteroids. Both topical and oral formulations of NSAIDs are available. However, unlike antihistamines, the side effects of oral NSAIDs are consistent. Therefore their use should be confined only to severe cases of VKC. Topical NSAIDs have been reported to be an alternative effective and safe option to corticosteroids in VKC and CLC, also showing analgesic properties. However, the safety profile of topical NSAIDs in aspirin – intolerant patients has not yet been documented and their efficacy in severe forms of VKC and AKC seems far less than that of corticosteroids. Cyclosporin A 2% in olive oil (5 mg/kg/day) has been used in the treatment of severe cases of AKC and VKC. The potential area of application of this and other immunomodulators is still to be defined. Immunotherapy (hyposensitization) is usually effective in reducing eye symptoms when administered for correct indications in allergic patients. Therefore, some SAC and PAC patients can benefit from it. On the contrary, the efficacy of immunotherapy in VKC and AKC is controversial, even in the presence of a documented sensitization to allergens for which hyposensitization is indicated and usually effective.

Prognosis

Although signs and symptoms of allergic conjunctivitis can be recurrent in SAC or persistent in PAC, these forms of allergic conjunctivitis are easily controlled by treatment and do not cause significant eye damage or visual impairment. On the contrary, several complications have been reported in VKC and AKC patients, including corneal ulcers (3–11% of cases) and iatrogenic damages from steroid treatment with permanent reduction in visual acuity. Although VKC has been reported to spontaneously subside with puberty, 52%

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of our patients followed up for a mean period of 5 years had persistent symptoms at the end of the follow-up period. 9.7% of them experienced corneal ulcers, 2% steroid-induced glaucoma and 6% had visual impairment as a result of permanent corneal damage.

References 1 2 3 4 5

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Bonini Se, Bonini St: Studies of allergic conjunctivitis. Chibret Int J 1987;5:12. Bielory L, Bonini Se, Bonini St: Allergic eye disorders; in Zweiman S, Schwartz LB (eds): Inflammatory Mechanisms in Allergic Diseases. Marcel Dekker, 2002, pp 311–323. Bonini Se, Bonini St, Tomassini M: Les allergies oculaires; in Charpin J, Vervloet D (eds): Allergologie, ed 3. Paris, Med Sci Flammarion, 1992, p 714. Bonini Se, Bonini St, Vecchione A, Naim DM, Allansmith MR, Balsano F: Inflammatory changes in conjunctival scrapings after challenge provocation in humans. J Allergy Clin Immunol 1988;82:462. Bonini St, Bonini Se, Bucci MG, Berruto A, Adriani E, Balsano F, Allansmith MR: Allergen dose response and late symptoms in a human model of ocular allergy. J Allergy Clin Immunol 1990;86: 869. Bonini Se, Bonini St, Berruto A, Tomassini M, Carlesimo S, Bucci MG, Balsano F: Conjunctival provocation test as a model for the study of allergy and inflammation in humans. Int Arch Allergy Appl Immunol 1989;88:144. Lambiase A, Bonini St, Bonini Se, Micera A, Magrini L, Bracci-Laudiero L, Aloe L: Increased plasma levels of nerve growth factor in vernal keratoconjunctivitis and relationship to conjunctival mast cells. Invest Ophthalmol Vis Sci 1995;36:2127–2132. Lambiase A, Bonini St, Micera A, Tirassa P, Magrini L, Bonni Se, Aloe L: Increased plasma levels of substance P and nerve growth in vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 1997;38:2162–2164. Bonini Se, Lambiase A: Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma. Proc Natl Acad Sci USA 1996;93:10955–10960. Bonini St, Lambiase A, Schiavone M: Estrogen and progesterone receptors in vernal keratoconjunctivitis. Ophthalmol 1995;102:1374–1379. Bonini St, Bonini Se, Schiavone M, Centofanti M, Allansmith MR, Bucci MG: Conjunctival hyperresponsiveness to ocular histamine challenge in patients with vernal conjunctivitis. J Allergy Clin Immunol 1992;89:103. Bonini Se, Tomassini M, Bonini St, Capron M, Balsano F: The eosinophil has a pivotal role in allergic inflammation of the eye. Int Arch Allergy Immunol 1992;99:354–358. Magrini L, Metz D, Bacon A, Bonini Se, Bonini St, Lightman S: Immunocytochemistry and inflammation of vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 1993;34:857. Bonini Se, Bonini St, Lambiase A, Magrini L, Rumi C, Del Prete GF, Schiavone M, Rotiroti G, Onorati P, Rutella S: Vernal keratoconjunctivitis: A model of 5q cytokine gene cluster disease. Int Arch Allergy Immunol 1995;107:95–98. Bonini Se, Bonini St: Vernal keratoconjunctivitis: A model Th2 disease. ACI Int 1998;10:88–91. Bonini St, Bonini Se, Lambiase A, Marchi S, Pasqualetti P, Zuccaro O, Rama P, Magrini L, Juhas T, Bucci MG: Vernal keratoconjunctivitis revisited: A case series of 195 patients with longterm follow-up. Ophthalmology 2000;107:1157–1163. Bonini S: Atopic keratoconjunctivitis. Allergy 2004;59:71–73. Bonini Se, Bonini St: Pathogenesis of allergic conjunctivitis; in Denburg JA (eds): Mechanisms of allergic diseases. Basic and clinical aspects. Totowa NJ, Humana Press, 1998, pp 509–519. Magrini L, Bonini Se, Centofanti M, Schiavone M, Bonini St: Tear tryptase levels and allergic conjunctivitis. Allergy 1996;51:577–581. Tomasini M, Magrini L, De Petrillo G: Serum levels of eosinophil cationic protein in allergic diseases and natural allergen exposure. J Allergy Clin Immunol 1996;97:1350–1355.

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Tomassini M, Magrini L, Bonini St: Increased serum levels of eosinophil cationic protein and eosinophil-derived neurotoxin (protein X) in vernal keratoconjunctivitis. Ophthalmol 1994;101: 1808–1811. Micera A, Bonini Se, Lambiase A, Sgrulletta R, Bonini St: Targets in ocular allergy 2004; in Pleyer U, Mondino B (eds): Uveitis and Immunological Disorders. Springer Verlag, 2004, pp 1–9.

Prof. Sergio Bonini IRCCS San Raffaele Via della Pisana 235 IT–00136 Rome (Italy) Tel. ⫹39 06 66130403, Fax ⫹39 06 66130407, E-Mail [email protected]

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Molecular Aspects of Allergy and Asthma Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 121–133

Fungal Allergies: A Yet Unsolved Problem Reto Crameri, Michael Weichel, Sabine Flückiger, Andreas G. Glaser, Claudio Rhyner Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland

Abstract Airborne fungal spores have been implicated as causative factors in respiratory allergy, particularly asthma. However, the prevalence of fungal sensitization is not known mainly due to the lack of standardized fungal extracts and to the overwhelming number of fungal species able to elicit IgE-mediated reactions. Recent work based on high-throughput cloning of fungal allergens revealed that fungi are able to produce extremely complex repertoires of species-specific and cross-reactive allergens. There is evidence that fungal sensitization also contributes to auto-reactivity against self-antigens due to shared epitopes with homologous fungal allergens. Detailed studies at structural and immunological level indicate molecular mimicry as a basic mechanism involved in perpetuation of severe chronic allergic diseases. The real challenge at present is not related to cloning or production of a large number of different fungal allergens but rather to the assessment of the clinical relevance of each single structure. To date, substitution of complex extracts presently used in the diagnosis of fungal allergy by single, perfectly standardized components seems feasible in contrast to specific immunotherapy which is still not developed. Recombinant fungal allergens might create new perspectives in diagnosis and therapy of fungal allergy. Copyright © 2006 S. Karger AG, Basel

The exact description of the immunological pathways leading to the development of allergic instead of normal immune responses is, in spite of large efforts, still incomplete. Obviously, a combination of genetic predisposition and environmental factors leads to the pathological state of atopy. Once established, allergic responses affect different organs and can lead to symptoms including nasal congestion, shortness of breath, skin rash and in extreme cases to anaphylaxis and death. The clinical pictures are described as rhinoconjunctivitis, asthma, urticaria, angioedema or atopic dermatitis. In Western countries, more than 20–30% of the population is affected by at least one manifestation

of allergic disease [1]. However, IgE-mediated allergic reactions are always related to (environmental) allergens. A considerable number of allergenic molecules associated with different forms of allergy have been cloned and characterized during the last two decades, and allergen sequence information is retrievable from different databases [2, 3]. Among the airborne allergens, fungal allergens, together with tree and grass pollen, constitute a major source of outdoor exposure, while in combination with allergens from dust mites, cockroaches and animal dander mould allergens are an important cause of indoor sensitization as well [4]. Fungi are eukaryotic organisms with a heterogeneous phenotype from unicellular (yeasts) to dimorphic or filamentous appearance. They are usually spore-bearing organisms, giving them a prominent place as allergen-spreading agents. The lack of chlorophyll and various biochemical markers, together with dendrograms derived from rDNA sequence comparisons clearly defines a kingdom of ‘fungi’ with a widespread diversity of phenotypes, occupying basically all ecological niches [5]. However, all members of the kingdom of fungi are strictly heterotrophic and depending on external sources of nutrients, therefore, living as saprobes, parasites or symbionts. Including yeasts and moulds, over 80,000 species of fungi are described [5]. As a consequence thereof, only very limited information about the role of fungi as allergenic sources are available. Respiratory allergic symptoms, primarily of asthma, have clearly been correlated with fungal exposure to spores of Aspergillus, Alternaria, Cladosporium, Penicillium or basdiomycetes [6].

Epidemiology

The moulds and fungi bear a widespread potential of causing diseases. Mycotic diseases include infections which can be cutaneous, localized or of invasive nature. Mycotoxicoses following the ingestion of toxic metabolites of fungi present different clinical pictures, which can vary from (desired) neurotoxic effects for party-participants to fatal cases caused by confusion of edible and toxic mushrooms. Some fungi are also producers of potent mycotoxins acting as carcinogens and teratogens like aflatoxins and ochratoxins [7]. In terms of allergy, three groups of fungi include most of the species relevant for allergic disease. First, the Zygomycota, including Rhizopus and Mucor (the bread moulds); second the Ascomycota, which are soil-settled agents including the families of Penicillium and Aspergillus, endophytes living on plants like Alternaria and Cladosporium or common commensal skin-colonizing yeasts like Candida and Malassezia. The third group is the Basidiomycota, including smuts, plant rusts, puffballs and mushrooms like the edible Boletus,

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Cantharellus and Coprinus [6]. Furthermore, spores deriving from these three groups contribute at most to the major airborne fungal spore load. Allergic reactions associated with fungi include asthma, allergic sinusitis, rhinitis, hypersensitivity pneumonitis, allergic bronchopulmonary mycoses, and atopic eczema. Sensitization to Alternaria species represents a significant risk factor for the development of severe asthma [8]. Allergic bronchopulmonary aspergillosis (ABPA) is a lung disease which is characterized by various symptoms summarized later on. Hypersensitivity pneumonitis is a syndrome with a variety of immunological symptoms related to pulmonary inflammation after inhalation of dusts of organic origin, including fungal spores, and the exact course and severity of the disease is a function of the frequency and intensity of the exposure. Other mould-mediated disorders include sick building syndrome, pulmonary hemorrhage and hemosiderosis related to exposure to Stachybotrys atra. The term sick building syndrome summarizes the constellation of symptoms like rhinitis, difficult breathing, headache, flu-like symptoms and teary eyes, associated with poor indoor air quality.

Incidence and Clinical Relevance of Fungal Allergies

Of the approximately 20% of the population of Western countries afflicted with allergic inhalant diseases such as asthma or rhinitis, 10% suffer from significant or severe allergic diseases. Results of skin test surveys suggest that at least 3–10% of the worldwide population is affected by fungal sensitization [6]. The exact prevalence of fungal sensitization has not yet been reliably established since the available reports of skin test reactivity to fungi vary from 3 to 90%. The huge variability results from the studied population, the tested species of fungi, and especially from the fungal extracts used [9]. This is not astonishing because even commercially available fungal extracts for skin testing show an extremely high variation in their allergen content. The problems related to standardization of fungal extracts are best exemplified by two pivotal works. The first one by Nikolaizik et al. [10] shows that skin tests with commercial extracts in patients suffering from cystic fibrosis sensitized to A. fumigatus with commercial extracts yield an ambiguous picture (fig. 1). These results were obviously related to the quality of the applied extracts which was not assessable at that time; however, a second report by Vailes et al. [11] clearly showed that the concentration of the major allergen Asp f 1 [12] in A. fumigatus extracts from eight different companies varied between 0.1 and 64 ␮g/ml. Since A. fumigatus is able to produce more than 80 major and minor allergens [13], the standardization of such an extract is an almost impossible task. The problem of

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Patient group

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Fig. 1. Skin test reactivity of patients suffering from cystic fibrosis and A. fumigatus allergy against different commercially available A. fumigatus extracts.⫺, Skin prick test positive, ⫹ skin prick test negative patient groups clearly showing the variable responses to different extracts.

preparing standardized fungal extracts is not confined to A. fumigatus, as shown by the huge variation in the content of the major allergen Alt a 1 in commercially available A. alternata extracts, but rather related to the complex allergen repertoire of most of the fungi [14]. Therefore, the prevalence of fungal allergy will only be a very approximate assumption until standardized extracts or recombinant fungal allergens become available for the testing of welldefined populations.

Diagnosis of Fungal Allergy

The diagnosis of type I allergic reactions is always associated with direct or indirect detection of allergen-specific IgE or biological consequences thereof. Standard skin prick tests (SPT) or intradermal skin tests can both be used to directly assess the reactivity of a patient to an extract or to a single molecule [15]. SPT are simpler to perform, faster, more comfortable for the patient and considered to be at lower risk than intradermal skin test for provocation of anaphylactic reactions. Therefore, SPT are recommended for routine assessments, epidemiological studies and in surveys involving a large population [16]. SPT results in clinical practice are routinely confirmed by the determination of allergen-specific serum IgE. Many in vitro methods for the determination of allergen-specific IgE are used, ranging from enzyme-linked immunosorbent assays and Western blot analyses in specialized laboratories to fully automated devices. Among these the PharmaciaCAP system is considered

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the ‘golden standard’ for the in vitro determination of IgE. The major breakdown in the diagnosis of allergy is derived from the fact that the experimental results directly depend on the quality of the used extracts, and unfortunately the extracts immobilized on the ImmunoCAP system are not available as SPT solutions, hampering a direct correlation between in vitro and in vivo diagnostic outcomes. Moreover, the clinical history that represents the perhaps more important diagnostic criterion for an allergy is difficult to reconstruct because most of the patients suffering from fungal allergy might, in contrast to other allergies, not be aware of the exposure, except that symptoms are usually not seasonal like in pollen-derived allergies. An additional complication derives from the fact that the number of fungi able to induce allergic responses exceeds by far the panel of extracts that can reasonably be tested in routine assessments. Therefore, environmental sampling of fungi at the assumed places of exposure is a valuable tool to narrow down the number of possible allergy-causing species to a testable subset. As impressively shown by a very recent study, airborne fungal components from species previously uncharacterized as allergen sources can considerably contribute to the total load of airborne allergens [17]. Studies on the basis of aerobiological surveys, skin test studies and allergen characterization suggest that at least Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Epicoccum nigrum, Fusarium roseum, Penicillium chrysogenum as well as the yeasts Candida and Malassezia should be considered as a minimal testing set when fungal allergy is suspected.

The Dimension of the Problem

Most cases of fungal allergy are associated with severe asthma or in the case of sensitization to yeasts to atopic dermatitis. In the USA around 16 million self-reported cases of asthma have been recorded in 2002 [18]. Depending on the definition, about 10–20% of these patients might be classified as subjects suffering from severe asthma and in this group 30–70% can be expected to be sensitized to at least one fungal species. Extrapolating this figure to the industrialized countries we have to assume that several millions of asthmatic patients are affected by fungal allergy. However, with exception of special cases like workplace exposure or ABPA which are well documented, the contribution of fungal sensitization to the severity of asthma remains to be investigated. A. fumigatus is the only mould which, according to the literature, is involved in a well-documented broad variety of pulmonary complications ranging from benign colonization of the lung to life-threatening disease like invasive aspergillosis or ABPA. However, also in the case of A. fumigatus large variations in the incidence of A. fumigatus sensitization and ABPA have been

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reported and are probably mainly due to the lack of reliable extracts. Since most of the clinical criteria used for the diagnosis of ABPA are not specific for the disease, serological findings showing sensitization to A. fumigatus are essential to confirm or exclude an ABPA suspected from clinical signs. ABPA is a disease with a high risk for the development of irreversible end stage fibrosis. Therefore, Greenberger and Patterson [19] recommend to rule out sensitization to A. fumigatus in all asthmatic patients.

Recombinant Fungal Allergens

The first biochemically purified fungal allergen was Asp f 1, a potent fungal ribotoxin [20], subsequently cloned and expressed as recombinant protein in E. coli [12]. The availability of recombinant Asp f 1 allowed to show that the biochemically purified allergen from the mould was still contaminated with IgE-binding proteins with similar biochemical or biophysical properties [12]. This example indicates the potential of molecular biology approaches to generate highly pure recombinant allergens satisfying the requirements for the use in human diagnosis and therapy. Although screening of cDNA libraries constructed in bacteriophage ␭ with sera of patients sensitized to fungi yielded the primary structure of additional allergens, the major breakthrough in cloning allergens from complex allergen sources was achieved by screening of cDNA libraries displayed on filamentous phage [21]. To date, a vast variety of different fungal allergens has been identified, cloned and produced [14], among these 81 complete and partial coding sequences of IgE-binding proteins from A. fumigatus [13]. Large scale serological and skin test evaluation of some of the recombinant A. fumigatus allergens revealed the existence of disease-specific molecules [22], opening new perspectives for diagnosis and management of ABPA. Although the pathogenic significance of ABPA-specific allergens is uncertain, they are of considerable diagnostic value to determine this particular asthma phenotype. Four of these allergens allowing discrimination between A. fumigatus sensitization and ABPA are now commercially available as single ImmunoCAPs (Pharmacia Diagnostics, Uppsala, Sweden). Moreover, a growing panel of allergens cloned from the yeast M. sympodialis showed that sensitization to this yeast occurs almost exclusively in patients suffering from atopic dermatitis [B. Fischer, pers. commun.]. They allowed to study the pathogenesis of this disease in more detail [23] and to partly explain at molecular level IgEmediated reactivity to self-antigens in long-lasting chronic inflammatory allergy [24, 25]. However, the way from cloning single fungal allergens to the reconstruction of the whole allergenicity of a fungal extract by mixing up single components is still long, although there is no doubt that such an approach is

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technically feasible. At present it has been clearly shown that the specificity of recombinant allergens used in both, skin test and serology is clearly superior to those obtained using commercial extracts [15], and there is a realistic hope that the sensitivity of diagnostic procedures based on recombinant allergens can be increased using panels of highly pure allergens. Although cloning and characterization of single allergens may be time-consuming, the advantages are manifold. The biotechnological processes allow the production of large quantities of pure proteins with reproducible batch-to-batch consistency. Therefore, problems related to contamination of the preparations with natural IgE-binding components, altered allergenicity due to extraction procedures and variability of allergen contents encountered during the preparation of extracts from natural sources can be solved. The limitations for the widespread use of recombinant allergens for the in vivo diagnosis of allergic conditions are presently due to ethical and legal restrictions, as in many countries their application in patients is not yet legalized [15].

Cross-Reactivity

IgE-mediated cross-reactivity is a frequently observed phenomenon in clinical practice and results from shared T- and B-cell epitopes present in homologous molecules from different allergenic sources. Polysensitization, which is easy to demonstrate by simple determination of the RAST value of sera against different allergen extracts (table 1), is partly due to cross-reactivity. To differentiate between independent sensitization to different allergenic sources and cross-reactivity, inhibition experiments determining the degree to which an extract in fluid phase can inhibit IgE-binding to a different solidphase coated extract as exemplified in figure 2 are useful. Cross-reactivity results in most of the cases from phylogenetically conserved proteins, formally termed pan-allergens. Among the kingdom of fungi many proteins, including many allergens, show extended homology at the primary and tertiary structure level. Well-studied examples thereof are manganese-dependent superoxide dismutase (MnSOD) [24, 26], acidic ribosomal P2 protein [25], cyclophilins (CyP) [27] and thioredoxins [Limacher, pers. commun.]. All these proteins are able to elicit skin test reactions and peripheral blood mononuclear cells proliferation in patients with serum IgE raised against these molecular structures. The postulated mechanism of cross-reactivity at molecular level is molecular mimicry between conserved T- and B-cell epitopes present on the structurally related proteins, although sensitization to each single allergen cannot be excluded. However, at least in some cases there is circumstantial evidence for molecular mimicry. For example it has been shown that IgE antibodies of individuals

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Table 1. Sensitization to different fungi of patients with a clinical history of A. fumigatus allergy determined by ImmunoCAP Patient

m3 (kUA/l)

m6 (kUA/l)

m2 (kUA/l)

m70 (kUA/l)

1 2 3 4 5 6 7 8 9 10

A. fumigatus ⬎100 ⬎100 99.3 70.4 61.5 45.3 11.3 10.6 5.01 1.95

A. alternata 20.4 5.63 5.89 4.32 16.5 18.5 42.8 22.9 23.4 9.56

C. herbarum 14.5 5.82 6.61 1.98 5.35 56.4 25.2 8.26 7.02 7.48

M. sympodialis 2.71 0.62 2.18 0.37 10.1 7.53 nd nd ⬎0.35 ⬎0.35

nd ⫽ Not determined.

2

OD 405nm

1.5

1

0.5

0 0.0001

0.001

0.01 0.1 Inhibitor concentration

1

10

Fig. 2. Inhibition of IgE-binding to A. fumigatus extract coated to solid phase by C. herbarum extract in fluid phase (I) and of C. herbarum extract coated to solid phase by A. fumigatus extract in fluid phase (⫹). The inhibition ELISA shows an extended crossreactivity between the extracts.

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sensitized to MnSOD of A. fumigatus fully cross-react with MnSOD of Drosophila melanogaster, although natural exposure to D. melanogaster is very unlikely to occur under normal circumstances. Moreover, D. melanogaster MnSOD can be used to stimulate proliferation of peripheral blood mononuclear cells from patients sensitized to A. fumigatus MnSOD indicating molecular mimicry not only at B but also at T-cell level [28]. Cloning, production and characterization of cross-reactive structures do not only help to understand clinically observed polysensitization phenomena but may also have direct clinical applications. Due to structural homology and immunological behavior of cross-reactive allergens, the number of epitopes needed for diagnosis and therapy of allergic diseases may be much less diverse than initially expected. This assumption is corroborated by high-resolution analyzes of crystal structures, among these MnSOD [24]. Only amino acid residues that are at least partly exposed to solvent can contribute to antigen-antibody interactions in native proteins. When comparing the crystal structures of homologous allergens, only identical or structurally similar residues which are solvent-exposed can account for cross-reactive B-cell epitopes. Superimposition of homologous structures allows fast and reliable identification of patches of conserved residues which most probably represent cross-reactive B-cell epitopes. Based on these results the actual contribution of a conserved, solventexposed residue to the binding of IgE and to the cross-reactivity can be investigated by site-directed mutagenesis and might lead to the production of a molecule with reduced IgE-binding capacity useful as candidate vaccine. If such molecules have the potential to induce desensitization to a whole panel of pan-allergens remains to be investigated. From a structural point of view fungal allergens can be subdivided into species-specific and cross-reactive allergens. Interestingly the major allergens of A. fumigatus, A. alternata and C. comatus, Asp f 1, Alt a 1, and Cop c 1, respectively, represent secreted species-specific proteins so far not found in any other organism. In contrast, cross-reactive allergens represent conserved intracellular proteins that show significant homology even between phylogenetically distant species. Representatives of this group are enolases, peroxisomal proteins, aldehyde dehydrogenases, heat shock proteins and the already mentioned MnSODs, acidic ribosomal P2 proteins, CyP, nuclear transport factors 2 and thioredoxins. All the cross-reactive allergens described so far have been found to share common features at the level of primary and tertiary structure. In contrast, not all proteins that share a similar fold or a similar molecular shape are necessarily cross-reactive. Similar protein folds can already be found with a sequence identity of as little as 25% while cross-reactivity is very rare below 50% of sequence identity [29]. However, the main problem related to fungal allergy results from the vast variety of different IgE-binding molecular

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structures, whether cross-reactive or not, that a single fungal species is able to produce. Apart from laborious cloning the real challenge is to assess the clinical relevance of each single structure. Sensitization to single structures among populations of patients suffering from a given fungal allergy range from more than 70% as in the case of Asp f 1 [12, 20] to a few percent as reported for example for the hydrophobin of C. herbarum [30]. Since very few contradictory reports about the efficacy of fungal immunotherapy are reported in the literature it is at present impossible to judge how many different molecular structures will be required to cover the allergen repertoire reflecting clinical symptoms after exposure.

Involvement of Fungal Allergens in the Pathogenesis of Severe Atopic Diseases

As already mentioned, many fungal allergens represent phylogenetically highly conserved structures. The sequence identity within these allergen families, which include also human proteins, range from 50 to more than 80% [23, 24, 26] and, therefore, cross-reactivity can be expected [29]. Many examples of structure-based cross-reactivity have been demonstrated not only between different fungal species but also between completely unrelated allergenic sources like A. fumigatus and H. brasiliensis [31]. Moreover, CyP, an allergenic structure first described in fungal species [32], has been reported as an allergen also present in pollens [33] and foods [34]. Interestingly, many of these phylogenetically highly conserved structures show cross-reactivity also to the corresponding human homologous proteins. This is quite surprising because the presence of a human protein homologous to an allergen accessible for the immune system would be expected to induce immune tolerance at least to the shared epitopes. Moreover, the highly conserved proteins like MnSOD, CyP and thioredoxin are intracellular enzymes and as such not expected to be accessible to the immune system under normal circumstances. However, in some pathological situations like chronic inflammation these proteins can become available to the immune system as a consequence of tissue damage caused by inflammatory processes [35]. Therefore, exposure to self-antigens containing cross-reactive determinants can occur in vivo and contribute to the perpetuation of allergic symptoms. A direct evidence for the possible involvement of MnSOD in the pathogenesis of atopic dermatitis has been recently shown [23]. Specific IgE antibodies against human MnSOD could be detected in 36% of tested sera from AD patients. All these patients were concomitantly sensitized to the skin-colonizing yeast M. sympodialis and strongly reacted in

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skin tests to M. sympodialis extract, to recombinant human MnSOD and to other structurally related MnSODs. Application of highly pure recombinant human MnSOD on healthy skin of patients suffering from severe atopic eczema in atopy patch tests was sufficient to elicit an eczematous reaction [23]. Moreover, histochemical investigations of skin sections obtained from lesional areas showed a local upregulation of MnSOD expression. MnSOD as well as CyP and thioredoxin are stress-inducible proteins, and therefore expected to be overexpressed under stress conditions. Such conditions are likely to occur in lesional skin areas due to mechanical irritation derived from scratching as a consequence of pruritus. Local release of self-antigens at the site of inflammation due to mechanical damage and IgE-mediated reactions with the cross-reactive antibodies raised against highly homologous environmental allergens is thus likely to contribute to exacerbation and/or perpetuation of the allergic symptoms also in absence of external exposure. Similar mechanisms have been proposed to be involved in the pathogenesis of ABPA [24, 25], another severe chronic and inflammatory form of allergy, although evidences for upregulation of stress-inducible proteins in the lung are lacking. Further studies based on the solved crystal structures of the CyPs from A. fumigatus and M. sympodialis as well as the crystal structure of thioredoxin from M. sympodialis [A. Limacher, pers. commun.] will further contribute to the elucidation of the pathophysiological mechanisms involved in chronic inflammatory allergies.

Conclusions

In spite of the relevant progress achieved during the past decade, fungal allergy is still an unsolved problem. There is no doubt that fungi are involved in allergic diseases, especially in asthma and, as best documented example, in ABPA. However, even in the case of ABPA and in spite of several efforts, the prevalence of the disease remains unknown [36]. If indeed fungal allergy is a distinct clinical entity, its prevalence will not be known until several standards are met. Development of standardized diagnostic criteria and laboratory tests will be essential to provide more reliable statistical data. Our best understanding of fungal sensitization under existing limitations suggests rates around 3%, whereas in selected patients with asthma, the sensitization rate might increase up to 30%. These projections should be relevant enough to encourage intensification of research in the field of fungal allergy, which is slowly moving from the stage of descriptive biology to a discipline based on modern molecular biology.

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Acknowledgements We are grateful to all former members of the Molecular Allergology Group and to Prof. Kurt Blaser for his continuous support. Work supported by the Swiss National Science Foundation grants 3100–063381.00 and 3600–063381.00/2, by the OPO-Stiftung Zürich and by the EMDO-Stiftung, Zürich.

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6 7 8 9

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Casolaro V, Georas SN, Song Z, Ono SJ: Biology and genetics of atopic disease. Curr Opin Immunol 1996;8:796–803. http://www.allergen.org/List.htm. http://www.allergome.org. Erwin EA, Platts-Mills TA: Allergens. Immunol Allergy Clin North Am 2005;25:1–14. Prillinger H, Lopandic K, Schweigkofler W, Deak R, Aarts HJM, Baur R, Sterflinger K, Kraus GF, Maraz A: Phylogeny and systematics of the fungi with special reference to the Ascomycota and Basidiomycota. Chem Immunol 2002;81:207–295. Horner WE, Helbling A, Salvaggio JE, Lehrer SB: Fungal allergens. Clin Microbiol Rev 1995;8: 161–179. Mishra HN, Das C: A review on biological control and metabolism of aflatoxin. Crit Rev Food Sci Nutr 2003;43:245–264. Neukirch C, Henry C, Leynaert B, Liard R, Bousquet J, Neukirch F: Is sensitization to Alternaria alternata a risk factor for severe asthma? J Allergy Clin Immunol 1999;103:709–711. Mari A, Schneider P, Wally V, Breitenbach M, Simon-Nobbe B: Sensitization to fungi: Epidemiology, comparative skin tests, and IgE reactivity of fungal extracts. Clin Exp Allergy 2003;33: 1429–1438. Nikolaizik WH, Crameri R, Blaser K, Schöni MH: Skin test reactivity to recombinant Aspergillus fumigatus allergen I/a in patients with cystic fibrosis. Int Arch Allergy Immunol 1996;111: 403–408. Vailes L, Sridhara S, Cromwell O, Weber B, Breitenbach M, Chapman M: Quantitation of the major fungal allergens, Alt a 1 and Asp f 1, in commercial allergenic products. J Allergy Clin Immunol 2001;107:641–646. Moser M, Crameri R, Menz G, Schneider T, Dudler T, Virchow C, Gmachl M, Blaser K, Suter M: Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE binding and type I skin test activity. J Immunol 1992;149:450–460. Kodzius R, Rhyner C, Konthur Z, Buczek D, Lehrach H, Walter G, Crameri R: Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays. Comb Chem High Throughput Screen 2003;6:147–154. Weichel M, Flückiger S, Crameri R: Molecular characterisation of mould allergens involved in respiratory complications. Recent Res Dev Respir Critical Care Med 2002;2:29–45 (ISBN: 81–7736–143–0). Schmid-Grendelmeier P, Crameri R: Recombinant allergens for skin testing. Int Arch Allergy Immunol 2001;125:96–111. The European Academy of Allergology and Clinical Immunology: Position paper. Allergen standardization and skin tests. Allergy 1993;48:48–82. Green BJ, Sercombe JK, Tovey ER: Fungal fragments and undocumented conidia funcion as new aeroallergen sources. J Allergy Clin Immunol 2005;115:1043–1048. http://www.cdc.gov/asthma/brfss/02/current/tableC1.htm. Greenberger PA, Patterson R: Allergic bronchopulmonary aspergillosis and the evaluation of the patient with asthma. J Allergy Clin Immunol 1988;81:646–650. Arruda LK, Platts-Mills TA, Fox JW, Chapman MD: Aspergillus fumigatus allergen I, a major IgEbinding protein, is a member of the mitogillin family of cytotoxins. J Exp Med 1990;172:1529–1532.

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Rhyner C, Weichel M, Flückiger S, Hemmann S, Kleber-Janke T, Crameri R: Cloning allergens via phage display. Methods 2004;32:212–218. Crameri R, Hemmann S, Ismail C, Menz G, Blaser K: Disease-specific recombinant allergens for the diagnosis of allergic bronchopulmonary aspergillosis. Int Immunol 1998;10:1211–1216. Schmid-Grendelmeier P, Flückiger S, Disch R, Trautmann A, Wüthrich B, Blaser K, Scheynius A, Crameri R: IgE-mediated ant T-cell-mediated autoimmunity against manganese superoxide dismutase in atopic dermatitis. J Allergy Clin Immunol 2005;115:1068–1075. Crameri R, Faith A, Hemmann S, Jaussi R, Ismail C, Menz G, Blaser K: Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus. J Exp Med 1996;184:265–270. Mayer C, Appenzeller U, Seelbach H, Achatz G, Oberkofler H, Breitenbach M, Blaser K, Crameri R: Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus. J Exp Med 1999;189:1507–1512. Flückiger S, Mittl PRE, Scapozza L, Fijten H, Folkers G, Grütter MG, Blaser K, Crameri R: Comparison of the crystal structures of the human manganese superoxide dismutase and the homologous Aspergillus fumigatus allergen at 2-Å resolution. J Immunol 2002;168:1267–1272. Flückiger S, Fijten H, Whitley P, Blaser K, Crameri R: Cyclophilins, a new family of cross-reactive allergens. Eur J Immunol 2002;32:10–17. Flückiger S, Scapozza L, Mayer C, Blaser K, Folkers G, Crameri R: Immunological and structural analysis of IgE-mediated cross-reactivity between manganese superoxide dismutases. Int Arch Allergy Immunol 2002;128:292–303. Aalberse RC: Structural biology of allergens. J Allergy Clin Immunol 2000;106:228–238. Weichel M, Schmid-Grendelmeier P, Rhyner C, Achatz, Blaser K, Crameri R: Immunoglobulin Ebinding and skin test reactivity to hydrophobin HCh-1 from Cladosporium herbarum, the first allergenic cell wall component of fungi. Clin Exp Allergy 2003;33:72–77. Wagner S, Sowka S, Mayer C, Crameri R, Focke M, Kurup VP, Scheiner O, Breiteneder H: Identification of Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a crossreactive allergen. Int Arch Allergy Immunol 2001;125:120–127. Horner W, Reese G, Lehrer SB: Identification of the allergen Psi c 2 from the basidiomycete Psilocybe cubensis as a fungal cyclophilin. Int Arch Allergy Immunol 1995;107:298–300. Cadot P, Diaz JF, Proost P, Van Damme J, Engelborghs Y, Stevens EA, Ceuppens JL: Purification and characterization of an 18 kD allergen of birch (Betula verrucosa) pollen: Identification as cyclophilin. J Allergy Clin Immunol 2000;105:286–291. Fujita C, Moriyama T, Ogawa T: Identification of cyclophilin as an IgE-binding protein from carrots. Int Arch Allergy Immunol 2001;125:44–55. Zhao ZS, Granucci F, Yeh L, Schaffer PA, Cantor H: Molecular mimicry by herpes simplex virustype 1: Autoimmune disease after viral infection. Science 1998;279:1344–1347. Novey HS: Epidemiology of allergic bronchopulmonary aspergillosis. Immunol Allergy Clin North Am 1998;18:641–653.

Prof. Reto Crameri, PhD Swiss Institute of Allergy and Asthma Research (SIAF) Obere Strasse 22 CH–7270 Davos (Switzerland) Tel. ⫹41 81 410 0848, Fax ⫹41 81 410 0840, E-Mail [email protected]

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Molecular Aspects of Allergy and Asthma Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 134–146

Structural Features of Allergenic Molecules Rob C. Aalberse Sanquin Research at CLB and Karl Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Abstract In this paper the relation between protein allergenicity (the capacity to induce IgE antibody production or the capacity to activate mast cells sensitized with IgE antibodies induced by a cross-reactive allergen) and protein structure is discussed. While cross-reactivity is to a large degree predictable from primary sequence comparisons, the IgE-inducing capacity is mostly determined by factors other than the primary sequence. Two routes to IgE are discussed: (1) the atopic route (used by allergens from pollen and mites) in which a direct switch from ␮ to ␧ is common and (2) the ‘modified Th2’ route (used by allergens from pets) in which the class switch to IgE is often preceded by a switch to IgG4. According to this working hypothesis, the choice between these two routes is determined at the level of the germinal center activity. Copyright © 2006 S. Karger AG, Basel

Allergenic activity reflects two properties: (1) the potential to induce IgE antibodies and (2) the potential to induce symptoms (or a positive skin test) in a sensitized subject. These two properties are not necessarily linked. Some proteins induce symptoms in a substantial number of subjects, but rarely induce IgE antibodies. A prototypic protein in this category is the apple allergen Mal d 1. This protein induces symptoms largely in patients that have become sensitized by inhalation of birch pollen grains. The birch allergen Bet v 1 in these pollen grains induce IgE antibodies, some of which cross-react with the homologous protein in, for example, apple. The consumption of apples rarely, if ever, induces IgE antibodies in immunologically naïve subjects (i.e. Mal d 1 has low

‘primary allergenicity’). It is not clear yet to what extent the consumption of apples by subjects with pre-existing IgE antibodies to Bet v 1 triggers naïve, Mal d 1-specific B cells (‘secondary allergenicity’). This issue of ‘secondary allergenicity’ is closely related to the phenomenon of epitope spreading and its converse: the ‘original antigenic sin’. These concepts will be discussed in more detail below. These two aspects of allergenic activity (IgE immunogenicity and crossreactivity) can conveniently be discussed in the context of the two cells that are central in these two phenomena: the B cell and the mast cell, respectively. The simple, but important, message is that it is easy to trigger a sensitized mast cell, whereas it is difficult to induce B cells to produce IgE. Crossreactivity is dictated by structural similarity and is thus largely predictable if structural information is available for the two allergens involved (logic dictates that one needs at least two allergens to discuss cross-reactivity). On the other hand, IgE immunogenicity is determined largely by factors unrelated to the primary structure of the protein (such as the ‘context’ in which the allergen enters the body) and is thus much more difficult to predict. Crossreactivity is relevant not only in relation to mast cell activation, but also in relation to allergen-induced B- and T-cell activation. For this reason, this presentation is given under 3 main headings below: Cross-reactivity in relation to mast cell triggering, cross-reactivity in relation to B-cell activation and IgE immunogenicity.

Cross-Reactivity in Relation to Mast Cell Triggering

In this paragraph I will only discuss cross-reactivity in relation to mast cell triggering. This is relatively straightforward, because it concerns only B-cell epitopes. A much more complicated issue is cross-reactive priming, i.e. the role of cross-reactivity on a subsequent immune response (for which both T- and B-cell epitopes need to be considered). This will be discussed later. In contrast to T-cell cross-reactivity, B-cell cross-reactivity is very much a 3D (conformational) feature: the antigen has to have some intrinsic 3D structure for the antibody to bind. The concept of an antigen being very adaptable to the shape of the antibody or vice versa (i.e. the extreme end of the spectrum of ‘induced fit’ that was suggested in the early concepts of antigen-antibody interactions) is thermodynamically not compatible with the combination of high specificity and high avidity that is the striking feature of the antigen-antibody interactions that we are discussing here.

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Cross-reactivity requires similarity in 3D structure, not only in the strictly architectural sense, but also in the physicochemical properties such as similar charge distribution, hydrogen-bonding potential and hydrophobicity patterns. In addition to being similar to the cross-reacting partner, the 3D structure should not be too similar to structures in the human subject, because many such auto-reactive B cells will have been deleted from the repertoire. For crossreactivity prediction, a 3-way comparison is therefore relevant: (1) the target (potentially cross-reactive) allergen; (2) the sensitizing allergen and (3) the human homolog(s) (fig. 1). Cross-reactivity can conveniently be subdivided into reactivity to peptide and nonpeptide epitopes (typically post-translational modifications of proteins such as glycosylation or haptenization). The information regarding the first type can be found from DNA sequence data on the allergen, whereas post-translational modification to a large degree depends on the host (host organism and/or host tissue type) in which the allergen resides before it hits the sensitized mast cell. A hybrid situation arises when a post-translational modification changes the structure of the protein (for example, removal of a pro-peptide). IgE epitopes generated by glycosylation have been relatively well studied. The types of glycosylation are in general well conserved within phylogenetic classes. This explains why it is rare to find immune reactivity to vertebrate glycan chains: all vertebrates have largely the same spectrum of glycan-generating enzymes. There are a few exceptions: enzymes that are found (or absent) in a subset of vertebrates, but there have been no reports on IgE epitopes on the resulting glycans. On the other hand, nonvertebrate glycans have a common ‘strangeness’ to the human immune system, which explains why widespread cross-reactivity (cross-reactive carbohydrate determinant [1]) is relatively frequent. It is of note that it has been proven hard to find murine monoclonal antibodies with the wide cross-reactivity spectrum found in many human sera. This might reflect the preferential stimulation of B cells to widely cross-reactive carbohydrate determinant epitopes as opposed to B cells to more restricted cross-reactive carbohydrate determinant epitopes. For peptide epitopes reliable in silico prediction of cross-reactivity is well within reach. An important observation is that cross-reactive proteins belong to similar protein fold families, whereas not all proteins belonging to the same protein fold family are cross-reactive. Knowledge on protein fold families is not quite complete, but it is already rare to find a completely new fold for a protein (particularly for soluble, relatively stable proteins such as most allergens). Since algorithms for prediction of the protein fold family for a novel protein are

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Bovine albumin

76.3

Dog albumin

Birch profilin

Grass profilin

74.8

46 71

56

95

31

27 42

399 4

6

68 43 87 75 Human albumin

75.8

Mite GST

36.7

Schisto GST

Human profilin

34.6

Shrimp tropomyosin

80.7

36.6

Mite tropomyosin

79

15

102

80.0

107

39 41

65

142 24

31

37

12

84 106

47.0

Human GST

44.2

56.2

Human tropomyosin

58.5

Fig. 1. Venn diagrams showing the amino acid identity between 4 protein triples: serum albumin, profilin, glutathione S-transferase (⫽GST) and tropomyosin. For all 4 proteins one of the 3 proteins in the comparison is the human protein. The other 2 proteins are from allergenic sources (Schisto ⫽ Schistosoma). The 7 numbers within each Venn diagram indicate the number of identical amino acids. The three numbers around each Venn diagram indicate the pair-wise percent identity. The message is that sequence identity between two allergens is not the only factor in relation to cross-reactivity, because a substantial number of the amino acids that are identical between two allergens are also identical in the human homologue. For examples: bovine albumin and dog albumin share 445 amino acids, but of these only 46 are not found in human serum albumin.

already very efficient, cross-reactivity targets can be identified. The major current limitation is that not all allergens have been sequenced yet, so the database is incomplete. This is true particularly for allergens to which less than 50% of the patients have substantial IgE antibodies.

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Cross-Reactivity in Relation to B-Cell Activation: Cross-Reactive Priming, Epitope Spreading and ‘The Original Antigenic Sin’

What happens when the immune system is first triggered by one allergen and subsequently exposed to a related (cross-reactive) allergen; in other words: what are the potential effects of cross-reactive priming? If the two allergens are very similar, the response will be undistinguishable from a regular anamnestic response. In general, such a response will be largely derived from memory cells: these are more numerous and more easily triggered than naïve B cells. Moreover, immune complexes formed between the allergen and the IgG antibodies induced by the priming reaction will have a negative effect on naïve B cells (due to negative signals from the Fc␥2b receptor on naïve B cells). Based on this scenario, the antibody response would be expected to be mainly of the same specificity as found during the priming reaction: the cross-reacting allergen preferentially stimulates the cross-reacting B cells rather than B cells specific for the second allergen. While this effect (‘the original antigenic sin’, first described in relation to influenza immunity [2]) is seen in many instances, the opposite is not at all uncommon: the induction of new specificities (‘epitope spreading’). In this context it is relevant to mention some of the features that make Th2 responses distinct from Th1 antibody responses such as to influenza. (1) Neither IgE nor IgG4 antibodies do efficiently interact with the Fc␥2b receptor; (2) IgE antibodies may engage naïve B cells via the low-affinity IgE receptor, which enhances rather than suppresses B-cell activity; (3) as will be discussed below, IgE-switched memory B cells are rare, which makes naïve B cells more likely to compete effectively; (4) IgE-allergen interactions create a milieu that favors Th2 B-cell responses, including responses to allergens for which no prior immune response had occurred (resulting in IgE to ‘bystander’ allergens): IgE breeds IgE [3].

IgE Immunogenicity

In the immunogenicity part of this paper, the focus will be very much on B-cell development, without much attention to the Th2 cell. This does not imply that the Th2 cell is considered to be irrelevant. There is no doubt that Th2 cells are essential for the process. However, it is much more a matter of debate which is the major limiting factor: the B cell or the Th2 cell. It could be argued that during the initial phase of sensitization, only a limited extent of Th2 activation is required, while a much more extensive Th2 activation develops secondary to

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the initiation of IgE antibody production, via the IgE-facilitated T-cell activation pathway [4–7].

The ‘Healthy’ Antiallergen Immune Response and Its Relation to the Modified Th2 Response

A fundamental immunological question in relation to IgE immunogenicity is how the majority of the human population avoids making IgE antibodies to specific allergens. Akdis and colleagues [8, 9] formulated the concept of the ‘healthy’ immune response, in which it is assumed that allergen exposure induces an immune response in everybody, but that this response does not necessarily develop into an allergic response. The development of an allergic response is proposed to be actively suppressed, largely by the activity of regulatory T cells. The modified Th2 response is a related concept. This was originally described as a Th2 response without IgE antibodies [10, 11]. IgG4 antibodies were used as a read-out for a Th2 response. This definition might suggest (1) that the modified Th2 response is an aborted Th2 response (i.e. aborted at an IgG4 ‘intermediary’ stage) and that a ‘nonmodified’ Th2 response not only should include IgE antibodies, but also IgG4 antibodies. I will be using the ‘modified Th2’ nomenclature in a slightly different way. In my view the nonmodified Th2 response rather than the modified Th2 response is an aborted reaction. The nonmodified Th2 response reflects an immune response to a classical atopic allergen exemplified by a pollen or mite allergen. In contrast, the modified Th2 response reflects a conventional immune response to relatively high dose of antigen that fails to induce a strong Th1 response, typically ‘clean’ protein antigens lacking microbial components that might be recognized by Toll-like receptors. A prototypic example is the major mouse allergen Mus m 1 (mouse urinary protein), but tetanus toxoid (from the vaccine) might also be taken as an example. It is of note that in this view of the modified Th2 response, such a response may occasionally include IgE antibodies (whereas these are absent by definition in the original description). The ‘healthy’ immune response mentioned above is in many ways similar to the ‘modified’ Th2 response (even if the former concept, which is largely focused on T-cell responses, does not actually exclude pure Th1 immune responses, whereas the latter concept obviously does). The spectrum of immune reactivity that can be induced by potential allergens is, however, much broader. For the sake of discussion I will distinguish six categories of immune responses to environmental proteins: (1) No response (2) T-independent response: no IgE

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(3) (4) (5) (6)

T-cell only (Th1, Th2 and/or TReg) Th1 ⫹ B cell Th2 ⫹ B cell (modified Th2) Th2 ⫹ B cell (nonmodified ⫽ atopic Th2) Category 2 is presumably not relevant for IgE allergenicity, as it seems likely that the switch to IgE in vivo requires Th2 support. An important issue is how common it is to find category 1 (no immune response, which I equate to immunological ignorance) or category 3 (T-cell priming without noticeable activation of allergen-specific B cells) in the general population. I assume that this is common for classical atopic allergens, which in this area of the world are allergens such as those from pollen and mites. Many investigators in this field do not share my view that there is little or no B-cell response to allergens from pollen or mite in nonatopic subjects. This discrepancy is largely due to technical issues related to the measurement of IgG antibodies to allergens: reports on the presence of substantial levels of IgG antibodies to pollen or mite allergens in nonatopic subjects are mainly based on solid-phase assays (enzyme-linked immunosorbent assay or immunoblot), often with total allergen extracts. In these reports the prevalence of such antibodies is not different between atopic and nonatopic subjects. In contrast, a striking difference between atopic and nonatopic subjects is found when 125I-labelled, purified allergens are used in fluid-phase assays. With the latter type of assay little or no antibody production is demonstrable in nonatopic subjects. This is the hallmark difference between antigens inducing responses of categories 5 and 6, respectively (for example, the grass pollen allergen Lol p 1 vs. Mus m 1): the prevalence of high-affinity IgG responses in the absence of IgE responses. For classical atopic allergens (which induce a ‘nonmodified’ Th2 response ⫽ category 6), such as Dac g 1, it is rare to find substantial levels of IgG antibodies in the absence of IgE antibodies (fig. 2). In contrast, for antigens that induce ‘modified’ immune responses, such as Fel d 1 (fig. 2) or Mus m 1, it is much less unusual to find IgG antibodies in the absence of detectable IgE antibodies. In both instances, IgG1 antibodies are initially predominating, but IgG4 antibodies may overtake the response upon prolonged heavy exposure (more common in the ‘modified’ Mus m 1 situation than in the ‘atopic’ Lol p 1 situation). The dichotomy at the antigen level suggested by the above description of immune responses in the categories 5 and 6 is an oversimplification. In reality it is much more likely to be a spectrum of reactivity, both among subjects and among antigens. However, for the sake of simplicity I will focus on the extremes of this spectrum.

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Working Hypothesis: the ‘Nonmodified’ Th2 Response is an Incomplete B-Cell Response Which Fails to Induce Mature Germinal Centers

Which immunological mechanism could explain the difference between these two responses (and thus between these two categories of antigens)? As a working hypothesis the following scenario is envisaged [12]. It is assumed that the atopic, nonmodified Th2 response is induced by a weak immunological stimulus, which just manages to initiate a B-cell reaction but fails to trigger a full-blown response. In particular, it fails to induce a mature germinal center (fig. 3a). This incomplete response permits the development of small numbers of isotype-switched B cells, without much preference for or against a particular isotype (including IgE). Because mature germinal centers are assumed not to develop, no major clonal expansion will occur. Moreover, B memory formation

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also depends on germinal centers, so no significant B memory is generated. These B cells may develop into plasma cells, some of which may survive for extended periods (years), provided that suitable survival niches are available [13–17]. In contrast, the modified Th2 response involves a full-blown B-cell response, including the formation of germinal centers (fig. 3b). In these germinal centers IgE-switched B cells are deleted, either by negative selection or by neglect. Why would an IgE-switched B-cell have a hard time to survive? One possible route leading to deletion of IgE-switched B cells in active germinal centers is the low level of expression of surface-anchored immunoglobulin [18]. For B cells to survive it is essential to express sufficient membrane-anchored immunoglobulin. Deletion of B cells lacking surface immunoglobulin is an important quality check following isotype switching and somatic hypermutation in the germinal center, because B cells in which these processes lead to nonproductive genes will be removed. An alternative explanation for the poor survival of IgE-switched B cells might be a negative signal via the interaction between IgE anchored in the membrane of the IgEswitched B-cell and CD23 expressed on follicular dendritic cells in the germinal center [19]. A testable prediction from this scenario is that the IgE-producing cells in the ‘atopic’ situation (e.g. untreated pollen- or mite allergy) are unlikely to have switched their isotype more than once, so analysis of switch regions of IgEswitched cells should indicate a predominance of direct (␮ to ␧) rather than a sequential (␮ to ␥4 to ␧) class switches. For this analysis it is crucial to exclude class-switching in vitro, so this analysis should not be performed on in vitro cultured cells. Experiments in rodents have shown that increasing the antigen dose increases the proportion of IgE produced via sequential rather than direct switching [20].

To What Extent Is IgG4 Special in This Context?

In the original description of the ‘modified Th2 response’ [10, 11], the emphasis was on IgG4, because IgG4 is a Th2-dependent isotype. However, IgG1 rather than IgG4 antibodies are the dominating IgG isotype in untreated allergic patients. It is therefore a misconception to associate IgG1 antibodies in the human with a Th1 response. Due to technical issues the measurement of allergen-specific IgG4 is less error-prone than that of allergen-specific IgG1. It is relevant to stress that the IgG assays mentioned in this presentation have all been performed with trace amounts of iodinated purified allergens, thus ensuring the selective measurement of high-affinity antibodies. IgG results

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obtained with enzyme-linked immunosorbent assays or immunoblots tend to be significantly different, and usually show a much less clear-cut distinction between atopic and nonatopic subjects.

Consequences for the Allergenicity Issue

The debate on ‘what makes an allergen an allergen’ is still ongoing. After a review of structural features of the allergens for which such information was available at the time (2000), I reached the conclusion that ‘allergens have no characteristic structural features other than that they need to be able to reach (and stimulate) immune cells and mast cells. Within this constraint, any antigen may be allergenic particularly if it avoids activation of Th2-suppressive mechanisms (CD8 cells, Th1 cells)’ [21]. At the present time, I would have added ‘or other regulatory T cells, or the production of regulatory cytokines such as IL-10 or TGFb’. Structural information on additional allergens has become available, but this does not really alter the message. For a protein to be allergenic it obviously needs to be immunogenic, i.e. it has to be an antigen. More specifically, since the IgE response is being discussed, it has to stimulate not only Th2 cells, but also B cells. Furthermore, early deletion of IgE-switched B cells (as described above) should be avoided. These features are to a large degree dictated by the ‘context’ of the potential allergen rather than by structural features of the allergen: how does the antigen enter the body and which cofactors are present? An interesting example is tetanus toxoid. This antigen is not in general viewed as an allergen, but is nevertheless capable of inducing IgE antibodies, usually without clinical consequences [22, 23]. If B. pertussis is administered at the same time as the tetanus toxoid, the production of IgE antibodies to tetanus is markedly suppressed [24]. This illustrates how the allergenicity of a protein is dependent on its context. Another (theoretical) example is the apple allergen, Mal d 1. As mentioned before, IgE antibodies reactive with this protein are seldom, if ever, induced by eating apples, but by inhaling pollen containing a closely related protein, such as Bet v 1. It is likely that the intrinsic allergenicity of Mal d 1 is not substantially different from that of its birch homolog Bet v 1, but it is not presented ‘properly’ to the immune system. How does allergenicity relate to the ‘modified Th2’ concept? Despite its name, the ‘modified Th2’ immune response is a ‘normal’, well-controlled immune response. Even if IgE antibodies are occasionally induced, these tend to be less problematic, possibly because of their clonal relation to IgG antibodies (which, because these IgG antibodies are produced in excess, may effectively compete with these clonally related IgE antibodies for allergen).

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In contrast, if allergens induce a ‘nonmodified’, or ‘atopic’ Th2 response, there is little, if any, clonal relation between IgE and IgG and the amount of IgG is also likely to be lower. IgG-producing plasma cells induced by immunotherapy are usually also not clonally related to the IgE-producing cells and may therefore be less effective in blocking the IgE-allergen interaction. The difference between these two classes of allergens is, by lack of full understanding, referred to as ‘antigenic strength’. The level of allergen exposure is one, but certainly not the only determinant of this difference. References 1 2 3 4 5 6 7

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Aalberse RC, Koshte V, Clemens JG: Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom. J Allergy Clin Immunol 1981;68:356–364. Fazekas de St Groth B, Webster RG: Disquisitions on original antigenic sin. I. Evidence in man. J Exp Med 1966;140:2893–2898. Aalberse RC: The IgE response and atopy. Eur Resp J 1991;4:S78–S84. Mudde GC, Bheekha R, Bruijnzeel-Koomen CA: Consequences of IgE/CD23-mediated antigen presentation in allergy. Immunol Today 1995;16:380–383. Mudde GC, Bheekha R, Bruijnzeel-Koomen CA: IgE-mediated antigen presentation. Allergy 1995;50:193–199. Mudde GC, Hansel TT, Reijsen FC, Osterhoff BF, Bruijnzeel-Koomen CAFM: IgE: An immunoglobulin specialized in antigen capture. Immunol Today 1990;11:440–443. Santamaria LF, Bheekha R, Van Reijsen FC, Perez S, Suter M, Bruijnzeel-Koomen CAFM, et al: Antigen focusing by specific monomeric immunoglobulin E bound to CD23 on Epstein-Barr virus-transformed B cells. Hum Immunol 1993;37:23–30. Akdis M, Verhagen J, Taylor A, Karamloo F, Karagiannidis C, Crameri R, et al: Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med 2004;199:1567–1575. Taylor A, Verhagen J, Akdis CA, Akdis M: T regulatory cells in allergy and health: A question of allergen specificity and balance. Int Arch Allergy Immunol 2004;135:73–82. Platts-Mills T, Vaughan J, Squillace S, Woodfolk J, Sporik R: Sensitisation, asthma, and a modified Th2 response in children exposed to cat allergen: A population-based cross-sectional study. Lancet 2001;357:752–756. Platts-Mills TA, Woodfolk JA, Erwin EA, Aalberse R: Mechanisms of tolerance to inhalant allergens: The relevance of a modified Th2 response to allergens from domestic animals. Springer Semin Immunopathol 2004;25:271–279. Aalberse RC, Platts-Mills TA: How do we avoid developing allergy: Modifications of the TH2 response from a B-cell perspective. J Allergy Clin Immunol 2004;113:983–986. Holt PG, Leivers S: Radiation-resistant IgE-secreting cells in the mouse: Susceptibility to suppressor T cells. Int Arch Allergy Appl Immunol 1983;71:188–190. Holt PG, Sedgwick JD, O’Leary C, Krska K, Leivers S: Long-lived IgE- and IgG-secreting cells in rodents manifesting persistent antibody responses. Cell Immunol 1984;89:281–289. Holt PG, Turner KJ: Persistent IgE-secreting cells which are refractory to T-cell control. Int Arch Allergy Appl Immunol 1985;77:45–46. Manz RA, Arce S, Cassese G, Hauser AE, Hiepe F, Radbruch A: Humoral immunity and longlived plasma cells. Curr Opin Immunol 2002;14:517–521. Manz RA, Radbruch A: Plasma cells for a lifetime? Eur J Immunol 2002;32:923–927. Karnowski A, Yu P, Achatz G, Lamers MC: The road to the production of IgE is long and winding. Am J Respir Crit Care Med 2000;162(3 Pt 2):S71–S75. Rabah D, Conrad DH: Effect of cell density on in vitro mouse immunoglobulin E production. Immunology 2002;106:503–510.

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Sudowe S, Rademaekers A, Kolsch E: Antigen dose-dependent predominance of either direct or sequential switch in IgE antibody responses. Immunology 1997;91:464–472. Aalberse RC: Structural biology of allergens. J Allergy Clin Immunol 2000;106:228–238. Aalberse RC, Van Ree R, Dannemann A, Wahn U: IgE antibodies to tetanus toxoid in relation to atopy. Int Arch Allergy Immunol 1995;107:169–171. Dannemann A, Van Ree R, Kulig M, Bergmann RL, Bauer P, Forster J, et al: Specific IgE and IgG4 immune responses to tetanus and diphtheria toxoid in atopic and nonatopic children during the first two years of life. Int Arch Allergy Immunol 1996;111:262–267. Gruber C, Lau S, Dannemann A, Sommerfeld C, Wahn U, Aalberse RC: Down-regulation of IgE and IgG4 antibodies to tetanus toxoid and diphtheria toxoid by covaccination with cellular Bordetella pertussis vaccine. J Immunol 2001;167:2411–2417.

Prof. Rob C. Aalberse, PhD Department of Immunopathology Sanquin Research at CLB, Plesmanlaan 125 NL–1066 CX Amsterdam (The Netherlands) Tel. ⫹31 20 5123158/3171, Fax ⫹31 20 5123170 E-Mail [email protected], www.sanquin.nl

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Molecular Aspects of Allergy and Asthma Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 147–158

Regulation of Human T Helper Cell Differentiation by Antigen-Presenting Cells:The Bee Venom Phospholipase A2 Model José M. Carballido, Nicole Carballido-Perrig, Christoph Schwärzler, Günther Lametschwandtner Novartis Institutes for Biomedical Research, Vienna, Austria

Abstract Whereas some individuals develop immunity to bee sting and mount protective IgG4mediated antibody responses to bee venom phospholipase A2 (PLA), others produce large amounts of PLA-specific IgE antibodies and become allergic to this, otherwise, innocuous antigen. PLA-specific IgE responses are the result of imbalanced T helper (Th)2-cell differentiation. There are multiple mechanisms driving the differentiation of naïve CD4⫹ T cells into Th1- or Th2-cell phenotypes. Most of them are linked to the conditions occurring during initial or repeated encounters with the allergen, in the context of an antigen-presenting cell (APC). The different types of APC and their availability to display particular cytokine production profiles, pattern recognition receptors, costimulatory molecules and specific HLA haplotypes are key determinants for human Th1- and Th2-cell polarization. Copyright © 2006 S. Karger AG, Basel

A bee-sting is often a painful experience characterized by redness, swelling and itching of the affected skin. These reactions are caused by a combination of at least 18 active substances, including glycoproteins, poly- and oligopeptides and other small compounds, which are present in bee venom [1]. Approximately 12% of the dry weight of this mixture corresponds to phospholipase A2 (PLA), a 14- to 16-kDa glycoprotein of known crystal structure [2], consisting of 134 amino acids [3] and containing a single carbohydrate unit attached to Asn13 [4]. PLA represents the main immunogenic and allergenic component of honeybee venom [5]. Low affinity, anti-PLA immunoglobulin (Ig)G1 antibodies are generally elicited following initial responses to bee sting.

With repeated exposures, as is often the case with bee keepers, most subjects mount protective responses involving the generation of high-affinity IgG4 antibodies to PLA. Conversely, some individuals generate PLA-specific IgE antibodies and develop bee sting hypersensitivity [6]. Isotype switching is under the control of T helper (Th) cells and is particularly dependent on the cytokine milieu in which B cells are activated. In humans, interleukin (IL)-4, which is a key cytokine secreted by Th2 cells, promotes IgE and IgG4 synthesis [7]. On the contrary, interferon (IFN)-␥, a major product of activated Th1 cells, predominantly suppresses IgE synthesis and contributes to the IgG4 switching generally associated with nonallergic responses to PLA [8, 9]. Thus, the immune responses elicited by bee venom PLA represent an excellent model to study the regulation of human Th1/Th2-cell differentiation. Th cell differentiation is a multifactorial process highly influenced by genetic modifiers, type and dose of the antigen, availability of costimulatory signals and, above all, presence of soluble cytokines. Here, we discuss the contribution of antigen-presenting cells (APC) to the generation of PLA-specific Th1- and Th2-cell responses.

Th-Cell Differentiation Is Largely Controlled by Cytokine Signaling

Antigen stimulation of naïve T cells in the presence of IFN-␥ induces the expression and activation of the T-box transcription factor T-bet [10]. The effects of IFN-␥ are mediated by signal transducer and activator of transcription 1 (STAT1) [11]. T-bet, in turn, induces remodeling of the repressed IFN-␥ locus and promotes the expression of the inducible IL-12R␤2 subunit, which forms the functional IL-12 receptor heterodimer together with the IL-12R␤1 chain. IL-12 signaling, which is mediated by STAT4, stimulates the de novo expression of IL-18R. Together, IL-12 and IL-18 act synergistically to promote Th1cell polarization. In the mouse, T-bet also induces the expression of the homeobox transcription factor HLX, which cooperates with T-bet to generate high level of IFN-␥ production [12]. In addition, the transcription factor E26 transformation specific-1 has been recently identified as a cofactor of T-bet, which is required for the T-bet-mediated production of IFN-␥ and essential for the development of Th1-cell responses [13]. Other members of the IL-12 family of cytokines have been recently implicated in the process of Th1-cell differentiation. Particularly, IL-27 synergizes with IL-12 early during Th1 commitment by activating the STAT1 pathway [14]. In contrast, IL-23, which signals through a receptor complex consisting of IL-23R␣ and IL-12R␤1 chains, acts predominantly on memory T cells [15], using STAT3 and STAT4 pathways. Interestingly, IL-23 is able to generate a yet different subpopulation of Th cells, termed ThIL-17 cells [16, 17], which are

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characterized by the production of high levels of IL-17 and tumor necrosis factor-␣ and are major inducers of autoimmune reactions [17, 18]. In general, all cytokines inducing Th1 cell polarization are provided by APC (fig. 1). Th2-cell differentiation is thought to be highly dependent on IL-4 but, in contrast to the Th1 pathway, the source of IL-4 required for the initial Th2-cell differentiation steps is not fully determined. Currently, it is not known whether the low levels of IL-4 produced by naïve T cells following antigenic stimulation are sufficient to initiate Th2-cell polarization or whether an additional source of IL-4 production by other cell types implicated in allergic responses, like mast cells, eosinophils and basophils, is also required. IL-4 signals are conveyed through STAT6 and result in rapid induction of GATA3 [19], which is only present at very low levels in naïve T cells. GATA3 induces remodeling of the IL-4 locus [20] and transactivates the promoters of IL-5 and IL-13 [21, 22]. GATA3 also increases the expression of c-MAF, which is a transcription factor involved in the production of high levels of IL-4 by Th2 cells [23]. In addition, IL-6, which could be provided by activated dendritic cells (DC), has been reported to enhance the expression of IL-4 and to suppress IFN-␥ production by upregulating

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suppressor of cytokine signaling-1 [24]. Furthermore, IL-25, a Th2-cell product, has been recently described as another important mediator of allergic diseases by inducing MHC class II⫹, CD11c⫹, non-T/non-B, accessory cells to produce IL-4, IL-5, IL-13 and eotaxin [25] (fig. 1). Th cell differentiation is further complicated by the fact that cytokines and transcription factors capable of promoting a particular Th-cell phenotype exert antagonistic effects on the differentiation of the opposite Th-cell type. For example, T-bet directly represses Th2 lineage commitment through ITK tyrosine-kinase-mediated interaction with GATA-3, which prevents binding of GATA-3 to regulatory sequences in the Th2 cytokine locus [26]. On the other hand, GATA-3 suppresses Th1 development by downregulation of STAT4 [27]. In addition, once differentiated, human Th cells still display a high degree of plasticity and their cytokine secretion patterns could be further modulated by subsequent antigen stimulation in a different APC and/or cytokine context. Particularly, T-cell receptor stimulation of polarized Th2 cells in the presence of IL-12 [28] or anti-SLAM [29] antibodies induce Th0/Th1 cytokine secretion profiles. In addition, anti-SLAM antibodies induce T-bet expression in human Th2 cells [Schwärzler et al., unpubl.] and exposure of human Th2 cells to T-bet and IL-12 results in a complete reversal into Th1 cytokine production and chemokine receptor expression [30]. Furthermore, effector responses of both Th1- and Th2-cell subsets are under the tight control of regulatory T cells (discussed by Akdis et al. elsewhere in this issue). Systemic allergic reactions to bee sting are not characteristically observed in individuals suffering common atopic manifestations, such as reactivity to multiple allergens, episodes of hay fever, allergic bronchial asthma or atopic dermatitis [31, 32]. Thus, it is not very likely that polarization of bee venom PLA-specific Th2 cells is forced by the pre-existence of an allergic environment rich in Th2 cytokines. Rather, it seems that undifferentiated Th cells are instructed by signals released during local interactions with APC and other accessory cells.

Th Polarizing Signals Generated by DC Are Regulated by Danger Signals

DCs are the key sentinels of the immune system. DCs recognize immunological insults either by initiating T-cell activation and mounting effective immune responses or by promoting a stage of peripheral tolerance. Presently, it is believed that these decisions depend largely on the maturation stage of the DC. DC maturation is initiated following the encounter with the antigen. DC express on their surfaces an array of molecules, including Toll-like receptors

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(TLR), which are able to recognize danger signals mediated by conserved microbial structures or pathogen-associated molecular patterns [33]. TLR activation on DC has been classically associated with the production of IL-12 and with the instruction to generate Th1-cell responses. However, recently it has been shown that activation of DC by aeroallergens requires simultaneous presence of pathogen-associated molecular patterns [34]. In addition, the type of Th-cell responses are also dependent on the intensity of TLR stimulation [35]. Particularly, low levels of TLR4 signaling favored the development of Th2-cell responses, whereas stronger TLR4 activation supported Th1-cell polarization. This idea is more attractive than the notion of having Th1 and Th2 responses induced by already programmed type 1 and type 2 DC. Thus, DC are highly flexible to adapt to the environmental conditions and to determine the most adequate response [36]. Low ratios of DC to T cells during initial T-cell activation in draining lymph nodes have also been reported to promote Th2-cell differentiation [37]. Whereas this mechanism might be relevant in chronic situations of low antigen exposure, it would be less significant for the induction of PLA-specific responses, which normally follow an acute and localized exposure of antigen in areas normally rich in DC. Under these circumstances, histamine, which is a potent allergic mediator secreted by mast cells and basophils, might also modulate the cytokine production of DC and promote the polarization of naïve CD4⫹ T cells towards the Th2 phenotype [38].

Costimulatory Molecules Displayed on DC Surfaces Modulate Th-Cell Differentiation

Activated DC express a large repertoire of costimulatory molecules, which in most cases are required for successful initiation of both Th1- and Th2-cell responses. A few of these molecules have been particularly suggested to play a role in the induction of Th2-cell polarization. OX40L (CD134L) is a member of the tumor necrosis factor-␣ family expressed on the surface of DC. OX40L/OX40 interactions were implicated in the development of naïve T cells into Th2-like effectors using anti-OX40 monoclonal antibodies in vitro [39]. Subsequently, it was shown that OX40-deficient mice have impaired Th2 cytokine production and mount reduced allergic responses following airway challenge with ovalbumin [40]. On the other hand, anti-CD134L monoclonal antibody are effective in inhibiting the development of colitis induced by adoptive transfer of CD45RBhigh cells into severe combined immunodeficient mice [41], which is normally considered a predominantly Th1-mediated response.

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B7H (ICOSL) is a member of the B7 family of costimulatory molecules expressed by APC. B7H was shown to be induced on DC surfaces following stimulation with GM-CSF and IL-4 [42]. B7H interaction with ICOS on T cells enhances predominantly IL-10 [43] production, and therefore this costimulatory pathway has been implicated with the generation of both Th2- and regulatory T-cell responses. In humans, ICOS deficiency results in impaired B-cell memory and decreased Ig production [44]. ICOS-deficient mice also have defects on Ig switching particularly on those isotypes induced by IL-4 [45]. ICOS⫺/⫺ mice were also shown to have reduced levels of IL-17 and to be completely resistant to collagen-induced arthritis [45]. This response could be associated with a particular role of B7H/ICOS interaction in the development of IL-23-mediated Th17 responses [46]. CD11c⫹ DC express the receptor for thymic stromal lymphopoietin, consisting of IL-7R␣ and thymic-stromal-lymphopoietin-binding chains. Thymic stromal lymphopoietin is a cytokine expressed by the keratinocytes of atopic dermatitis patients, which confers DC with the capacity to instruct Th-cells to secrete IL-4, IL-5 and IL-13, but not IL-10 [25]. TIM-4 is a member of the Ig mucin proteins which is predominantly expressed on APC. TIM-4 was recently identified as the ligand for TIM-1 [47]. TIM-1 is expressed on naïve CD4⫹ T cells following activation and its expression is sustained predominantly on memory Th2 cells [48]. Costimulation of T cells through TIM-1 induces T-cell proliferation and enhances IL-4 production, providing a further mechanism to enhance Th2-cell polarization [47, 48]. The role of costimulatory signals in Th-cell differentiation has been often controversial. In general, costimulatory conditions favoring low-intensity interactions between the APC and T cells tend to promote Th2-cell polarization. However, these signals should not be regarded in isolation, since often it is their particular combination with different antigens and different cytokine and/or tissue environments which determines the generation of distinct polarizing instructions.

Antigen Presentation by Nonprofessional APC Influences Th-Cell Differentiation

DCs are recognized as the most efficient APC able to stimulate CD4⫹ T cells. However, other cell types can perform these functions, especially to activate already experienced T cells. Among these, monocytes/macrophages and B cells represent the leukocyte lineages more capable to replace DC in their antigen presentation role. The fact that B cells could recognize and internalize antigens using their specific surface Ig receptors suggested that under low antigen

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concentration these cells could be the most efficient APC [49]. Alternatively, in the case of allergens, these antigenic molecules could be captured by specific IgE antibodies and focused to DC and Langerhans cells through Fc␧ receptors [50]. These interactions are important in the context of memory responses but they are not very relevant during the initial steps of Th-cell polarization, where most likely only little amounts of specific antibodies in soluble form or expressed as surface Igs on B cells will be present. Furthermore, in the case of bee venom PLA, it was shown that PLA specific and non-specific B cells are equally able to present the antigen and induce proliferation and cytokine production by PLA-specific Th cells [51]. These results were not related to different levels of MHC expression by the different B-cell lines used as APC. Instead, they could be explained by the high affinity of PLA (⬍10⫺13 M) [52] for its phospholipid substrates, which are universal constituents of the cellular membranes. On the other hand, using a mouse asthma model, it has been recently shown that large amounts of eosinophils can be found in draining lymph nodes at early times after allergen challenge [53]. Eosinophils, once activated are able to present antigens, including PLA [54], to T cells and to secrete IL-4 [55]. Thus, these cells, which are efficiently recruited during the initial encounter with the antigen, could also be regarded as potential inducers of Th2-cell differentiation.

Affinity of Interaction between MHC Class II Molecules and Antigen Peptides Might Determine the Type of Immune Response

Th-cell activation requires recognition of immunogenic peptides presented in the context of MHC class II molecules displayed on the surface of APC. The molecule of bee venom PLA contains three peptidic and one glycosylationdependent T-cell epitopes [56–58]. These peptides, which were identified using PLA-specific Th-cell clones, correspond to the amino acid regions PLA45–62, PLA82–92 and PLA113–124, and to the glycopeptide structure around Asn13 of PLA. All T-cell epitopes elicit proliferative cell responses of unfractionated peripheral blood mononuclear cells isolated from hyperimmune, allergic and hyposensitized individuals [56, 58], indicating that there is no preferential recognition of a distinct epitope by a particular population of individuals. On the other hand, the cytokine production profiles of these PLA-specific Th-cell clones were found to be highly dependent on the dose of antigen [59]. Particularly, the production of IL-4 by Th-cell clones of both hyperimmune and allergic individuals required a lower threshold of specific antigen, or crosslinked anti-CD3 monoclonal antibodies, than the production of IFN-␥ [51].

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However, a major difference between allergic and nonallergic Th-cell clones was observed by comparing their ratios of IL-4/IFN-␥ production. The IL4:IFN-␥ ratios were generally 10-fold higher in allergic individuals than in hyperimmune subjects, but they decreased dramatically with increased antigen concentrations [51]. The relevance of this observation was accentuated by the fact that increasing antigen concentration reduced simultaneously the ratios of IL4:IFN-␥ production and IgE:IgG4 secretion by peripheral blood mononuclear cells of bee-sting-sensitized individuals [8, 60]. Based on these results, it was proposed that the dose of antigen could be a driving force for IgE or IgG production and consequently, a determinant for allergy or immunoprotection [59]. It was hypothesized that individuals expressing HLA-D haplotypes with low affinity for PLA epitopes will have low density HLA-D/PLA-peptide complexes on the surfaces of their APC. These APC will induce weak T-cell activation and will generate high ratios of IL-4:IFN-␥ production. This could be the case in bee-sting-allergic individuals, who normally produce little IFN-␥ and have high levels of IgE. Stronger T-cell activation, resulting from higher affinities of HLA-D molecules for PLA-immunogenic peptides, and consequently, higher densities of HLA-D/PLA-peptide complexes on APC, will result in enhanced IFN-␥ production and reduced IgE synthesis [61]. This model has not been formally validated, for example, the association of particular HLA-D haplotypes with allergy or protection to PLA has not been investigated, but it is consistent with the results obtained in the clinic during allergen hyposensitization. During immunotherapy, high doses of allergen will compensate for low affinities of HLA-D/PLA-peptide and revert the ratios of IL4:IFN-␥ production, and subsequently the IgE:IgG4 secretion in the allergic patients [9, 62]. In practice, immunotherapy results in a clear raise of IgG4 antibodies, although shifts from Th2- into Th1- cytokine production patterns are controversial. A possible explanation might be the difficulty to measure antigen-specific cytokine production in vivo, compared to the simplicity of detecting antigen-specific antibody responses. In addition, human Th-cell cytokine responses, including those to bee venom PLA, are not restricted to the canonic Th1- and Th2-cytokine expression profiles and other types of responses, including regulatory T-cell responses, are also elicited during allergen-specific immunotherapy [63, 64].

Conclusions

The well-known physicochemical properties of bee venom PLA, together with its capacity to induce both, Th1- and Th2-cell responses make this antigen a very attractive model to study the processes that control human Th-cell differentiation. To date, the main emphasis of this research field has been placed on

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the study of cytokine production by already differentiated PLA-specific Th cells and on the reversal of these responses using antagonistic cytokines or altered peptide ligands. Little is known about the role played by professional and nonprofessional APC in the generation of naïve and memory responses to PLA. A better understanding of these innate interactions will provide significant highlights on the mechanisms that regulate human Th-cell differentiation, and simultaneously, will open new possibilities for therapeutic intervention in bee-sting-allergy and related disorders.

Acknowledgements Nicole and José M. Carballido worked at SIAF from the time of its foundation, in 1988, until 1993. They would like to express their gratitude to Kurt Blaser for providing them with a friendly and stimulating scientific environment and for the continuous support during their time in Davos.

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Hansel TT, De Vries IJ, Carballido JM, Braun RK, Carballido-Perrig N, Rihs S, Blaser K, Walker C: Induction and function of eosinophil intercellular adhesion molecule-1 and HLA-DR. J Immunol 1992;149:2130–2136. Gessner A, Mohrs K, Mohrs M: Mast cells, basophils, and eosinophils acquire constitutive IL-4 and IL-13 transcripts during lineage differentiation that are sufficient for rapid cytokine production. J Immunol 2005;174:1063–1072. Carballido JM, Carballido-Perrig N, Kagi MK, Meloen RH, Wuthrich B, Heusser CH, Blaser K: T cell epitope specificity in human allergic and nonallergic subjects to bee venom phospholipase A2. J Immunol 1993;150:3582–3591. Dhillon M, Roberts C, Nunn T, Kuo M: Mapping human T cell epitopes on phospholipase A2: The major bee-venom allergen. J Allergy Clin Immunol 1992;90:42–51. Dudler T, Altmann F, Carballido JM, Blaser K: Carbohydrate-dependent, HLA class II-restricted, human T cell response to the bee venom allergen phospholipase A2 in allergic patients. Eur J Immunol 1995;25:538–542. Carballido JM, Carballido-Perrig N, Terres G, Heusser CH, Blaser K: Bee venom phospholipase A2-specific T cell clones from human allergic and non-allergic individuals: Cytokine patterns change in response to the antigen concentration. Eur J Immunol 1992;22:1357–1363. Akdis CA, Blesken T, Wymann D, Akdis M, Blaser K: Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants. Eur J Immunol 1998;28:914–925. Carballido JM, Carballido-Perrig N, Heusser C, Blaser K: Regulation of the cytokine production of allergen-specific human T-cell clones by the allergen. Int Arch Allergy Immunol 1992;99: 366–369. Muller U, Akdis CA, Fricker M, Akdis M, Blesken T, Bettens F, Blaser K: Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol 1998;101:747–754. Bellinghausen I, Metz G, Enk AH, Christmann S, Knop J, Saloga J: Insect venom immunotherapy induces interleukin-10 production and a Th2-to-Th1 shift, and changes surface marker expression in venom-allergic subjects. Eur J Immunol 1997;27:1131–1139. Akdis CA, Blesken T, Akdis M, Wuthrich B, Blaser K: Role of interleukin 10 in specific immunotherapy. J Clin Invest 1998;102:98–106.

Dr. José M. Carballido Novartis Institutes for Biomedical Research Autoimmunity and Transplantation Disease Area, Brunner Strasse 59 AT–1235 Vienna (Austria) Tel. ⫹43 1 866 34 545, Fax ⫹43 1 866 34 518, E-Mail [email protected]

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Molecular Aspects of Allergy and Asthma Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 159–173

T Regulatory Cells in Allergy Mübeccel Akdis, Kurt Blaser, Cezmi A. Akdis Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland

Abstract Activation-induced cell death, anergy and/or immune response modulation by T regulatory cells (TReg) are essential mechanisms of peripheral T-cell tolerance. There is growing evidence that anergy, tolerance and active suppression are not entirely distinct, but rather, represent linked mechanisms possibly involving the same cells and multiple suppressor mechanisms. Skewing of allergen-specific effector T cells to TReg cells appears as a crucial event in the control of healthy immune response to allergens and successful allergen-specific immunotherapy. The TReg cell response is characterized by abolished allergen-induced specific T-cell proliferation and suppressed T helper 1 (Th1)- and Th2-type cytokine secretion. The increased levels of interleukin-10 (IL-10) and transforming growth factor-
(TGF-
) that are produced by TReg cells, potently suppress IgE production, while simultaneously increasing production of noninflammatory isotypes IgG4 and IgA, respectively. In addition, TReg cells directly or indirectly suppress effector cells of allergic inflammation such as mast cells, basophils, and eosinophils. In conclusion, peripheral tolerance to allergens is controlled by multiple active suppression mechanisms. It is associated with regulation of antibody isotypes and effector cells to the direction of a healthy immune response and opens a window for novel therapies of allergic diseases. Copyright © 2006 S. Karger AG, Basel

Anergy,Tolerance and Active Suppression Are Not Fully Distinct Events

T-cell tolerance is characterized by functional inactivation of the cell-toantigen encounter, which remains alive for an extended period of time in an unresponsive state. In recognition of the importance of the phenomenon of immunological tolerance, the Nobel Prize in Physiology and Medicine was awarded in 1960 to Medawar for discovering that skin allografts in mice and chicken can be accepted if they had been pre-inoculated during embryonic development with allogeneic lymphoid cells [1], and to Burnet for first proposing that

exposure to antigens before the development of immune response, specifically abrogates the capacity to respond to that antigen in later life [2]. During the last decade, this area of immunology research had become so popular and promiscuous. The overall evaluation of the studies on T-cell unresponsiveness suggest that anergy, tolerance, and active suppression are not entirely distinct, but rather, represent linked mechanisms possibly involving the same molecular events. The term anergy was first coined by Von Pirquet in 1908 to describe the loss of delayed-type hypersensitivity to tuberculin in individuals infected with measles virus [3]. The term was clinically accepted since then to describe negative tuberculin skin test results in conditions, where it is expected to be positive. In 1980, the term ‘anergy’ was used to describe the specific inactivation of B cells in mice by high doses of antigen [4]. It was subsequently used for T cells, to describe a phenomena in which antigen presentation to T-cell clones in the absence of professional antigen-presenting cells (APC), induced a hyporesponsive state affecting subsequent IL-2 production and proliferation upon restimulation [5]. A variety of reversible functional limitations characterize the anergic state, including cell division, cell differentiation, and cytokine production. It is important to note here that in early studies, which are referred to as a basis for the definition of anergy/tolerance, functional unresponsiveness was analyzed by nonsophisticated assays such as antigen-induced [3H] thymidine incorporation, IL-2 and total IgG production. In addition, antigens used in mouse models until the last few years contained high amounts of impurities, such as lipopolysaccharides and other innate immune response stimulating substances, which may influence the outcome of the experiments. Although some of the biochemical steps overlap with anergy, activation-induced cell death, induced by trigger of the death receptors and caspase activation, represents a distinct physiological response [6].

Essential Features of Allergic Inflammation

It is still not understood why exposure to allergens causes atopic disorders in some individuals, but not in others; however, it is clear that strong interaction of environmental and genetic factors is involved. The four cardinal events during allergic inflammation, can be classified as activation of memory/effector T cells and other effector cells such as mast cells, eosinophils and basophils, their organ-selective homing, prolonged survival and reactivation inside the allergic organs, and effector functions (fig. 1) [7]. T cells are activated by aeroallergens, food antigens, autoantigens, and bacterial superantigens in allergic inflammation [8]. They are under the influence of skin-, lung- or nose-related chemokine network and they show organ-selective homing [9]. A prolonged survival of the

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Activation of immune cells aeroallergens, food antigens, autoantigens and superantigens Organ homing organ-selective and under the influence of chemokine network

Allergic inflammation

Survival and reactivation in subepithelial tissue

Effector functions cell interaction and effector functions (epithelial apoptosis, remodeling, IgE, eosinophilia)

Fig. 1. The four sequential processes characterizing allergic inflammation. Various antigens, superantigens, or yet unidentified factors, activate T cells. These cells then undergo organ-selective homing according to the influence of organ-related chemokine networks. T cells within subepithelial tissues show increased survival and are continuously stimulated. These activated T cells then trigger effector functions, including apoptosis, remodeling, hyper IgE, and eosinophilia.

inflammatory cells and strong interaction with resident cells of the allergic organ, and consequent reactivation is observed in the subepithelial tissues [10]. T cells play important effector roles in atopic dermatitis and asthma with induction of hyper IgE, eosinophil survival, and mucus hyperproduction [7] (fig. 1). In addition, activated T cells induce bronchial epithelial cell and keratinocyte apoptosis as major tissue injury events [11, 12]. Peripheral T-cell tolerance to allergens can overcome all of the above pathological events in allergic inflammation, because they all require T-cell activation. The initial event responsible for the development of allergic diseases is the generation of allergen-specific CD4 T helper cells. The current view is that under the influence of interleukin-4 (IL-4), naive T cells activated by APC differentiate into T helper (Th) 2 cells [13]. Once generated, effector Th2 cells produce IL-4, IL-5 and IL-13, and mediate several regulatory and effector functions. These cytokines induce the production of allergen-specific IgE by B cells, development and recruitment of eosinophils, production of mucus, and contraction of smooth muscles. Furthermore, the degranulation of basophils and mast cells by IgE-mediated cross-linking of receptors is the key event in type I hypersensitivity, which may lead to chronic allergic inflammation. Importantly, although Th2 cells are responsible for the development of allergic diseases, Th1 cells may contribute to chronicity and effector phase in allergic

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diseases [11, 12]. Distinct Th1 and Th2 subpopulations of T cells counter-regulate each other and play a role in distinct diseases [13]. In addition, recent studies have demonstrated that peripheral T-cell tolerance is crucial for a healthy immune response and successful treatment of allergic disorders [14–16]. A further subtype of T cells, with immunosuppressive function and cytokine profiles distinct from either Th1 and Th2 cells, termed regulatory/suppressor T cells (TReg) has been described [17–19] and evidence for their existence in humans has been demonstrated [15]. In addition to Th1 cells, TReg cells are able to inhibit the development of allergic Th2 responses and play a major role in allergen-specific immunotherapy (SIT) [15, 16]. This review will examine allergenspecific peripheral tolerance mechanisms in humans and discuss novel ways of T-cell suppression.

TReg Cells

T cells that were able to suppress immune responses were described first in the early 1970s [20]. Suppressor T cells were thought to be a specialized subpopulation, the effects of which were mediated in an antigen-specific fashion. The concept of T-cell-mediated immune suppression is being strongly explored starting from the mid 1990s. Many types of suppressor T cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which suppressor cells exert their effects remain to be elucidated, it is well established that TReg cells suppress immune responses via cell-to-cell interactions and/or the production of suppressor cytokines. Suppression is antigen-specific, and can be overcome by high T-cell receptor triggers and innate immune response triggers such as those by toll-like receptors [21, 22] (table 1). Tr1 Cells Type-1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-
[19, 23]. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4 T cells. Tr1 cells have a low proliferative capacity, which can be overcome by IL-15 [24]. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL10 and TGF-
[23]. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance in allergy and autoimmunity, can be foreseen. The generation in vitro of a TReg cell subset by stimulating naive CD4 T cells in the presence of IL-10, IFN- or a combination of IL-4 and IL-10, has previously been reported [19, 23]. To overcome the problems in cytokine

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Table 1. Regulatory/suppressor cells and their subsets Regulatory/suppressor cells T cells

B-cell subset DC NK cell subset1 Macrophages Resident tissue cells1

Tr1 Th3 CD4 CD25 TReg CD8 CD25 CD28 TReg CD4CD8 TReg TCR  TReg BReg? DCReg? NKReg? ? ?

Suppressor mechanism* IL-10, TGF-
TGF-
IL-10, TGF-
, CTLA-4, PD-1, GITR same as CD4 CD25 induction of apoptosis IL-10, TGF-
IL-10 IL-10 IL-10 IL-10, TGF-
IL-10, TGF-


1 NK cells and resident tissue cells (astrocytes, keratinocytes, glomerular mesangial cells etc.) are included in the table because of expression of suppressive cytokines by these cells.

profiles of TReg cells, it has been demonstrated that a combination of vitamin D3 and dexamethasone, induced human and mouse naive CD4 T cells to differentiate in vitro into TReg cells [25]. In contrast to the previously described in vitro-derived CD4 T cells, these cells produced only IL-10, but no IL-5 and IFN-, and furthermore, retained strong proliferative capacity and prevented central nervous system inflammation in an IL-10 dependent manner. There is now clear evidence that IL-10- and/or TGF-
-producing Tr1 cells are generated in vivo in humans during the early course of allergen-SIT, suggesting that high and increasing doses of allergens induce Tr1 cells in humans [15, 16]. Analysis of responses to various food and inhalant antigens has shown that healthy immune response to mucosal antigens displays a similar mechanism of active regulation [21]. A recent study has been performed using interferon (IFN)--, IL-4- and IL-10-secreting allergen-specific CD4 T cells that resemble Th1-, Th2- and Tr1-like cells, respectively. Healthy and allergic individuals exhibit all three subsets, but in different proportions. In healthy individuals Tr1 cells represent the dominant subset for common environmental allergens, whereas a high frequency of allergen-specific IL-4-secreting T cells (Th2-like) is found in allergic individuals. Hence, a change in the dominant subset may lead to either the development of allergy or recovery (fig. 2) [21]. Th3 Cells Regulatory/supressor T-cell clones have been induced by oral feeding of low doses of antigen in a TCR-transgenic experimental encephalitis model [17].

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Suppression of Th0/Th1 effector cells

Epithelial apoptosis Th0 Th1

IFN-, TNF- FasL

TReg Mast cell

IL-

10

G ,T

IL-


F-

10

,T

GF

Eosinophil Suppression of effector cells and mucus production

B cell

3

-1

IL-

3,

IL 4,

IL-

IL4

, IL

-5 Th2

Mucus production

-


-13 IL-9, IL

IgE

n

tio

uc

d pro

Induction of IgG4, IgA suppression of IgE

Decreased Th2 cytokines

Fig. 2. TReg cells in allergy, specific immunotherapy and healthy immune response. Immune deviation towards TReg cell response is an essential step in allergen-specific immunotherapy and natural allergen exposure of nonallergic individuals. TReg cells utilize multiple suppressor factors, which influence the final outcome of allergen-specific immunotherapy. In addition, IL-10 and TGF-
induce IgG4 and IgA respectively from B cells as noninflammatory Ig isotypes and suppress IgE production. These two cytokines directly or indirectly suppress effector cells of allergic inflammation such as mast cells, basophils and eosinophils. In addition, Th2 cells, which are dominated by TReg cells cannot induce IgE by IL-4 and IL-13 any further, and also cannot provide cytokines such as IL-3, IL-4, IL-5 and IL-9, which are required for the differentiation, survival, and activity of mast cells, basophils and eosinophils, and mucus-producing cells. Furthermore, the suppressed Th0/Th1 compartment of allergic inflammation cannot mediate essential tissue injury mechanisms via IFN-, TNF- and Fas-ligand (L) such as apoptosis of skin keratinocytes and bronchial epithelial cells (grey line: suppression, black line: stimulation).

CD4 T-cell clones isolated from mesenteric lymph nodes in animals tolerating oral induction, produced high levels of TGF-
, and variable amounts of IL-4 and IL-10 upon activation with appropriate antigen or anti-CD3 antibody [17]. These cells functioned in vivo to suppress encephalitis induction with myelin basic protein and were designated as Th3 cells. TGF-
and IL-10 seemed

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critical, as treatment with neutralizing antibodies abrogated the disease-protective effects of these cells. These TReg cells also exerted bystander immune suppression in vitro. Th3 cells and Tr1 cells may apparently represent the same cells in humans. CD4 CD25 TReg Cells There is clear evidence from various animal models and human studies for an active mechanism of immune suppression, whereby a distinct subset of T cells inhibits the activation of conventional T cells in the periphery [26]. This TReg cell population has been determined as CD4CD25 T cells. The CD4 CD25 T cells constitute 5–10% of peripheral CD4 T cells and express the IL-2 receptor  chain (CD25) [26]. They can prevent the development of autoimmunity, indicating that the normal immune system contains a population of professional TReg cells. Elimination of CD4CD25 T cells leads to spontaneous development of various autoimmune diseases, such as gastritis or thyroiditis, in genetically susceptible hosts. In mice, these cells have been shown to express CD45RBlow [18]. The CD38– CD25 CD4 CD45RBlow subpopulation contains T cells, which respond to recall antigens and produce high levels of cytokines in response to polyclonal stimulation. There are two major hypotheses concerning the generation of CD4 CD25 TReg cells. One of these, suggests that TReg cells emerge from the thymus as a distinct subset of mature T cells with defined functions [26]. On the other hand, several studies have shown that TReg cells may differentiate from naive T cells in the periphery upon encountering antigens present at high concentrations [19, 25]. It can be proposed that thymic differentiation accounts for TReg cells that are specific for self peptides and are devoted to the control of autoimmune responses, whereas peripheral differentiation may be required for environmental antigen-specific T cells for which an undesired immune response results in pathology. Currently there is a tendency to discriminate CD4CD25FoxP3 TReg cells as constitutive TReg cells whereas Tr1 cells as inducible TReg cells. However, since FoxP3 is downregulated in activated T cells, these subsets can be overlapping and even represent the same cells in adult humans. In addition, recent studies have shown non-thymus-dependent in vivo induction of CD4 CD25 FoxP3 TReg cells in mice [27].

Other Regulatory Cells

It has been proposed that, in addition to CD4 T cells, CD8 TReg cells may have a role in oral tolerance [28]. Recent efforts to generate suppressor cell lines in vitro resulted in a population of CD8 CD28 T cells, restricted by

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allogeneic HLA class I antigens, which were able to prevent upregulation of B7 molecules induced by Th cells on APCs [29]. This resulted in the suppression of CD4 T cells in an HLA-nonrestricted fashion [29]. Interestingly, the magnitude of a CD8 T-cell-mediated immune response to an acute viral infection is also subject to control by CD4 CD25 TReg cells. Double negative (CD4 CD8) TCR
 TReg cells that mediate tolerance in several experimental autoimmune diseases have been described. These double-negative T cells are specific for MHC class I molecules and the suppressive effect of these cells on the proliferation and cytotoxic activity of CD8 T cells with the same antigen specificity was not mediated by cytokines, but instead was attributed to Fas-mediated apoptosis of alloreactive T cells [30].  T cells with regulatory functions have also been described. A population of TReg cells with a cytokine profile reminiscent of Tr1 cell clones has been isolated from tumor-infiltrating lymphocytes [31]. These TReg cells could play a role in the inhibition of immune responses to tumors. It has also been shown that aerosol delivery of protein antigens resulted in the differentiation of  T cells with regulatory functions [31]. BReg Cells: Do They Exist? A regulatory role for IL-10-secreting B cells has been recently proposed [32]. These B cells prevented the development of arthritis and their suppressive effect was particularly IL-10 dependent, because the B cells isolated from IL10-deficient mice failed to protect from arthritis. Dendritic Cells That May Play a Regulatory Function It is generally thought that immature dendritic cells (DC) do not appropriately activate T cells, which may lead to tolerance. In normal immunity, DC should not have any restriction in antigen presentation and they should appropriately receive maturation signals given by the surroundings of the antigen, T cells and other tissue cells, such as co-stimulatory ligands, cytokines, and innate immune response stimulating (i.e. Toll-like receptor triggering) substances. However, there are some indications that DC can induce peripheral T-cell tolerance and a regulatory DC subset may exist. Pulmonary dendritic cells from mice exposed to respiratory antigen transiently produce IL-10 [33]. These phenotypically mature pulmonary DCs, which were B7high, stimulated the development of CD4 Tr1-like cells that also produced high amounts of IL-10. Adoptive transfer of pulmonary DCs from IL-10/, but not IL-10/, mice exposed to respiratory antigen, induced antigen-specific unresponsiveness in recipient mice. In accordance with these findings, IL-10 inhibited the development of fully mature DC, which induced a state of alloantigen-specific anergy in CD4 T cells [34]. These studies show that IL-10 production by DCs is

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critical for the induction of tolerance, and that phenotypically mature DCReg may exist under certain circumstances. Other Cells with Possible Regulatory Function It has been clearly demonstrated that natural killer cells, epithelial cells, macrophages, glial cells etc., express suppressor cytokines such as IL-10 and TGF-
. Although their role has not been coined as professional regulatory cells, these cells may efficiently contribute to the generation and maintenance of a regulatory/suppressor type immune response [35]. The expression of suppressor cytokines in resident tissue cells may additionally contribute to this process.

Suppression Mechanisms of TReg Cells

A great deal of uncertainity remains about mechanisms of action of TReg cells. Initial studies have shown that TReg cells act as suppressor T cells, which downregulate effector cells and inflammation models in chronic infection, organ transplantation, and autoimmunity [35]. Most studies have failed to find a soluble factor as a suppressive mechanism of CD4 CD25 TReg cells. Antigen-induced proliferation of CD4 T cells was dramatically reduced following co-culture with activated TReg clones, which had been separated from the responding T cells by a Transwell insert. However, in Transwell membrane cultures that separate suppressor cells and target cells, the distance between the two populations is approximately 2 mm and this may influence the concentration of suppressor cytokines. Accordingly, it cannot be possible to rule out an effect of a cytokine that acts in short distances or a membrane-bound cytokine. Indeed, membrane-bound TGF-
might be one of the mechanisms of suppression of CD4CD25 TReg cells [36]. In contrast to CD4 CD25 TReg cells, suppressive effects of Tr1 cells was reversed by addition of neutralizing mAb, directed against TGF-
and IL-10, implicating the role of suppressive cytokines in the mechanism of immune suppression both in vitro and in vivo in different settings and different autoimmune as well as allergy models [16, 35]. One group of CD4 CD25 TReg cells originate from the thymus as a distinctive subset [26]. Thymectomy at a very early stage of animal development induces various autoimmune diseases in genetically susceptible animals. Furthermore, induction of autoimmune diseases in an immunodeficient animal model was prevented by adoptively transferred CD4 T cells or CD4CD8 thymocytes isolated from normal syngeneic animals. In a rat model, CD4 TReg cells were found to be of the CD45RClow phenotype, and to produce IL-2 and IL-4, but not IFN-, upon in vitro stimulation. IL-4 and TGF-
are critical in preventing autoimmunity, as neutralization of either of these two cytokines

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abrogates the protective response. In another study, CD4 CD25 TReg cells from thymus were shown to exert their suppressive function via the inhibition of IL-2R-chain in target T cells, induced by the combined activity of CTLA-4 and membrane TGF-
1 [26]. Studies of this activated CD4 T-cell subpopulation have shown that they do not proliferate upon normal TCR-mediated stimulation and suppress proliferation of other T cells. TCR stimulation was required for these cells to exert suppression of other T cells; such suppression, however, was not confined to T cells specific for the same antigen. CD4 CD25 T cells are the only lymphocyte subpopulation in both mice and humans that express CTLA-4 constitutively. The expression apparently correlates with the suppressor function of CTLA-4. The addition of anti-CTLA-4 antibody or its Fab (fragment of antigen binding) reverses suppression in co-cultures of CD4 CD25 and CD4 CD25 T cells [26]. Similarly, the treatment of mice which are recipients of CD4 CD45RBlow T cells with these agents, abrogated the suppression of inflammatory bowel disease [18]. These studies indicate that signals that result from the engagement of CTLA-4 by its ligands, CD80 or CD86, are required for the induction of suppressor activity. Under some circumstances, the engagement of CTLA-4 on the CD4 CD25 T cells by antibody or by CD80/CD86 might lead to inhibition of the TCR-derived signals that are required for the induction of suppressor activity. Programmed death-1 (PD-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor, expressed upon T-cell activation. PD-1-deleted mice develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses [37]. Members of the B7 family, PD-L1 and PDL2, are ligands for PD-1. PD-1:PD-L engagement on murine CD4 and CD8 T cells results in inhibition of proliferation and cytokine production. T cells stimulated with anti-CD3/PD-L1 display dramatically decreased proliferation and IL-2 production. PD-1:PD-L interactions inhibit IL-2 production even in the presence of costimulation and, thus, after prolonged activation, the PD-1: PD-L inhibitory pathway dominates. Exogenous IL-2 is able to overcome PD-L1-mediated inhibition at all times, indicating that cells maintain IL-2 responsiveness. Glucocorticoid-induced tumour-necrosis factor receptor family-related gene (TNFRSF18, GITR) is expressed by CD4 CD25 alloantigen-specific and naturally occurring circulating TReg cells. Stimulation of CD25 CD4 TReg cells through GITR breaks immunological self-tolerance [26]. GITR is upregulated in CD4 CD25 T cells after T-cell receptor stimulation and it also functions as a survival signal for activated cells. In addition, CD103 (E
7 integrin) and CD122 (
chain of IL-2 receptor) are highly expressed on CD4 CD25 TReg cells, which correlates with their suppressive activity.

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An X-linked forkhead/winged helix transcription factor, FoxP3 (Scurfin) is essential for the suppressive function of CD4 CD25 TReg cells. It is highly expressed in CD4 CD25, but not CD4 CD25 T cells. It acts as a silencer of cytokine gene promoters and programs the development and function of CD4 CD25 TReg cells. Mutations in the FoxP3 gene in humans leads to a severe immune dysregulation with poly endocrinopathy, enteropathy and hyper IgE known as IPEX syndrome [38]. The failure of TReg cells to proliferate after TCR stimulation in vitro has suggested that they are naturally anergic. However, TReg cells expressing a transgenic TCR were shown to proliferate and accumulate locally in response to transgenically expressed tissue antigen, whereas their CD25 counterparts are depleted at such sites [26]. CD4 CD25 TReg cells population is composed of two Treg subsets that have distinct phenotypes. Some TReg remain quiescent and have a long lifespan, in the order of months, whereas the other TReg cells (mainly the autoantigen-specific ones) divide extensively and express multiple activation markers [26].

Clinical Relevance of TReg Cells

Since the concept of professional suppressor cells is recovering interest among the immunological community, it is now time to consider how the manipulation of regulatory/suppressor T cells might be used clinically. As tumour antigens are an important group of autoantigens, the depletion of TReg cells should result in an enhanced immune response to tumour vaccines. It has been shown that the antibody-mediated depletion of CD25 T cells facilitates the induction of tumour immunity [26]. Currently, the relationship between the different TReg cell populations is unclear with respect to their development and activation. However, numerous animal experiments have clearly shown that TReg cells can suppress both Th1 and Th2 responses in vivo and thereby, actively suppress the development of autoimmune and allergic responses. In humans, there is circumstantial evidence to suggest that TReg cells play a major role in the inhibition of allergic disorders (fig. 2). It has been reported that IL-10 levels in the bronchoalveolar-lavage fluid of asthmatic patients are lower than in healthy controls, and that T cells from children suffering from asthma also produce less IL-10 mRNA than T cells from control children. These findings indicate that increased IL-10 production is associated with decreased allergic reactions. As TReg cells are a major source of IL-10, it has been speculated that TReg cells secreting IL-10 are involved in the suppression of allergic Th2 responses in humans. Several human allergen-SIT studies

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supported this hypothesis. In contrast, some studies demonstrated that increased IL-10 levels are not associated with less allergic disease. IL-10 may also promote airway hyperresponsiveness and even eosinophilia in allergy models. In contrast to its known T cell suppressive activity, some reports imply a role for TGF-
in the pathogenesis of asthma, particularly in remodeling of injured lung tissue in humans. A recent report indicated that the increased allergic inflammation observed after blocking of CTLA-4 is clearly associated with decreased TGF-
levels in the bronchoalveolar-lavage fluid of these animals (reviewed in [35]). To analyze human in vivo existence of TReg cells, lymphocyte populations in human lymph nodes with a special emphasis on the CD4 CD25 TReg cells have been investigated. CD4 CD25 T cells constitutively co-expressed high levels of CD152. Similar to TReg cells from peripheral blood, TReg cells from lymph nodes were in vitro anergic and efficiently inhibited other CD4 and CD8 lymphocyte proliferation. TReg cells may play destructive roles in cancer and chronic infectious diseases [35]. Further studies are needed to demonstrate in the clinic, whether in vivo generation or adoptive transfer of TReg cells and/or their related suppressive cytokines may change the course of allergy and asthma. Small molecular weight compounds, that may generate TReg cells or increase their suppressive properties, are important targets not only for the use in allergy and asthma, but also for transplantation and autoimmunity.

Conclusions

Peripheral T-cell tolerance is the key immunological mechanism in healthy immune response to self and noninfectious, nonself antigens. This phenomenon is clinically welldocumented in allergy, autoimmunity, transplantation, tumor and infection. There is growing evidence supporting the role for TReg cells and/or immunosuppressive cytokines as a mechanism, by which allergen-SIT and healthy immune response to allergens is mediated (fig. 2). In addition to the treatment of established allergy, it is essential to consider prophylactic approaches before initial sensitization has taken place. Preventive vaccines that induce TReg responses can be developed. Allergen-specific TReg cells may in turn dampen both the Th1 and Th2 cells and cytokines, ensuring a well-balanced immune response. Enhancement of the number and activity of TReg cells could be an obvious goal for the suppression of allograft rejection, graft versus host disease, and autoimmunity. TReg cells may not be always responsible for healthy immune response, because several studies have shown that they may be responsible for the chronicity of infections and tumour tolerance. TReg cell populations have proven possible, but difficult, to grow, expand and clone in vitro. A crucial

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area for future studies is the identification of drugs, cytokines or co-stimulatory molecules that induce the growth while preserving the suppressor function of the TReg cells. In this context, by the application of the recent knowledge in peripheral tolerance mechanisms, more rational and safer approaches are awaiting for the future prevention and cure of allergic diseases.

Acknowledgements The authors’ laboratories are supported by the Swiss National Foundation grants.

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PD Dr. Mübeccel Akdis Swiss Institute of Allergy and Asthma Research (SIAF) Obere Strasse 22 CH–7270 Davos (Switzerland) Tel. 41 81 4100848, Fax 41 81 4100840, E-Mail [email protected]

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The Role of Histamine in Regulation of Immune Responses Marek Jutela,b, Kurt Blasera, Cezmi A. Akdisa a

Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland; Wroclaw University of Medicine, Wroclaw, Poland

b

Abstract Histamine is not only the major mediator of the acute inflammatory and immediate hypersensitivity responses, but has also been demonstrated to affect chronic inflammation and regulate several essential events in the immune response. It can influence numerous functions of the cells involved in the regulation of immune response and hematopoiesis including macrophages, dendritic cells, T lymphocytes, B lymphocytes and endothelial cells. These cells express histamine receptors and also secrete histamine, which can selectively recruit the major effector cells into tissue sites and affect their maturation, activation, polarization, and effector functions leading to chronic inflammation. Histamine regulates antigenspecific Th1 and Th2 cells, as well as related antibody isotype responses. Histamine acting through its receptor (HR) type 2, positively interferes with the peripheral antigen tolerance induced by T regulatory (TReg) cells in several pathways. The diverse effects of histamine on immune regulation are due to differential expression and regulation of 4 histamine receptors and their distinct intracellular signals. In addition, differences in affinities of these receptors are highly decisive on the biological effects of histamine and agents that target histamine receptors. Although substantial evidence has been accumulated about histamine metabolism, receptors, signal transduction, physiological and pathological effects, the complex interrelationship and cross-talk by histamine, its receptors and other G-protein coupled receptors remain to be understood. Copyright © 2006 S. Karger AG, Basel

Since the discovery of histamine (2-[4-imodazole]-ethylamine) as an uterine stimulant more than 100 years ago, it has become one of the most intensely studied molecules in medicine. Its smooth muscle stimulating and vasodepressor action was demonstrated in the first experiments by Dale and Laidlaw [1], who also found that the effects of histamine mimicked those

occurring during anaphylaxis. In 1927, histamine was isolated from liver and lung tissue followed by several other tissues demonstrating that it is a natural constituent of the body, hence the name histamine was given after the Greek word for tissue, histos. Histamine is a low-molecular-weight amine synthesized from L-histidine exclusively by histidine decarboxylase (HDC). It is produced by various cells throughout the body, including central nervous system neurons, gastric mucosa parietal cells, mast cells, basophils and lymphocytes [2, 3]. Histamine is involved in the regulation of many physiological functions including cell proliferation and differentiation, hematopoiesis, embryonic development, regeneration, and wound healing [2–6]. Within central nervous system it affects cognition and memory, the regulation of sleep cycle, energy and endocrine homeostasis [4]. In human pathology, histamine triggers acute symptoms due to its very rapid activity on vascular endothelium, and bronchial and smooth muscle cells leading to the development of such symptoms as acute rhinitis, bronchospasm, cramping, diarrhea or cutaneous wheal and flare responses. Moreover, in addition to these effects on the immediate type response histamine also significantly modulates chronic phase inflammatory events [2, 3]. In this article the findings leading to a change of perspective in histamine immunobiology are highlighted.

Cellular Sources of Histamine

Mast cells and basophils, gastric enterochromaffin-like cells, platelets and histaminergic neurons are the classical sources of histamine. Remarkably, other cells in the immune system which do not store histamine show high HDC activity and are capable of production of high amounts of histamine, which is secreted immediately after synthesis [5]. These cells include platelets, monocytes/macrophages, dendritic cells, neutrophils and T and B cells.

Synthesis and Metabolism of Histamine

Histamine is synthesized by decarboxylation of histidine by L-HDC, which is dependent on the cofactor pyridoxal-5⬘-phosphate [6]. Mast cells and basophils are the major source of granule-stored histamine, where it is closely associated with the anionic proteoglycans and chondroitin-4-sulfate. Histamine is released when these cells degranulate in response to various immunologic and nonimmunologic stimuli. In addition, several myeloid and lymphoid cell types (dendritic cells [DCs] and T cells), which do not store histamine, show

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high HDC activity and are capable of production of high amounts of histamine [5]. HDC activity is modulated by cytokines, such as IL-1, IL-3, IL-12, IL-18, granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor, TNF-␣ and calcium ionophore, in vitro [7]. More than 97% of the histamine is metabolized in 2 major pathways before excretion [8]. Histamine N-methyltransferase metabolizes the majority of histamine to N-methylhistamine, which is further metabolized to the primary urinary metabolite M-methylimidazole acetic acid by monoamine oxidase. Diamine oxidase metabolizes 15% to 30% of histamine to imidazole acetic acid.

Histamine Receptors

The pleiotropic effects of histamine are triggered by activating one or several of histamine membrane receptors on different cells. Four subtypes of receptors (histamine receptor (HR)1, HR2, HR3, and HR4) have been described (table 1). All of these receptors belong to the G-protein-coupled receptor family. They are heptahelical transmembrane molecules that transduce extracellular signal by using G-proteins and intracellular second messenger systems [2, 3]. The active and inactive states of HRs exist in equilibrium. However, it has been shown in recombinant systems that HRs can trigger downstream events in the absence of receptor occupancy by an agonist, which accounts for constitutive spontaneous receptor activity [9]. HRs agonists stimulate the active state in the receptor, and inverse agonists, the inactive one. An agonist with a preferential affinity for the active state of the receptor stabilizes the receptor in its active conformation leading to continuous activation signal. An inverse agonist with a preferential affinity for the inactive state stabilizes the receptor in this conformation and consequently induces an inactive state, which is characterized by blocked signal transduction via the HR [9]. In reporter gene assays, constitutive HR1-mediated nuclear factor (NF)-␬B activation has been shown to be inhibited by many of the clinically used H1-antihistamines, indicating that these agents are inverse HR1agonists [9]. Constitutive activity has now been shown for all four histamine receptors [9]. Specific activation or blockade of HRs showed that they differ in expression, signal transduction or function and improved the understanding of the role of histamine in physiology and disease mechanisms. Histamine receptors were first distinguished into HR1 and HR2 by Ash and Schild in 1966. It has long been recognized that the most positive effects of histamine are mediated by HR1, while HR2 is mostly involved in its suppressive activities. The human Gq/11-coupled HR1 is encoded by a single exon gene located on the distal short

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Table 1. Histamine receptors, expression, coupled G-proteins and activated intracellular signals Histamine receptors

Expression

Activated intracellular signals

G-proteins

HR1

nerve cells, airway and vascular smooth muscles, hepatocytes, chondrocytes, endothelial cells, epithelial cells, neutrophils, eosinophils, monocytes, DC, T and B cells nerve cells, airway and vascular smooth muscles, hepatocytes, chondrocytes, endothelial cells, epithelial cells, neutrophils, eosinophils, monocytes, DC, T and B cells, histaminergic neurons, eosinophils, DC, monocytes low expression in peripheral tissues High expression on bone marrow and peripheral hematopoietic cells, eosinophils, neutrophils, DC, T cells, basophils, mast cells, low expression in nerve cells, hepatocytes peripheral tissues, spleen, thymus, lung, small intestine, colon and heart

Ca2⫹, cGMP, phospholipase D, phospholipase A2, NF-␬B

Gq/11

adenylate cyclase, cAMP, c-Fos, c-Jun, PKC, p70S6K

G␣s

enhanced Ca2⫹, MAP kinase, inhibition of cAMP

Gi/o

enhanced Ca2⫹, inhibition of cAM

Gi/o

HR2

HR3

HR4

arm of chromosome 3p25b and contains 487 aminoacids. The HR1 is expressed in numerous cells including airway and vascular smooth muscle cells, hepatocytes, chondrocytes, nerve cells, endothelial cells, dendritic cells, monocytes, neutrophils, T and B cells [2, 3]. Histamine binds to transmembrane domains 3 and 5. Activation of the HR1-coupled Gq/11 stimulates the inositol phospholipid signaling pathways resulting in formation of inositol-1,4,5-triphosphate (IP3)

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and diacylglycerol and an increase in intracellular calcium [10]. The rise in intracellular calcium accounts for nitric oxide production, liberation of arachidonic acid from phospholipids and increased cyclic AMP. The HR1 also activates phospholipase D and phospholipase A2 and the transcription factor NF-␬B through Gq/11 and G␤␥ upon agonist binding. Constitutive activation of NF-␬B occurs only through G␤␥ [10]. The HR1 is responsible for the development of many symptoms of allergic disease. Targeted disruption of the H1receptor gene in mice results in the impairment of neurologic functions such as memory, learning, locomotion, and nocioperception, and in aggressive behavior. Immunologic abnormalities have also been described in HR1-deleted mice, with impairment of both T and B cell responses [11]. Activation of HR1 is responsible for many symptoms of allergic disease. In humans the intronless gene encoding HR2 is located on chromosome 5. The human HR2 is a protein of 359 aminoacids coupled to both adenylate cyclase and phosphoinositide second messenger systems by separate GTPdependent mechanisms including G␣s, and also induces activation of c-Fos, c-Jun PKC and p70S6 kinase [12]. Studies in different species and several human cells demonstrated that inhibition of characteristic features of the cells by primarily cAMP formation dominates in HR2-dependent effects of histamine. Human HR3, encoded by a gene which consists of four exons on chromosome 20, has been demonstrated in 1987 and cloned recently [13]. HR3 has initially been identified in the central and peripheral nervous system as pre-synaptic receptors controlling the release of histamine and other neurotransmitters (dopamine, serotonine, noradrenaline, GABA, and acetylocholine). HR3 signal transduction involves Gi/o of G proteins leading to inhibition of cAMP and accumulation of Ca⫹⫹ and activation of mitogen-activated protein kinase pathway. R-␣-methyhistamine and imetit are agonists, thioperamide and clobenpropit are antagonists of HR3. The control of mast cells by histamine acting on HR3 involves neuropeptide-containing nerves and might be related to a local neuron-mast cell feedback loop controlling neurogenic inflammation. Dysregulation of this feedback loop may lead to excessive inflammatory responses and suggests a novel therapeutic approach by using HR3 agonists. Probably more than one HR3 subtype exist, which differ in central nervous system localization and signaling pathways. Human HR4, which is encoded by a gene containing three exons, is separated by two large introns located in chromosome 18q11.2. It has 37–43% homology to HR3 (58% in the transmembrane region). HR4 is functionally coupled to G protein Gi/o, inhibiting forskolin-induced cAMP formation like the HR3 [14]. HR4 shows high expression in the bone marrow and peripheral hematopoietic cells, neutrophils, eosinophils and T cells, and basophils and

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mast cells, and moderate expression in spleen, thymus, lung, small intestine, colon, and heart [14]. Until now relatively little is known about the biological function of HR4. It seems to be involved in the immune regulatory functions including chemotaxis and cytokine secretion [2, 3]. HRs form dimers and even oligomers, which allow cooperation between HRs and other G protein-coupled receptors. Thus, the effects of histamine upon receptor stimulation can be very complex.

Regulation of Immune Response

Antigen-Presenting Cells DCs are often located in the vicinity of various histamine sources such as connective tissue mast cells. They are potent antigen-presenting and cytokineproducing cells. Therefore, histamine may effectively influence the immune response through DC. This professional antigen-presenting cells mature from monocytic and lymphoid precursors and acquire DC1 and DC2 phenotypes, which in turn facilitate the development of Th1 and Th2 cells, respectively. Endogenous histamine is actively synthesized during cytokine-induced DC differentiation, which acts in autocrine and paracrine fashion and modifies DC markers [15]. Histamine actively participates in function and activity of DC precursors as well as their immature and mature forms (fig. 1). Immature and mature DCs express all four HR, however comparison of their levels of expression has not yet been studied [16]. In the differentiation process of DC1 from monocytes, HR1 and HR3 act as positive stimulants that increase antigen-presentation capacity, and proinflammatory cytokine production and Th1 priming activity. In contrast, HR2 acts as a suppressive molecule for antigen-presentation capacity, enhances IL-10 production and induces IL-10-producing T cells or Th2 cells [16]. In monocytes stimulated with Toll-like receptor-triggering bacterial products, histamine inhibits the production of pro-inflammatory IL-1-like activity, TNF-␣, IL-12 and IL-18, but enhances IL-10 secretion, through HR2 stimulation [17]. Histamine induces intracellular Ca⫹⫹ flux, actin polymerization, and chemotaxis in immature DCs due to stimulation of HR1 and HR3 subtypes. Maturation of DCs results in loss of these responses. In maturing DCs, however, histamine dose-dependently enhances intracellular cAMP levels and stimulates IL-10 secretion, while inhibiting production of IL-12 via HR2 [16]. Interestingly, although human monocyte-derived dendritic cells have both histamine H1 and H2 receptors and can induce CD86 expression by histamine, human epidermal Langerhans cells express neither H1 nor H2 receptors [3].

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HR2 Induction of IL-10, suppression of IL-12, Th2 or tolerance inducing DC

Monocyte dendritic cell HR1–4

DC

HR1/HR3 Proinflammatory activity, increased APC capacity

High HR2 Low HR1 suppressed Th2 cytokines tolerance

HR2: induction of humoral immunity and suppression of cellular immunity. HR2-deficient mice show suppressed specific IgE

Th2

H

H

B cell

Th1

High HR1 Low HR2 Increased IFN-␥ autoimmunity

HR1: blocking of humoral immunity and induction of cellular immunity. HR1-deficient mice show increased specific IgE

Fig. 1. Histamine regulates monocyte, dendritic cell (DC), T cells and B-cell functions. Monocytes and DCs express all four HRs. Activation of HR1 and HR3 triggers proinflammatory events and increases APC capacity. HR2 plays a suppressive role on monocytes and monocyte-derived DC. Th1 cells show predominant, but not exclusive, expression of HR1, whereas Th2 cells show upregulation of HR2. Histamine induces increased proliferation and IFN-␥ production in Th1 cells. Th2 cells express predominant HR2, which acts as the negative regulator of proliferation, and IL-4 and IL-13 production. Histamine enhances Th1-type responses by triggering the HR1, whereas both Th1- and Th2-type responses are negatively regulated by HR2, showing an essential role for immune regulation for this receptor. Distinct effects of histamine suggest roles of HR1 and HR2 on T cells for autoimmunity and peripheral tolerance, respectively. Histamine also modulates antibody production. Histamine directly effects B-cell antibody production as a co-stimulatory receptor on B cells. HR1 predominantly expressed on Th1 cells may block humoral immune responses by enhancing Th1 type cytokine IFN-␥. In contrast, HR2 enhances humoral immune responses. Allergen-specific IgE production is differentially regulated in HR1- and HR2-deficient mice. HR1deleted mice show increased allergen-specific IgE production, whereas HR2-deleted mice show suppressed IgE production.

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T Cells and Antibody Isotypes Histamine has been shown to intervene in the Th1, Th2, and TReg cell balance and consequently antibody formation. Differential patterns of histamine receptor expression on Th1 and Th2 cells determine reciprocal T-cell responses following histamine stimulation (fig. 1) [18]. Th1 cells show predominant, but not exclusive, expression of HR1, while Th2 cells show increased expression of HR2. Histamine enhances Th1-type responses by triggering the HR1, whereas both Th1- and Th2-type responses are negatively regulated by HR2, due to activation of different biochemical intracellular signals [18]. In mice, deletion of HR1 results in suppression of IFN-␥ and dominant secretion of Th2 cytokines (IL-4 and IL-13). HR2-deleted mice show upregulation of both Th1 and Th2 cytokines. Recently, Bphs a non-major histocompatibility complex-linked gene involved in the susceptibility to many autoimmune diseases has been identified as HR1 gene in mice. HR1-deleted mice showed delayed disease onset and decreased disease severity when immunized to develop experimental allergic encephalomyelitis [19]. It has also been shown that histamine stimulation induced IL-10 secretion through HR2 [2, 3]. Increased IL-10 production in both DC and T cells may account for an important regulatory mechanism in the control of inflammatory functions through histamine. Various cytokines regulate the production of histamine and its receptor expression. IL-3 stimulation significantly increases HR1 expression on Th1, but not on Th2 cells [18]. In mice, histamine enhances anti-IgM-induced proliferation of B cells, which is abolished in HR1-deleted mice. In HR1-deleted mice, antibody production against a T cell-independent antigen, TNP-Ficoll, is decreased [11], suggesting an important role of HR1 signaling in responses triggered from B-cell receptors. Antibody responses to T-cell-dependent antigens like ovalbumin (OVA) show a different pattern [11]. HR1-deleted mice produced high OVAspecific IgG1 and IgE in comparison to wild type mice. In contrast, HR2deleted mice showed decreased serum levels of OVA-specific IgE in comparison to wild type mice and HR1-deficient mice. Although T cells of HR2-deficient mice secreted increased IL-4 and IL-13, OVA-specific IgE was suppressed in the presence of highly increased IFN-␥. Thus, HR1 and related Th1 response may play a dominant role in the suppression of humoral immune response. Peripheral T-cell tolerance characterized by immune deviation to regulatory/suppressor T cells represents a key event in the control of specific immune response during allergen-specific immunotherapy [20]. Although, multiple suppressor factors including contact dependent or independent mechanisms might be involved, IL-10 and TGF-␤, predominantly produced by allergen-specific T cells, play an essential role [20]. Histamine interferes with the peripheral tolerance induced during specific immunotherapy in several pathways. Histamine induces the production of IL-10 by dendritic cells. In addition, histamine

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induces IL-10 production by Th2 cells [3]. Furthermore, histamine enhances the suppressive activity of TGF-␤ on T cells [21]. All three of these effects are mediated through HR2, which is relatively highly expressed on Th2 cells and suppresses IL-4 and IL-13 production and T-cell proliferation [18]. Apparently, these recent findings suggest that HR2 may represent an essential receptor that participates in peripheral tolerance or active suppression of inflammatory/ immune responses.

Histamine and Chronic Inflammatory Responses

Over the course of the pollen season, there might be even a 10-fold increase in numbers of nasal epithelial submucosal mast cells. Histamine released from these cells might not only induce acute allergic symptoms, but also be crucial for sustaining this response into a chronic phase, as increasing evidence suggests that it influences several immune/inflammatory and effector functions [2, 3]. Histamine contributes to the progression of allergic-inflammatory responses by enhancement of the secretion of proinflammatory cytokines like IL-1␣, IL-1␤, IL-6 as well as chemokines like Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES) or IL-8, both in several cell types and local tissues [2, 3]. Histamine induces the CC chemokines, monocyte chemotactic protein 1 and 3, RANTES and eotaxin in explant cultures of human nasal mucosa through HR1, suggesting a prolonged inflammatory cycle in allergic rhinitis between the cells that release histamine and their enhanced migration to nasal mucosa. Recently, it has been shown that the histamine receptor responsible for the selective recruitment of eosinophils is HR4 [14]. Histamine possesses all the properties of a classical leukocyte chemoattractant (i.e., agonist-induced actin polymerization, mobilization of intracellular calcium, alteration in cell shape, and upregulation of adhesion molecule expression). Triggering of HR4 also induces chemotaxis of mast cells [22]. Experiments in mice showed that mast cells from wild type and HR3-receptordeleted mice migrated in response to histamine, while mast cells from the HR4deleted mice did not. Thus, chemotaxis of eosinophils and mast cells by histamine is triggered mainly through the HR4. The HR4-mediated chronic inflammatory effects of histamine may be aborted by administration of HR4 antagonists, and combination therapies with the HR1 antagonists are a promising approach. Downregulation of NF-␬B, which acts as a potent transcription factor in initiating inflammation may represent a possible mechanism for H1-antihistamines to inhibit inflammatory cell accumulation [23].

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Effects of Histamine and Antihistamines on Airway Function

Although the use of H1-antihistamine in persistent asthma is currently not recommended, some recent evidence might finally lead to re-evaluation of this approach. The level of histamine in bronchoalveolar lavage fluid has been found to correlate with the severity of asthma and airway hyper-responsiveness [24]. Inhaled and intravenous histamine causes bronchoconstriction as one of the first recognized properties of histamine, which is inhibited by H1-antihistamines. In lavage fluid of the patients treated with H1-antihistamines, decreased levels of proinflammatory cytokines, and mediators (e.g., histamine, leukotrienes, prostaglandin, cell adhesion molecules [e.g., intercellular adhesion molecules and vascular cell adhesion molecules],) cells (e.g., eosinophils and neutrophils), and plasma exudation along with reduced symptom score has been found [25]. The potential efficacy of H1-antihistamines in asthma has been intensively investigated [25]. It has been shown that inhalation, intravenous, or oral administration of clemastine or chlorpheniramine, induced significant bronchodilatation. However, second generation H1-antihistamines induce only a very limited increase of FEV1 (5–10% over baseline) by recommended doses [25]. The mechanisms of the beneficial effect of HR1-antihistamines in asthma have been investigated in a mice model. Fexofenadine was found to suppress allergic immune/inflammatory responses in sensitized mice [26]. Treatment with fexofenadine diminished Th2-like response that typically follows sensitization and challenge with allergen. Decreased secretion of IL-4 and IL-5, prevention of allergen-specific IgE increase, and reduced eosinophilia in lung tissue and BALF, as well as normalization of airway response to metacholine was observed. Importantly, in an adoptive transfer model, it was demonstarated that the target mechanism was T-cell mediated. Lung T cells from sensitized mice when transferred to naïve recipient mice triggered airway hyper-responsiveness and allergic inflammatory features after allergen challenge. In contrast, naïve mice which received T cells from sensitized mice treated before with fexofenadine showed no such responses to allergen challenge [26]. The inability of T cells from HR1 antihistamine-treated allergen-sensitized mice to transfer allergic sensitivity to naïve recipients resulted from an alteration in the cytokine production profile of the transferred cells. Recent studies suggest that histamine may play an important role in the modulation of the cytokine network in the lung through HR2, HR3 and HR4 that are expressed in distinct cells and cell subsets [27]. Apparently, due to the same signal transduction patterns, ␤2 adrenergic receptors may function similar to HR2 in humans. The role of histamine and other redundant G-protein-coupled receptors in the regulation of immune/inflammatory pathways in the lung remain to be intensely focused in future studies.

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Histamine and HR1 in Autoimmunity

The role of histamine in autoimmune reactions was often questioned in the past [2,3]. Because of inadequate model systems and reagents, the roles and mechanisms of histamine in autoimmunity remained unclear. Recently, the gene encoding HR1 was identified as Bphs, which represents an autoimmune disease locus [19]. HR1 differs at 3 amino acid residues in autoimmune orchitis- and allergic encephalomyelitis-susceptible and resistant mice. T cells from HR1-deficient mice produce significantly less IFN-␥ associated with significantly less severe autoimmune disease. Apparently, the IFN-␥-inducing capacity of HR1 on T cells might play a role in tissue injury mechanisms of several other diseases of allergic, infectious, and autoimmune origin, as well as allograft rejection.

Histamine Signal in Malignancies

Histamine might play a major role in the growth of normal and malignant tissue as a regulator of proliferation and angiogenesis. Specific HRs have been identified on the surface of bone marrow cells, immune competent cells, endothelial cells, fibroblasts, and also malignant cells. This has prompted tumor treatment by specific HR agonists and antagonists [28]. Results from such studies are currently accumulating and suggest that the HR2 antihistamines have potential beneficial effects in the treatment of certain malignant diseases, either alone or in combination with other drugs [29]. The beneficial effect of HR2 antihistamines as adjuvant single drugs to reduce trauma-, blood transfusion-, and sepsis-induced immunosuppression has indicated combined treatment regimens in major surgery, particularly in patients operated on for malignant diseases. Two different mechanisms are probably acting concordantly: direct inhibition of tumor cell proliferation by the HR2 antihistamines and activation of the local immune response characterized by IFN-␥ production. These findings might help to elucidate the possibility of a rationally designed antihistamine strategy in tumor therapy.

Conclusions

The recent developments in histamine research have led to the change in the approach to role of this mediator and its receptors in the allergic inflammatory reactions. Histamine and its different HR display a complex system with distinct functions of receptor subtypes and their differential expression, which

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changes according to the stage of cell differentiation as well as microenvironmental influences. It has been commonly acknowledged that HR1 activates the immune system cells potentiating a proinflammatory activity for higher migration to inflammation area as well as increased effector functions. HR2 on the other hand seems to be a potent suppressor of inflammatory and effector functions. Moreover a new light was shed by the demonstration of histaminecytokine cross-talk, the ability to newly synthesize and liberate histamine without storage by immunocompetent cells, discovery of a novel receptor HR4, preferentially expressed on hematopoietic and immunocompetent cells, and the demonstaration of T cells as the target for H1-antihistamine in an adoptive transfer model. These developments open a window for therapeutic approaches based on combined anti-HR1/anti-HR4 blockade or development of selective dual HR1/HR4 antagonists. Whether such therapies will provide further benefit to suppression of allergic inflammatory responses as observed after HR1 blockade remains to be elucidated.

Acknowledgements The authors’ laboratories are supported by the Swiss National Foundation Grants: 31–65436 and 32–105865 and Polish National Science Committee grant No 1387/PO5/ 2000/19.

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Dale HH, Laidlaw PP: The physiological action of beta-imidazolylethylamine. J Physiol (London) 1910;41:318–344. Jutel M, Watanabe T, Akdis M, Blaser K, Akdis CA: Immune regulation by histamine. Curr Opin Immunol 2002;14:735–740. Akdis CA, Blaser K: Histamine in the immune regulation of allergic inflammation. J Allergy Clin Immunol 2003;112:15–22. Haas H, Panula P: The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci 2003;4:121–130. Kubo Y, Nakano K: Regulation of histamine synthesis in mouse CD4⫹ and CD8⫹ T lymphocytes. Inflamm Res 1999;48:149–153. Endo Y: Simultaneous induction of histidine and ornithine decarboxylases and changes in their product amines following the injection of Escherichia coli lipopolysaccharide into mice. Biochem Pharmacol 1982;31:1643–1647. Yoshimoto T, Tsutsui H, Tominaga K, Hoshino K, Okamura H, Akira S, Paul WE, Nakanishi K: IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc Natl Acad Sci USA 1999;96:13962–13966. Abe Y, Ogino S, Irifune M, Imamura I, Fukui H, Wada H, Matsunaga T: Histamine content, synthesis and degradation in human nasal mucosa. Clin Exp Allergy 1993;23:132–136. Leurs R, Church MK, Taglialatela M: H1-antihistamines: Inverse agonism, anti-inflammatory actions and cardiac effects. Clin Exp Allergy 2002;32:489–498.

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Leurs R, Smit MJ, Timmerman H: Molecular pharmacological aspects of histamine receptors. Pharmacol Ther 1995;66:413–463. Banu Y, Watanabe T: Augmentation of antigen receptor-mediated responses by histamine H1 receptor signaling. J Exp Med 1999;189:673–682. Del Valle J, Gantz I: Novel insights into histamine H2 receptor biology. Am J Physiol 1997;273: G987–G996. Lovenberg TW, Roland BL, Wilson SJ, Jiang X, Pyati J, Huvar A, Jackson MR, Erlander MG: Cloning and functional expression of the human histamine H3 receptor. Mol Pharmacol 1999;55: 1101–1107. Nakamura T, Itadani H, Hidaka Y, Ohta M, Tanaka K: Molecular cloning and characterization of a new human histamine receptor, HHR4. Biochem Biophys Res Commun 2000;279:615–620. Szeberenyi JB, Pallinger E, Zsinko M, Pos Z, Rothe G, Orso E, Szeberenyi S, Schmitz G, Falus A, Laszlo V: Inhibition of effects of endogenously synthesized histamine disturbs in vitro human dendritic cell differentiation. Immunol Lett 2001;76:175–182. Mazzoni A, Young HA, Spitzer JH, Visintin A, Segal DM: Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization. J Clin Invest 2001;108: 1865–1873. Elenkov IJ, Webster E, Papanicolaou DA, Fleisher TA, Chrousos GP, Wilder RL: Histamine potently suppresses human IL-12 and stimulates IL-10 production via H2 receptors. J Immunol 1998;161:2586–2593. Jutel M, Watanabe T, Klunker S, Akdis M, Thomet OA, Malolepszy J, Zak-Nejmark T, Koga R, Kobayashi T, Blaser K, Akdis CA: Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors. Nature 2001;413:420–425. Ma RZ, Gao J, Meeker ND, Fillmore PD, Tung KS, Watanabe T, Zachary JF, Offner H, Blankenhorn EP, Teuscher C: Identification of Bphs, an autoimmune disease locus, as histamine receptor H1. Science 2002;297:620–623. Jutel M, Akdis M, Budak F, Aebischer-Casaulta C, Wrzyszcz M, Blaser K, Akdis C: IL-10 and TGF-beta cooperate in the regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur J Immunol 2003;33:1205–1214. Kunzmann S, Mantel P-Y, Wohlfahrt J, Akdis M, Blaser K, Schmidt-Weber C: Histamine enhances TGF-beta1-mediated suppression of Th2 responses. FASEB J 2003;17:1089–1095. Hofstra CL, Desai PJ, Thurmond RL, Fung-Leung WP: Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells. J Pharmacol Exp Ther 2003;305:1212–1221. Bakker RA, Schoonus SB, Smit MJ, Timmerman H, Leurs R: Histamine H(1)-receptor activation of nuclear factor-kappa B: Roles for G beta gamma- and G alpha(q/11)-subunits in constitutive and agonist-mediated signaling. Mol Pharmacol 2001;60:1133–1142. Jarjour NN, Calhoun WJ, Schwartz LB, Busse WW: Elevated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with increased airway obstruction. Am Rev Respir Dis 1991;144:83–87. Malick A, Grant JA: Antihistamines in the treatment of asthma. Allergy 1997;52:55–66. Gelfand EW, Cui ZH, Takeda K, Kanehiro A, Joetham A: Fexofenadine modulates T-cell function, preventing allergen-induced airway inflammation and hyperresponsiveness. J Allergy Clin Immunol 2002;110:85–95. Sirois J, Menard G, Moses AS, Bissonnette EY: Importance of histamine in the cytokine network in the lung through H2 and H3 receptors: Stimulation of IL-10 production. J Immunol 2000;164: 2964–2970. Mavligit GM: Immunologic effects of cimetidine: Potential uses. Pharmacotherapy 1987;7: S120–S124. Szincsak N, Hegyesi H, Hunyadi J, Martin G, Lazar-Molnar E, Kovacs P, Rivera E, Falus A, Juhasz I: Cimetidine and a tamoxifen derivate reduce tumour formation in SCID mice xenotransplanted with a human melanoma cell line. Melanoma Res 2002;12:231–240.

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Dr. Marek Jutel Department of Internal Medicine and Allergy Wroclaw Medical University, Traugutta 57 PL–50-417 Wroclaw (Poland) Tel. ⫹48 71 344 2264, Fax ⫹48 71 370 0129, E-Mail [email protected]

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Molecular Aspects of Allergy and Asthma Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 188–194

Gene Expression Profiling in Allergy and Asthma Carsten B. Schmidt-Weber Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland

Abstract DNA array technique was applied for the simultaneous analysis of multiple genes for the characterization of diseases and for cellular analysis. This technology implicated genes in the pathogenesis of allergic disease, which were previously not discussed in this context. This article summarizes results of gene expression profiling of allergic disease and immunologically relevant cells, using DNA array technology. The results of these studies are searchable in public databases and represent exciting tools to study gene expression of diseases on a global base. Copyright © 2006 S. Karger AG, Basel

The DNA array technology allows monitoring of expression changes of the expected 30–100,000 mRNA species present in a single (human) cell. This entire pool of expressed mRNAs is also called ‘transcriptome’. Moreover, uncharacterized open reading frames revealed by the Human Genome Project may be characterized in context of cellular activity. In combination with knowledge of gene loci, which are known to correlate with susceptibility to allergic disorders and asthma [1–3], it is likely that DNA array analysis will catalyze the research for key regulatory genes and molecules that are related to these diseases.

The DNA Array Technology

The DNA array consists of a DNA antisense probe specific for genes of interest, which are blotted, printed or synthesized on a solid carrier. As DNA

probes, plasmids, PCR products, or oligonucleotides can be used. Generally it is assumed that longer DNA probes are more specific and display better annealing properties. Nylon membranes, plastic, or glass are used as solid carrier. Depending on the density of spotted DNA probes, the arrays are distinguished in ‘macroarrays’, ‘microarrays’ and ‘DNA chips’. DNA chips are now commercially available carrying essentially the whole human or mouse genome, overcoming the limitations given by preselection of genes. The density can be accomplished by photolithographic synthesis of DNA on the carrier. The limitation of the system is on one hand the high costs, but on the other hand also the difficulty to work with a large number of genes, which may introduce errors during the array production [4]. The high number of genes also requires the use of sophisticated bioinformatics to bring interesting results out of the overwhelming flood of information. The array-generated data require subsequent validation and investigation on whether the gene is more/less abundantly expressed or whether the gene-expressing cell is more/less frequent. Although the array reaches full genome size, it should be realized that genes with major relevance in disease may still be missed. An example is the G-protein-coupled receptor A (GPRA or GPR 154), which has been genetically identified to be strongly associated with asthma [3]. However, none of the array studies identified this gene as a major contributor to asthma pathogenesis. Considering this example, limitations of this technology become clear, which in the case of GPRA is dictated by the importance of the B-isoform, which is only expressed in smooth muscle cells of asthma patients, and which may not be detected by the array probes generated from other mRNA sources because smooth muscle cells are difficult to access for such studies. Furthermore, important genes may have relatively low mRNA-turnover, as is the case for many membrane receptors such as the G-protein-coupled receptors, and thus fall under the statistical thresholds which are applied to filter DNA-array data.

Application of DNA Arrays

DNA arrays have been used for genomic profiling which is the detection of mutations and single nucleotide polymorphisms, and for pathogen detection, chromatin analysis, identification of genetically manipulated organisms, and expression profiling [5]. Gene expression profiling of disease relates multiple genes to complex clinical phenotypes. In particular, immunological disorders such as allergic conditions have been investigated and provide information, which will improve our pathological understanding of this disease. Using this technology, different allergic conditions and various inflammatory diseases displaying the same clinical symptoms, but differing in their expression profile

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can be discriminated. In turn, this will allow narrowing down the causes of the disease and in addition provide new possibilities for an optimized therapy based on an individual patient’s condition.

Gene Expression Profiles in Allergy

The gene expression profiling has been applied for human and murine systems and can be assessed in public databases (table 1 shows an allergy relevant selection). The GEO database (http://www.ncbi.nlm.nih.gov/geo/) at the National Center for Biotechnology carries datasets on intratracheal IL-13 treatment (the set is called allergen-induced goblet cells), allergic asthma models, allergen-triggered lung, T cells from atopic and asthma patients, allergen provocation in IL-13 knockouts, asthma exacerbation factors, and studies which are indirectly related to allergy such as effects of glucocorticoids etc. Looking into these datasets is most useful, if the potential of a gene of interest is searched, whereas the view on allergen-induced effects for example is difficult and should be guided by functional definitions. A great overview over such functions is provided for phenotypes such as apoptosis, chemotaxis and cytokine networks, which are affected by multiple genes. Genes regulating apoptosis were analyzed and revealed that peripheral blood cells display a net upregulation of prosurvival cytokines and the bcl-2 family of genes in allergic asthma and in atopic nonasthmatic patients [6]. Using peripheral blood mononuclear cells as cellular source they revealed that both atopic asthmatic and nonasthmatic patients show activated proinflammatory pathways, minimal requirement for B-cell isotype switching, and a net proIgE cytokine environment [7]. Similar studies were performed with PBMC isolated from atopic dermatitis patients [8]. It could also be shown that gene expression profiling allows quantification of phenotype-specific alteration in gene expression of atopic individuals, such as severity and extent of atopy [9]. Particularly helpful are DNA arrays for the understanding of the chemokine network, which was investigated in atopic dermatitis and psoriasis, both characterized by local infiltrates into the skin. Very distinctive gene expression pattern was observed in atopic dermatitis as compared with psoriasis, that may explain several features of cell infiltrations which are found in the skin, including Th2 cells, eosinophils, and mast cells in atopic dermatitis, and Th1 cells and neutrophils in psoriasis [10, 11]. Syed et al. [12] found downregulated CCR7 in steroid-resistant and steroid-sensitive asthma, and downregulated laminin receptor and integrin ␤5 in atopic and nonatopic steroid-resistant patients. The DNA array-based analysis of the cytokine network also improved the understanding of T cells activity in allergy. Gene expression profiling of

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Table 1. Searchable results of DNA arrays in allergy and asthma relevant tissues and cells (Datasets searchable in GEO database) Type of material

Dataset

Species

Category*

IL-13 exposed murine airways Analysis of the effect of inactivating interleukin 13 on the lung Lungs of ovalbumin-sensitized and -challenged A/J mice Lungs of ovalbumin-sensitized and -challenged C3H mice Lungs of ovalbumin-sensitized and -challenged C57BL/6 mice Response to ragweed

GDS245 GDS958, 959

M M

A A

GDS349 GDS347 GDS348

M M M

A A A

GDS42, 56, 12, 58, 14, 60 GDS64

M

A

R

A

GDS521, 350, 351

M

A

GDS266, 267

H

B

GDS261

H

B

GDS684

M

B

GDS877 GDS751

R H

B B/C

GDS308 GDS852

M H

C C

GDS498

H

C

GDS501

H

C

GDS601

H

C

Effects of large tidal volume ventilation and hyperoxia in lungs Sensitivity to pulmonary fibrosis induced by bleomycin by comparing strains which are susceptible (C57BL6/J) and resistant (Balb/c) CD4⫹ lymphocytes from patients with and without atopy, in combination with asthma Epithelial cells derived from asthmatic and normal bronchial airways; Effects of ozone and rhinovirus examined Regulatory and T effector cells from prediabetic pancreatic lesion Alveolar type I cell to that in type II cells Expression profiling of CD56dimCD16⫹, CD56brightCD16⫺, and in vitro-activated CD56⫹CD16⫹ natural killer cells Analysis of effects induced by ligand IL-4 in B cells Alveolar epithelial cells exposed to tumor necrosis factor-alpha, lipopolysaccharide, or cyclic stretch Interferon gamma, tumor necrosis factor alpha and interleukin 4 on primary lung endothelial cells Interferon gamma, tumor necrosis factor alpha and interleukin 4 on primary dermal endothelial cells T lymphocyte activation genes using an experimental compendium composed of activated T cells and non-T cells

*The categorization was performed as previously described [20]: The category A involves studies based on mRNAs isolated from biopsies. Category B covers approaches, which take advantage of pure, isolated cell populations and category C holds those analysis, which include an additional stimulation step following isolation of the cells.

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purified and in vitro activated CD4⫹ T cells of patients with atopic dermatitis or allergic asthma provided differential gene expression of T cells in distinct types of allergic diseases [13]. The in vitro stimulation has the advantage of compensating individual activation differences and provides an mRNA pool under maximal stimulatory conditions. This study stimulated the analysis of the family of Ephrins, which were identified for the first time during this study and which were confirmed to be involved in asthma pathology in a subsequent study [14]. Particularly Ephrin A1 was found to be downregulated on T cells of asthma patients, which normally may act suppressive on T cells. Interestingly, its receptors are abundantly expressed by epithelial cells of the airways and are therefore likely to affect immune reactions in the lung [14]. The immune response in allergy is dominated by effector T cells mostly of the Th2 phenotype, which are controlled by regulatory T cells. Both subsets were subjected to expression profiling. Differences in gene expression profiles of Th1 and Th2 were analyzed by Rogge et al. [15], by comparing cord blooddifferentiated Th1 and Th2; they show that from 6,000 investigated genes 215 are differentially expressed among these T-cell subtypes. This study demonstrates that a high number of differentially expressed genes contribute to the Th1/Th2 phenotypes. The expression pattern of regulatory T cells in a model of diabetes revealed an important role of ICOS. CD4⫹CD25⫹CD69⫺ TRegs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state [16]. This study has been published (GDS684 record) in the GEO database and it can be seen that only 5 genes are 3-fold higher expressed in TRegs compared to effector T cells, which is lectin-like receptor subfamily G, member 1 (Klrg1), chemokine receptor 5 (CCR5), suppressor of cytokine signaling 2 (SOCS2), granzyme B, integrin, alpha E, lectin, galactose binding, soluble 1 (Lgals1). Interesting is the circumstance that 2 genes occur, which were described in the context of killer cells (Klrg1 & granzyme B), suggesting that TRegs may also have lytic functions. Other allergy-related studies elucidate the gene expression profile of mast cells [17] and eosinophils [18]. The effect of contact allergens and their effect on dendritic cells was investigated on a transcriptome level and confirmed that contact allergens promote the differentiation of dendritic cells [19]. Only a small portion of gene-expression-profiled genes can be integrated into the current immunologic knowledge, because the function of many transcription factors, enzymes, or other signaling molecules is not yet fully understood. Therefore many investigators limit the number of the analyzed genes by using theme-oriented DNA arrays, which are focused on genes involved in defined processes such as apoptosis, cytokine-regulation, chemotaxis etc. This approach is useful for restricted experimental systems such as in vitro manipulated cells.

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Conclusions

The DNA array technology has been intensively applied in the field of allergology and was the starting point for subsequent novel and original findings. However, as in most complex diseases, the array technology provided many new puzzle pieces which need to be set into the big picture. Thus, it becomes more important to efficiently use bioinformatics along with genetic, proteomic, and cellular biology technologies to translate the array data into pathophysiologic knowledge.

Acknowledgements This article is dedicated to Kurt Blaser on the occasion of his 65th birthday for his personal and professional support and most importantly for his friendship.

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Cookson WO, Ubhi B, Lawrence R, Abecasis GR, Walley AJ, Cox HE, Coleman R, Leaves NI, Trembath RC, Moffatt MF, Harper JI: Genetic linkage of childhood atopic dermatitis to psoriasis susceptibility loci. Nat Genet 2001;27:372–373. Lee YA, Wahn U, Kehrt R, Tarani L, Businco L, Gustafsson D, Andersson F, Oranje AP, Wolkertstorfer A, v Berg A, Hoffmann U, Kuster W, Wienker T, Ruschendorf F, Reis A: A major susceptibility locus for atopic dermatitis maps to chromosome 3q21. Nat Genet 2000;26:470–473. Laitinen T, Polvi A, Rydman P, Vendelin J, Pulkkinen V, Salmikangas P, Makela S, Rehn M, Pirskanen A, Rautanen A, Zucchelli M, Gullsten H, Leino M, Alenius H, Petays T, Haahtela T, Laitinen A, Laprise C, Hudson TJ, Laitinen LA, Kere J: Characterization of a common susceptibility locus for asthma-related traits. Science 2004;304:300–304. Knight J: When the chips are down. Nature 2001;410:851–1008. Wilgenbus KK, Lichter P: DNA chip technology ante portas. J Mol Med 1999;77:761–768. Brutsche MH, Brutsche IC, Wood P, Brass A, Morrison N, Rattay M, Mogulkoc N, Simler N, Craven M, Custovic A, Egan JJ, Woodcock A: Apoptosis signals in atopy and asthma measured with cDNA arrays. Clin Exp Immunol 2001;123:181–187. Brutsche MH, Brutsche IC, Wood P, Mogulkoc N, Custovic A, Egan J, Woodcock A: B-cell isotype control in atopy and asthma assessed with cDNA array technology. Am J Physiol Lung Cell Mol Physiol 2001;280:L627–L637. Heishi M, Kagaya S, Katsunuma T, Nakajima T, Yuki K, Akasawa A, Maeda M, Gunji S, Sugita Y, Tsujimoto G, Saito H: High-density oligonucleotide array analysis of mRNA transcripts in peripheral blood cells of severe atopic dermatitis patients. Int Arch Allergy Immunol 2002;129:57–66. Brutsche MH, Joos L, Carlen Brutsche IE, Bissinger R, Tamm M, Custovic A, Woodcock A: Array-based diagnostic gene-expression score for atopy and asthma. J Allergy Clin Immunol 2002;109:271–273. Nomura I, Gao B, Boguniewicz M, Darst MA, Travers JB, Leung DY: Distinct patterns of gene expression in the skin lesions of atopic dermatitis and psoriasis: A gene microarray analysis. J Allergy Clin Immunol 2003;112:1195–1202. Nomura I, Goleva E, Howell MD, Hamid QA, Ong PY, Hall CF, Darst MA, Gao B, Boguniewicz M, Travers JB, Leung DY: Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes. J Immunol 2003;171:3262–3269.

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Syed F, Blakemore SJ, Wallace DM, Trower MK, Johnson M, Markham AF, Morrison JF: CCR7 (EBI1) receptor down-regulation in asthma: Differential gene expression in human CD4⫹ T lymphocytes. QJM 1999;92:463–471. Wohlfahrt JG, Kunzmann S, Menz G, Kneist W, Akdis CA, Blaser K, Schmidt-Weber CB: T cell phenotype in allergic asthma and atopic dermatitis. Int Arch Allergy Immunol 2003;131: 272–282. Wohlfahrt JG, Karagiannidis C, Kunzmann S, Epstein MM, Kempf W, Blaser K, Schmidt-Weber CB: Ephrin-A1 suppresses Th2 cell activation and provides a regulatory link to lung epithelial cells. J Immunol 2004;172:843–850. Rogge L, Bianchi E, Biffi M, Bono E, Chang SY, Alexander H, Santini C, Ferrari G, Sinigaglia L, Seiler M, Neeb M, Mous J, Sinigaglia F, Certa U: Transcript imaging of the development of human T helper cells using oligonucleotide arrays. Nat Genet 2000;25:96–101. Herman AE, Freeman GJ, Mathis D, Benoist C: CD4⫹CD25⫹ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 2004;199: 1479–1489. Saito H, Nakajima T, Matsumoto K: Human mast cell transcriptome project. Int Arch Allergy Immunol 2001;125:1–8. Brenner V, Lindauer K, Parkar A, Fordham J, Hayes I, Stow M, Gama R, Pollock K, Jupp R: Analysis of cellular adhesion by microarray expression profiling. J Immunol Methods 2001;250: 15–28. Ryan CA, Gildea LA, Hulette BC, Dearman RJ, Kimber I, Gerberick GF: Gene expression changes in peripheral blood-derived dendritic cells following exposure to a contact allergen. Toxicol Lett 2004;150:301–316. Schmidt-Weber CB, Wohlfahrt JG, Blaser K: DNA arrays in allergy and immunology. Int Arch Allergy Immunol 2001;126:1–10.

Dr. Carsten B. Schmidt-Weber Swiss Institute of Allergy and Asthma Research (SIAF) Obere Strasse 22 CH–7270 Davos (Switzerland) Tel. ⫹41 81 410 0853, Fax ⫹41 81 410 0840, E-Mail [email protected]

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Immunotherapy Crameri R (ed): Allergy and Asthma in Modern Society: A Scientific Approach. Chem Immunol Allergy. Basel, Karger, 2006, vol 91, pp 195–203

Mechanisms of Allergen-Specific Immunotherapy Cezmi A. Akdis, Kurt Blaser, Mübeccel Akdis Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland

Abstract Allergen-specific immunotherapy (SIT) is the only treatment, which leads to a life-long tolerance against allergens due to restoration of normal immunity. The induction of a tolerant state in peripheral T cells represents an essential step in allergen-SIT. Peripheral T-cell tolerance is characterized mainly by suppressed proliferative and cytokine responses against the major allergens and its T-cell recognition sites. It is initiated by autocrine action of IL-10 and/or TGF-␤, which are increasingly produced by the antigen-specific T Regulatory (TReg) cells. Tolerized T cells can be reactivated to produce either of the distinct Th1 or Th2 cytokine patterns, thus directing allergen-SIT towards successful or unsuccessful treatment. TReg cells directly or indirectly influence effector cells of allergic inflammation, such as mast cells, basophils and eosinophils. In addition, there is accumulating evidence that they may suppress IgE production and induce IgG4 and IgA production against allergens. By the application of the recent knowledge in mechanisms of allergen-SIT, more rational and safer approaches are awaiting in the future for the prevention and cure of allergic diseases. Copyright © 2006 S. Karger AG, Basel

Peripheral T-Cell Tolerance in Allergen-Specific Immunotherapy

The symptoms of IgE-mediated allergic reactions – such as rhinitis, conjunctivitis and asthma – can be ameliorated by temporary suppression of mediators and immune cells (by antihistamines, antileukotrienes, ␤2 adrenergic receptor antagonists and corticosteroids) [1, 2]. However, a more long term solution is allergen-specific immunotherapy (SIT) that specifically restores a normal immunity against allergens. Allergen-SIT is most efficiently used in allergy to insect venoms and allergic rhinitis [3–5]. Despite its usage in clinical practice for nearly a century, the underlying immunological mechanisms of allergen-SIT are slowly being elucidated. A rise in allergen-blocking IgG antibodies particularly of the IgG4 class, which supposedly block allergen and

Direct or indirect suppression of

Increased levels of

Th2 cells Th1 cells Cytokines of Th1 and Th2 cells (IFN-␥, IL-4, IL-5, IL-13) IgE Mast cells Basophils Eosinophils Macrophages Dendritic cells Mucus production

TReg cells Cytokines of TReg cells (IL-10, TGF-␤) IgG4 IgA

Fig. 1. Immunological changes after allergen-SIT. A healthy immune response to allergen after SIT might not depend on the balance between Th2 and Th1 cytokines, but rather on the balance between Th2 and TReg cells. The major immunological change is characterized by a decrease in allergen-specific Th2 cells and increased numbers of TReg cells.

IgE facilitated antigen presentation [6], the generation of IgE-modulating CD8⫹ T cells [7] and a reduction in the numbers of mast cells and eosinophils, including the release of mediators [8, 9] were shown to be associated with successful allergen-SIT. Furthermore, allergen-SIT was found to be associated with a decrease in IL-4 and IL-5 production by CD4⫹ T cells [10–12]. Also a shift from Th2 cytokine pattern towards increased IFN-␥ production in allergen-SIT of allergy to bee venom, wasp venom, grass pollen and house dust mite was observed [10, 13]. It appears however, that the induction of a tolerant state in peripheral T cells, represents an essential step in allergen-SIT (fig. 1) [14–17]. Peripheral T-cell tolerance is characterized mainly by suppressed proliferative and cytokine responses against the major allergens and its T-cell recognition sites [14]. T-cell tolerance is initiated by autocrine action of IL-10, which is increasingly produced by the antigen-specific T cells [15, 16]. Tolerized T cells can be reactivated to produce either of the distinct Th1 or Th2 cytokine patterns depending on the cytokine present in the tissue microenvironment, and thus directing allergen-SIT towards successful or unsuccessful treatment [14]. Immunotherapy using peptides (PIT) is another attractive approach for investigation of peripheral T-cell tolerance in humans. Short allergen peptides, either native sequences or altered peptide ligands, with amino acid substitutions do not contain epitopes for IgE cross-linking to induce anaphylaxis. There is considerable rationale for targeting T cells with synthetic peptides based on such T-cell epitopes. To date, clinical studies of PIT have been performed in two allergies [18–20]. Relatively long peptides of 27 and 35 amino acids of the major cat allergen Fel d 1 containing the T-cell epitopes or mixture of peptides spanning the whole protein sequence were used to treat allergy to cats, and

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resulted in the induction of tolerance in IL-4-producing cells [19, 20]. In the other trial, PIT of bee venom allergy was performed with a mixture of short peptides that directly represent the T-cell epitopes (17, 12, 11 amino acids) of the major allergen in bee venom, phospholipase A2 [18]. The study showed modulation of the immune response against the whole allergen, inducing specific T-cell tolerance and a decrease in the specific IgE:IgG4 ratio [18]. Single amino acid alteration in T-cell epitopes can modify specific T-cell activation and cytokine production [21]. Rodent studies suggest that, under highly controlled experimental conditions, allergic diseases can be inhibited by altered peptide ligand administration. Whether this is due to Th2 to Th1 immune deviation, or the induction of TReg cells remains to be elucidated [21]. Although PIT is theoretically attractive as a means to avoid IgE-mediated early phase reactions, it is important to note that serum IgE in allergic individuals may sometimes bind to relatively short linear epitopes of protein allergens [22]. A potential barrier to PIT of allergy is the apparent complexity of the allergenspecific T-cell response in terms of epitope usage and dominant epitopes in humans [23]. Analysis of responses to various food and inhalant antigens has shown that healthy immune response to mucosal antigens displays a similar mechanism of active regulation [24]. A recent study has been performed using interferon (IFN)-␥-, IL-4- and IL-10- secreting allergen-specific CD4⫹ T cells that resemble Th1, Th2 and Tr1-like cells, respectively. Healthy and allergic individuals exhibit all three subsets, but in different proportions. In healthy individuals Tr1 cells represent the dominant subset for common environmental allergens, whereas a high frequency of allergen-specific IL-4-secreting T cells (Th2-like) is found in allergic individuals. Hence, a change in the dominant subset may lead to either the development of allergy or recovery (fig. 2) [24].

Peripheral T-Cell Tolerance to Allergens is Associated with Regulation of Antibody Isotypes

The serum levels of specific IgE and IgG4 antibodies delineate allergic and normal immunity to allergen. Although peripheral tolerance was demonstrated in specific T cells, the capacity of B cells to produce specific IgE and IgG4 antibodies was not abolished during SIT [14]. In fact, specific serum levels of both isotypes increased during the early phase of treatment. However, the increase in antigen-specific IgG4 was more pronounced and the ratio of specific IgE to IgG4 decreased by 10- to 100-fold. Also the in vitro production of phospholipase A2-specific IgE and IgG4 antibodies by peripheral blood mononuclear cells, changed in parallel to the serum levels of specific isotypes.

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Th2 Th2 Th1

Th1

TReg

TReg

Before SIT Allergic

After SIT Healthy

Fig. 2. Direct or indirect suppression of mast cells, basophils and eosinophils by TReg cells is a crucial event for successful SIT. In addition, suppression of IgE and an increase in allergen-specific IgG4 and IgA levels might effectively contribute to long-term success.

A similar change in specific isotype ratio was observed in SIT of various allergies. Moreover, IL-10 which is induced and increasingly secreted by SIT, appears to counter-regulate antigen-specific IgE and IgG4 antibody synthesis (fig. 1) [15]. IL-10 is a potent suppressor of both total and allergen-specific IgE, while it simultaneously increases IgG4 production [15, 25]. Thus, IL-10 not only generates tolerance in T cells; it also regulates specific isotype formation and skews the specific response from an IgE to an IgG4-dominated phenotype (fig. 1). The healthy immune response to Der p1 demonstrated increased specific IgA and IgG4, small amounts of IgG1, and almost undetectable IgE antibodies in serum [17]. House dust mite-SIT did not significantly change specific IgE levels after 70 days of treatment; however, a significant increase in specific IgA, IgG1, and IgG4 was observed [17]. The increase of specific IgA and IgG4 in serum coincides with increased TGF-␤ and IL-10 respectively. This may account for the role of IgA and TGF-␤, as well as IgG4 and IL-10, in peripheral mucosal immune responses to allergens in healthy individuals [15, 26]. Supporting these findings, correction of low Der p 1-specific IgA levels after sublingual immunotherapy has been demonstrated in house dust mite allergic children [27].

Suppression of Effector Cells by Allergen-Specific Immunotherapy

Despite the fact that definite decrease in IgE antibody levels and IgEmediated skin sensitivity, normally requires several years of SIT, most patients

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are protected against bee stings already at an early stage of bee venom-SIT. The reason for this is that effector cells of allergic inflammation, such as mast cells, basophils and eosinophils require T-cell cytokines for priming, survival and activity, [28, 29] which are not efficiently provided by suppressed Th2 cells and TReg cells (fig. 1). SIT efficiently modulates the thresholds for mast cell and basophil activation and decreases immunoglobulin E-mediated histamine release (fig. 1) [30, 31]. In addition, IL-10 was shown to reduce proinflammatory cytokine release from mast cells [32]. Furthermore, IL-10 downregulates eosinophil function and activity, and suppresses IL-5 production by human resting Th0 and Th2 cells [33]. Moreover, IL-10 inhibits endogenous granulocyte macrophage – colony stimulating factor production and CD40 expression by activated eosinophils and enhances eosinophil cell death [34].

Mechanism of T-Cell Suppression by IL-10 and Its Relationship to Anergy

Inhibition of T-cell costimulatory molecules at the cell surface or their intracellular signal transduction, has been repeatedly reported to play an important role in T cell tolerance [35–37]. T cells could be anergized in experimental models that bypass costimulatory signals [38–40]. The interaction between B7 and CD28 may determine whether a T-cell response develops. Blocking antibodies to B7–2 inhibit the development of specific IgE, pulmonary eosinophilia and airway hyper-responsiveness in mice [41]. A molecule on activated T cells, cytotoxic T lymphocyte antigen 4 (CTLA4), seems to act on endogenous inhibitors of T-cell activation and CTLA4-Ig, is a soluble fusion-protein construct, and is also effective in blocking airway hyper-responsiveness in mice [42]. Anti-CD28, anti-B7–2 and CTLA4-Ig also block the proliferative response of T cells to allergen [43]. Furthermore cytokine production by memory/effector T cells, particularly those of Th2 subset, is highly dependent on costimulation via the ICOS/B7RP1 (ICOS-ligand) pathway. Blockage [44] or genetic disruption [45] of this costimulatory pathway markedly reduces allergen-induced asthma in mice. Thus blockage of B7–2/CD28 and ICOS/B7RP1 interaction may be a promising approach for the treatment of allergic disease in humans. One mechanism of direct T-cell suppression by IL-10 is due to the inhibition of CD28 costimulation. IL-10 inhibits the proliferative T-cell response in peripheral blood mononuclear cells to various antigens, and the superantigen staphylococcal enterotoxin B [46]. However, IL-10 does not affect the proliferative responses of T cells that were stimulated by anti-CD3. In contrast, IL-10 significantly inhibits the anti-CD28 stimulated proliferation. The analysis of

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T cell receptor (TCR) numbers on T cells demonstrated the essential requirement for costimulation in T-cell activation and its relation to the number of triggered TCRs [46]. IL-10 inhibited the T-cell proliferation within a certain range of triggered TCRs that require T-cell costimulation. T cells, which were stimulated by different concentrations of anti-CD3, and a constant amount of anti-CD28 showed that low numbers of triggered TCRs required CD28 costimulation. Thus, IL-10 suppressed only those T cells that had low numbers of TCRs triggered and which required CD28 for proliferation [46]. Stimulation of CD28 by B7 surface molecules leads to tyrosine phosphorylation of CD28. Ligation of IL-10 receptor at the time of CD28 stimulation inhibits tyrosine phosphorylation of CD28 as detected after 10 min [46, 47]. The inhibitory effect of IL-10 on CD28 appeared to be specific for the CD28 pathway, because IL-10 did not affect ZAP-70 tyrosine phosphorylation stimulated by CD3 cross-linking. As a consecutive event for signal transduction, the association of CD28 with the phosphatidylinositol 3-kinase (PI3-K) p85 molecule was inhibited by IL-10. This inhibition can be specifically blocked by preventing IL-10 binding to its receptor with an anti-IL-10R mAb. PI3-K is a heterodimer that comprises an 85 kD regulatory and a 110 kD catalytic subunit, possessing both protein serine-kinase and lipid-kinase activity [48]. Binding of PI3-K to CD28 occurs by direct interaction between SH2 domain motifs of p85 PI3-K and a (p)YXXM motif in the cytoplasmic part [49]. Taken together, after a century of its first usage, the term ‘anergy’ rests on an immunological basis. Containment and cure of tuberculosis requires an effective cell-mediated immune response and its absence during active tuberculosis infection has been demonstrated to be related to IL-10-mediated peripheral tolerance [50]. Accordingly, T-cell response in IL-10-mediated peripheral tolerance is the same as definition of anergy, because costimulatory signals are suppressed and T cells receive a weak signal only via the T-cell receptor without costimulation [38]. To date, there were no reports of clinical studies, which relates inhibition of costimulation to the specific treatment of allergy and asthma. Nevertheless, it remains as a fruitful approach for the treatment of both autoimmunity and allergy.

Conclusions

There is growing evidence supporting the role for TReg cells and/or immunosuppressive cytokine – IL-10 and TGF-␤ – as a mechanism by which SIT and healthy immune response to allergens is mediated, leading to both suppression of Th2 responses and ensuring a well-balanced immune response, and a switch from IgE to IgG4 antibody production (fig. 1). These mechanisms can

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be better used by improvement of current treatment using recombinant allergens or peptide therapy. The elaboration of more efficacious desensitization methods, including rapid protocols and targeting histamine receptors, [51] also holds a promise for further development.

Acknowledgements The authors’ laboratories are supported by the Swiss National Foundation Grants: 32–100266, 32–65436, 32–105865.

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Akdis CA, Blesken T, Akdis M, Wuthrich B, Blaser K: Role of interleukin 10 in specific immunotherapy. J Clin Invest 1998;102:98–106. Akdis CA, Blaser K: IL-10-induced anergy in peripheral T cell and reactivation by microenvironmental cytokines: Two key steps in specific immunotherapy. Faseb J 1999;13:603–609. Jutel M, Akdis M, Budak F, Aebischer-Casaulta C, Wrzyszcz M, Blaser K, Akdis AC: IL-10 and TGF-b cooperate in regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur J Immunol 2003;33:1205–1214. Müller UR, Akdis CA, Fricker M, Akdis M, Bettens F, Blesken T, Blaser K: Successful immunotherapy with T cell epitope peptides of bee venom phospholipase A2 induces specific T cell anergy in bee sting allergic patients. J Allergy Clin Immunol 1998;101:747–754. Marcotte GV, Braun CM, Norman PS, Nicodemus CF, Kagey-Sobotka A, Lichtenstein LM, Essayan DM: Effects of peptide therapy on ex vivo T-cell responses. J Allergy Clin Immunol 1998;101:506–513. Oldfield WL, Larche M, Kay AB: Effect of T-cell peptides derived from Fel d 1 on allergic reactions and cytokine production in patients sensitive to cats: A randomised controlled trial. Lancet 2002;360:47–53. Faith A, Akdis CA, Akdis M, Joss A, Wymann D, Blaser K: An altered peptide ligand specifically inhibits Th2 cytokine synthesis by abrogating TCR signaling. J Immunol 1999;162:1836–1842. Banerjee B, Kanitpong K, Fink JN, Zussman M, Sussman GL, Kelly KJ, Kurup VP: Unique and shared IgE epitopes of Hev b 1 and Hev b 3 in latex allergy. Mol Immunol 2000;37:789–798. Rolland JM, O’Hehir RE: Immunotherapy of allergy: Anergy, deletion, and immune deviation. Curr Opin Immunol 1998;10:640–645. Akdis M, Verhagen J, Taylor A, Karamloo F, Karagiannidis C, Crameri R, Thunberg S, Deniz G, Valenta R, Fiebig H, Kegel C, Disch R, Schmidt-Weber CB, Blaser K, Akdis CA: Immune responses in healthy and allergic individuals are characterized by a fine balance between allergenspecific T regulatory 1 and T helper 2 cells. J Exp Med 2004;199:1567–1575. Punnonen J, de Waal Malefyt R, van Vlasselaer P, Gauchat J-F, de Vries JE: IL-10 and viral IL-10 prevent IL-4-induced IgE synthesis by inhibiting the accessory cell function of monocytes. J Immunol 1993;151:1280–1289. Sonoda E, Matsumoto R, Hitoshi Y, Ishii T, Sugimoto M, Araki S, Tominaga A, Yamaguchi N, Takatsu K: Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production. J Exp Med 1989;170:1415–1420. Bahceciler NN, Arikan C, Taylor A, Akdis M, Blaser K, Barlan IB, Akdis CA: Impact of sublingual immunotherapy on specific antibody levels in asthmatic children allergic to house dust mites. Int Arch Allergy Immunol 2005;136:287–294. Walker C, Virchow JC Jr, Bruijnzeel PL, Blaser K: T cell subsets and their soluble products regulate eosinophilia in allergic and nonallergic asthma. J Immunol 1991;146:1829–1835. Schleimer RP, Derse CP, Friedman B, Gillis S, Plaut M, Lichtenstein LM, MacGlashan Jr DW: Regulation of human basophil mediator release by cytokines. I. Interaction with anti-inflammatory steroids. J Immunol 1989;143:1310–1327. Treter S, Luqman M: Antigen-specific T cell tolerance down-regulates mast cell responses in vivo. Cell Immunol 2000;206:116–124. Shim YK, Kim BS, Cho SH, Min KU, Hong SJ: Allergen-specific conventional immunotherapy decreases immunoglobulin E-mediated basophil histamine releasability. Clin Exp Allergy 2003; 33:52–57. Marshall JS, Leal-Berumen I, Nielsen L, Glibetic M, Jordana M: Interleukin (IL)-10 inhibits longterm IL-6 production but not preformed mediator release from rat peritoneal mast cells. J Clin Invest 1996;97:1122–1128. Schandane L, Alonso-Vega C, Willems F, Gerard C, Delvaux A, Velu T, Devos R, de Boer M, Goldman M: B7/CD28-dependent IL-5 production by human resting T cells is inhibited by IL-10. J Immunol 1994;152:4368–4374. Ohkawara Y, Lim KG, Glibetic M, Nakano K, Dolovich J, Croitoru K, Weller PF, Jordana M: CD40 expression by human peripheral blood eosinophils. J Clin Invest 1996;97:1761–1766. Knechtle SJ, Hamawy MM, Hu H, Fechner JH Jr, Cho CS: Tolerance and near-tolerance strategies in monkeys and their application to human renal transplantation. Immunol Rev 2001;183:205–213.

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Chambers CA: The expanding world of co-stimulation: The two-signal model revisited. Trends Immunol 2001;22:217–223. Schwartz RH: Models of T cell anergy: Is there a common molecular mechanism? J Exp Med 1996;184:1–8. Lamb JR, Skidmore BJ, Green N, Chiller JM, Feldman M: Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J Exp Med 1983;157:1434–1447. Faith A, Akdis CA, Akdis M, Simon H-U, Blaser K: Defective TCR stimulation in anergized type 2 T helper cells correlates with abrogated p56lck and ZAP-70 tyrosine kinase activities. J Immunol 1997;159:53–60. Hoyne GF, O’Hehir R, Wraith DC, Thomas WR, Lamb JR: Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice. J Exp Med 1993;178:1783–1788. Haczku A, Takeda K, Redai I, Hamelmann E, Cieslewicz G, Joetham A, Loader J, Lee JJ, Irvin C, Gelfand EW: Anti-CD86 (B7.2) treatment abolishes allergic airway hyperresponsiveness in mice. Am J Respir Crit Care Med 1999;159:1638–1643. Van Oosterhout AJ, Hofstra CL, Shields R, Chan B, Van Ark I, Jardieu PM, Nijkamp FP: Murine CTLA4-IgG treatment inhibits airway eosinophilia and hyperresponsiveness and attenuates IgE upregulation in a murine model of allergic asthma. Am J Respir Cell Mol Biol 1997;17:386–392. Van Neerven RJ, Van de Pol MM, Van der Zee JS, Stiekema FE, De Boer M, Kapsenberg ML: Requirement of CD28–CD86 costimulation for allergen-specific T cell proliferation and cytokine expression. Clin Exp Allergy 1998;28:808–816. Gonzalo JA, Tian J, Delaney T, Corcoran J, Rottman JB, Lora J, Al-garawi A, Kroczek R, Gutierrez-Ramos JC, Coyle AJ: ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses. Nat Immunol 2001;2:597–604. Dong C, Juedes AE, Temann UA, Shresta S, Allison JP, Ruddle NH, Flavell RA: ICOS co-stimulatory receptor is essential for T-cell activation and function. Nature 2001;409:97–101. Akdis CA, Joss A, Akdis M, Faith A, Blaser K: A molecular basis for T cell suppression by IL-10: CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding. FASEB J 2000;14:1666–1669. Joss A, Akdis M, Faith A, Blaser K, Akdis CA: IL-10 directly acts on T cells by specifically altering the CD28 co-stimulation pathway. Eur J Immunol 2000;30:1683–1690. Dhand R, Hara K, Hiles I, Bax B, Gout I, Panayotou G, Fry MJ, Yonezawa K, Kasuga M, Waterfield MD: PI 3-kinase: Structural and functional analysis of intersubunit interactions. EMBO J 1994;13:511–521. Prasad KVS, Cai Y-C, Raab M, Duckworth B, Cantley L, Shoelson SE, Rudd CE: T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-MetXaa-Met motif. Proc Natl Acad Sci USA 1994;91:2834–2838. Boussiotis VA, Tsai EY, Yunis EJ, Thim S, Delgado JC, Dascher CC, Berezovskaya A, Rousset D, Reynes JM, Goldfeld AE: IL-10-producing T cells suppress immune responses in anergic tuberculosis patients. J Clin Invest 2000;105:1317–1325. Jutel M, Watanabe T, Klunker S, Akdis M, Thomet OAR, Malolepszy J, Zak-Nejmarrk T, Koga R, Kobayashi T, Blaser K, Akdis AC: Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors. Nature 2001;413:420–425.

Prof. Dr. Cezmi A. Akdis Swiss Institute of Allergy and Asthma Research (SIAF) Obere Strasse 22 CH–7270 Davos (Switzerland) Tel. +41 81 410 0848, Fax +41 81 410 0840, E-Mail [email protected]

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Regulation of the IgE Response at the Molecular Level: Impact on the Development of Systemic Anti IgE Therapeutic Strategies Gernot Achatza, Gertrude Achatz-Straussbergera, Elke Lugerb, Rinus Lamersc, Reto Cramerid a

Department of Molecular Biology, University of Salzburg, Austria; bDRFZ, Berlin, MPI for Immunobiology, Freiburg, Germany; dSIAF, Davos, Switzerland

c

Abstract The classical allergic reaction starts within seconds or minutes after antigen contact and is induced by antibodies produced by a special subset of B lymphocytes. These antibodies belong to the IgE subclass and are responsible for Type I hyper-reactivity reactions. IgE plays a minor role in healthy individuals. In allergic individuals, however, IgE antibodies trigger allergic responses through allergen-mediated cross-linking on effector cells followed by mediator release. The mechanisms inducing a switch to IgE production are not fully understood with the consequence that allergies are mainly treated with antisymptomatic drugs. To develop basic therapies, many questions concerning the very complex regulation of IgE expression have to be understood. Positive and negative regulators influence the synthesis of IgE. Experiments in our laboratory could show that not only regulatory molecules, but also the membrane bound IgE itself controls the quantity and quality of the IgE produced. This fact becomes more and more interesting, because the signals generated by the B-cell receptor may be important targets for interference in allergic patients, in whom the titer and the affinity of the IgE antibodies for the allergen are directly related to disease activity. Copyright © 2006 S. Karger AG, Basel

Overview

The biological function of IgE [1] and the therewith-connected necessity of this special immunoglobulin (Ig)-isotype is so far unknown. Because IgE titers are elevated in helminthic infestations, it was thought to play a role in the defence against worms. The value of this notion is, however, debated because it

was shown that high titers of IgE may be even detrimental during infestations with worms [2, 3]. What remains, is the general acceptance that IgE has a pathological function as key molecule of the allergic response. The clinical manifestation in allergic diseases is reflected by the elevation of the allergenspecific serum IgE levels. Under normal conditions, the immune system tries to keep the level of IgE as low as possible. Compared to other Ig isotypes, total serum IgE levels are very low. However, if the decision to ‘force IgE expression’ occurred, the organism sets value on highest quality of the IgE molecules produced [4, 5]. The biological mechanism of this dangerous immunological response is unknown, but obviously several independent mechanisms regulate IgE levels. First: The short half-life of IgE. The half-lives of several sets of murine monoclonal antibodies spanning all Ig isotypes in the serum were determined by Vieira et al. [6]. The antibodies in each set shared the same V region which had the advantage that the differences in half-life observed were independent of the V region carried by the mAb’s, and therefore, must relate to each other in the same way as the half-lives of the corresponding isotype-specific constant regions. In this way, the half-life of IgM was calculated as 2 days, and that of IgG3 and IgG1 as 6–8 days; IgG2b has a half-life of 4–6 days. A polymeric form of IgA was found to be eliminated from the serum with a half-life of 17–22 h. Interestingly, IgE has a half-life of only 12 h, which contrasts significantly from other Ig classes that form a substantial component of serum proteins. Second: CD23, the low-affinity receptor for IgE (Fc␧RII) also acts as a buffer in negative feedback regulation of IgE synthesis [7]. CD23 knock-out mice over-express IgE, whereas transgenic mice over-expressing CD23 are deficient in IgE [8]. In isolated B cells, cross-linking of CD23 or IgE and CD23 resulted in the downregulation of IgE synthesis; thus, mCD23 and/or proteins associated with mCD23 may deliver signals to the B cell to inhibit IgE synthesis when IgE concentrations reach the same order as Kd (0.1 ␮M). Negative feedback regulation would thus occur in a much higher concentration range than that required for sensitization of mast cells and basophils (Kd 0.1 nM) when positive feedback mechanisms are dominant (reviewed in [1]). Murine IgE binds with low affinity to Fc␧RII, which contains an inhibitory motif; thus, murine IgE synthesis is inhibited when IgE concentrations approach the Kd of this interaction. Human IgE does not bind to IgG receptors but operates by way of an istoype-specific mechanism of feedback control through CD23. Third: mIgE knock-out mice indicate the necessity of the IgE receptor for an IgEmediated immune response, and clearly showed that the cytoplasmic tail determines not only the absolute amount of IgE produced, but also the quality of the antibodies. Fourth: in vitro data of our laboratories indicate an influence of an inefficient processing of the mRNA transcripts for the membrane form of IgE [9, 10]. The ratio of transcripts for the secreted and the membrane form of

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immunoglobulin reflects the usage of either polyadenylation signal. Posttranscriptional processing of the transcript coding for the membrane form of IgE, thus influences the signal transduction on the later recruitment of IgE secreting plasma cells (PCs), which in general are factories for the production of neutralizing antibodies in response to invading pathogens. PCs are interesting for several reasons. On a basic level, they show the general mechanisms involved in the commitment of somatic cells to a terminally differentiated, postmitotic state. As final mediators of the humoral immune response, they provide insights into the regulation of humoral immunity that may be helpful for the development of new vaccination strategies. Because PCs are long-living cells, residing in the body for many years, knowledge about the nature of their longevity, and in turn, the possibilities to affect this longevity, would be crucial to provide a rational basis for designing effective treatments for allergic diseases. The fundamental question is, whether PCs are still sensitive to antigen-induced cell death, which would have vast implications for the development of vaccination strategies and therapeutic approaches.

The Biological Function of the B-Cell Antigen Receptor

From the numerous surface markers of a B cell, the B-cell antigen receptor (BCR) is probably the most powerful one in influencing developmental processes of the cell. The BCR (fig. 1) consists of the membrane-bound immunoglobulin (mIg) but, depending on the state of differentiation, may be associated with a couple of other transmembrane proteins, most notably Ig-␣ (CD79␣) and Ig-␤ (CD79␤) [11]. The N-terminal domain of the mIg contains the antigen binding site that features an incredibly high potential for diversification, allowing the BCRs to respond to nearly every antigenic determinant. The transmembrane domain as well as the cytoplasmic portion vary in their amino acid sequence depending on which of the five Ig isotypes is expressed [12]. Though the IgG and IgD class of the BCR does not require the Ig-␣/Ig-␤ for surface expression [13], it could be shown that Ig-␣/Ig-␤ is the minimum requirement for signal transduction [14] and in the case of IgM, for the release from intracellular retention sites [15]. Ig-␣ is encoded by the mb-1 gene and is a 32 kDa glycoprotein. Ig-␤ (B29 gene) can be expressed in two different isoforms of 37 kDa and 39 kDa, respectively. Though all different mIg isotypes can potentially interact noncovalently with the same Ig-␣/Ig-␤, the Ig-␣, however, may have a different glycosylation pattern for each class. The BCR is permanently internalized from the cell membrane to the cytoplasm and endocytic compartments through clathrin-coated pits. This process of random BCR-internalization can be sufficient to effectively process and

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BCR-complex lg-heavy chain lg-light chain

ITAMs

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exon M1 extracellular

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exon M2 transmembrane

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Fig. 1. Model of the BCR complex: The mIg backbone, consisting of two Ig-heavy, two Ig-light chains, the transmembrane region and cytoplasmic domain, is associated with the two coat proteins Ig-␣ and Ig-␤. Both sheath proteins, inside their intracellular tail, harbour an immunoreceptor tyrosin based activation motif (ITAM). On antigen contact, the tyrosins of the ITAMs get phosphorylated by members of the Src-family kinases. Amino acid alignment of the C-terminal domains of mouse mIg-isotypes: Light shade: domains as predicted by the Kyte and Doolittle algorithm. Medium shade: domains as predicted by PHD topology. Dark shade: domains as predicted by the Klein algorithm. In the case of mIgA, the transmembrane and cytoplasmic domains are encoded by just one exon.

present bound antigen to activated T cells and hence to initiate a humoral response, without the need for any prominent BCR-mediated signalling [16]. Nonetheless, appropriate antigen mostly evokes receptor stimulation, resulting in cytoplasmic signalling and increased receptor endocytosis. This cytoplasmic signalling is never a decision between ‘aught or naught’. It is believed that the signalling cascade underlies a permanent stochastic fluctuation, which induces no cellular response, but is important for maintenance of cell viability. However, when the net activation energy of the signal strength reaches a certain threshold, the cell ‘jumps’ the activation barrier and acquires effector competence (reviewed in [17]).

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All the distinct cytoplasmic signals wired from the BCR upon engagement can differ not just in quantity but more surprisingly, even in quality. This means, the BCR is able to selectively turn on a specific signalling pathway, by keeping another quiescent. This capacity of the BCR allows the B cell to respond to antigenic challenge in a very specific way. In other words, the nature of the antigenic stimulus (which basically is determined by concentration, avidity and immunogenicity of the antigen as well as duration, place and timing of the encounter) decides what kind of physiologic response – basically B-cell activation or deactivation – is initiated. In this context it is important to note that the BCR is indispensable in warranting the survival of the B cell. Knock-out studies in mice show that loss of the BCR expression blocks B-cell development at the pre-B cell stage [18, 19] and the inducible deletion of secretory and membrane Ig molecules is followed by apoptotic cell death [20]. That impressively shows that the BCR guarantees the longevity of the B cell, probably through a constant low level stochastic signalling in the absence of antigen.

Regulation of the Membrane Expression of IgE

An additional step forward in understanding the IgE regulation in vivo was achieved with the two knock-out mice KVK⌬tail and ⌬M1M2 [4]. Two major conclusions were achieved from the study of the two knock-out mice: the transmembrane domain of mIgE is indispensable for T-cell-dependent IgE secretion and the cytoplasmic domain not only determines the absolute amount of IgE produced, but also influences the quality of the Igs. The mIg transmembrane segments are about 25 amino acids long and have the potential for interaction with other polypeptides [11]. The cytoplasmic domains of mIgs are different in length (fig. 1) and range from only three amino acid residues (KVK) in the case of mIgM and mIgD to 14–28 residues for the other mIg subclasses. The cytoplasmic tail of IgE is less conserved when compared to the other Ig classes. However, we assume that results obtained with the KVK⌬tail line can also be extended to other isotypes. B cells carrying mIgM as antigen receptor are easily induced to undergo apoptosis unless rescuing stimuli are present. mIgE carrying B cells in KVK⌬tail mice look from the cytoplasmic perspective rather like mIgM carrying cells. The low serum levels for IgE in KVK⌬tail mice could be the result of an elimination of mIgE cells by induction of apoptosis, an impaired signal transduction committed by the short tail, or an impaired antigen presentation of mIgE. Alternatively, the cytoplasmic tail normally interacts with factors that cooperate in processes like affinity maturation and somatic mutation, induction of memory cells, and induction of plasma

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IgM AATAAA

AATAAA

IgG1 AATAAA

AATAAA

IgG2a AATAAA

AATAAA

IgA AATAAA

AATAAA

IgE AATAAA

AGTAAA

ATTAAA

AAGAAA

Fig. 2. External and internal poly(A) signals in different Ig-isotypes: With the exception of IgE, for the ‘secreted’ and the ‘membrane’ polyadenylation signals, the consensus sequence AATAAA is used. However, the epsilon membrane locus uses three cryptic polyadenylation signals. These signals (AGTAAA, AAGAAA and ATTAAA) are in considerable disagreement with the consensus sequence and influence the stability of the mIgE transcript.

cells. Whether the cytoplasmic tail of IgE exerts a different role than those of the IgG and IgA isotypes, is yet not clear. As mentioned earlier, both, the secreted and membrane transcripts get polyadenylated by different polyadenylation signals, an internal that is used by the secreted form and an external which is used by the membrane transcript (fig. 2). This differential polyadenylation mechanism introduces a further regulatory possibility for in vivo IgE expression.

The Biochemical Process of Polyadenylation

The process of polyadenylation requires numerous factors responsible for the recognition, cleavage and addition of the poly(A) tail. Two multi-subunit complexes, designated cleavage-polyadenylation specificity factor and cleavage stimulation factor (CstF) [21], cooperate with each other to define the site of polyadenylation [22]. They recognize the highly conserved AAUAAA hexanucleotide [22] and a more divergent GU-rich sequence located downstream of the actual cleavage site [23]. Two additional factors, cleavage factor I and II (CFI and CFII), are additionally essential for the cleavage reaction [24]. For the poly(A) synthesis itself, cleavage-polyadenylation specificity factor and poly(A)

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polymerase [25], together with poly(A) binding protein II (PAB II), are sufficient, although CstF was recently shown to enhance this reaction [26]. A subunit of the CstF, CstF-64, was shown to be expressed in a stage-specific manner in B cells expressing IgM [27, 28]. It was speculated that in resting B cells, where CstF-64 is less abundant, the poly(A) site for the ␮m Ig is preferred over the ␮s Ig. The higher amount of CstF in activated plasma cells leads to the better recognition of the weaker ␮s poly(A) site. A supposed mechanism for this phenomenon is, that polyadenylation of the ␮s poly(A) site takes place before transcription over the ␮m poly(A) site can proceed. However, this phenomenon was found to be valid for IgM but not for IgG and IgA [29]. For IgG2b, not the increasing amount of CstF-64 during B-cell development, but the binding and polyadenylation efficiency is improved [30]. Yan et al. [44] postulated a specific activity in B-cell extracts that selectively impairs the formation of an Ig ␮s poly(A) site processing complex, suggesting that the function of this poly(A) site may be regulated by both positive- and negative-acting factors. Additionally, the sequence of the intron 5⬘- of the M1 exon influences the polyadenylation/splicing balance. For IgA, a specific sequence in the C␣3-␣M1 intron is recognized by a 58 kDa protein and leads to a predominant usage of the ␣s poly(A) site [31]. In this special case, the ratio of ␣s to ␣m IgA in resting B cells is about 2. Therefore it is possible that cis-acting elements unique to each CH gene act upon a common mechanism regulation Ig mRNA processing.

Alternative Polyadenylation of IgE

The third (IgD and IgGs) or fourth (IgM and IgE) constant exon (fig. 1) which is located 5⬘- of the transmembrane and the cytoplasmic exons, is a composite exon: it contains an internal splice donor site which is used when mRNA for membrane-bound Ig is made. It is also followed by an ‘internal’ polyadenylation-addition site that is used when mRNA for secreted Ig is made. A 3⬘ ‘external’ polyadenylation-addition site is found downstream of the membrane exon. With the exception of IgE, for the ‘internal’ and the ‘external’ polyadenylation signals, the consensus sequence AATAAA is used. However, the epsilon membrane locus uses three cryptic ‘external’ polyadenylation signals (fig. 2). These signals (AGTAAA, AAGAAA and ATTAAA) are in considerable disagreement with the consensus sequence. The ratio of transcripts for the secreted and the membrane form of Ig reflects the usage of either polyadenylation signal. The choice depends normally on the developmental stage of the B lymphocyte. Therefore, the production of the two types of mRNA are determined by alternative splicing or rather, alternative polyadenylation [30]. The ratio between the amount of secreted versus membrane-bound Ig that is produced by

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a single cell is therefore determined by the efficiency with which the internal or external polyadenylation sites are used and by the stability of the ensuing mRNA’s. In resting B cells the ratio is about 1, but in activated lymphoblasts more mRNA for secretory Ig can be found. This effect seems to be correlated with, but is not absolutely dependent on, a rise in concentration of a 64 kDa subunit of the CstF and an increase of the stability of the mRNA [32]. Whether the regulation of splicing for the IgE heavy chain mRNA is similar to that described for IgG and IgA remains to be established. Preliminary data from our laboratories suggest a different regulation: expression of mRNA for the secreted form of IgE is favored over that for the membrane form in resting B cells. Factors that influence the alternative polyadenylation are largely unknown. However, because expression of mIgE is essential for recruitment of IgE-producing plasma cells in the immune response, clarification of this issue is of great importance.

Plasma-Cells – A Therapy Resistant Population of Cells?

B lymphocytes derive from hematopoetic stem cells by a complex set of differentiation events. This process occurs in the fetal liver and, in adult life, in the bone marrow. The stem cells, which are capable of self-renewal give rise to the more identifiable members of the lineage which are pro-B, pre-B and immature B cells. The key events for the completion of this differentiation process are the successful rearrangement and expression of the BCR, consisting of Ig molecules specialized for the expression on the cell surface. The BCR on immature B cells belongs to the IgM subclass. The B cell completes its maturation process by expressing on its membrane a second class of Ig, designated IgD. The mature IgM-IgD double-positive mature B cell leaves the bone marrow and circulates through blood and lymph, homing to secondary lymphoid organs, like spleen and lymph nodes. B lymphocyte responses are initiated by the binding of antigen to the BCR, an event that triggers signalling cascades resulting in the transcription of a variety of genes associated with B-cell activation. In a complex interplay with other white blood cells, this activation process leads to the formation of a germinal centre (GC) reaction within secondary lymphoid organs that culminates in the transformation of the mature B cell into a terminally differentiated plasma cell (PC), whose only function is to produce (neutralizing) soluble Ig, termed antibodies [33]. In humans, protective antibody titers can last for more than 20 years, and in mice, high titers of specific antibodies are maintained for more than one year, which means protection for nearly a murine lifetime [34]. Since antibodies have a half-life of only a few days for IgGs and only 12 h for IgE, continuous production of antibodies is

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required to warrant a protective titer. Recently, it could be shown that persistent antibody titers can be provided by long-lived PCs [35]. In general, the lifespan of nondividing PCs varies from a few days up to years, and this appears to be determined by their developmental history and the quality of the B-cell activation. However, T-cell-dependent antigens mostly induce the formation of long-lived PCs, providing persistent protective antibody titers. Upon PC differentiation, there is a marked increase in the amounts of Ig mRNA transcripts, accompanied by an increase of secretory to membrane Ig ratio, as determined by differential use of poly(A) sites. Several B-cell specific markers are downregulated upon plasma cell differentiation, including major histocompatibility complex class II, CD19, CD21, CD22 and CD45 [36]. In contrast, the proteoglycan syndecan-1 (syn-1, also designated CD138), recognizing extracellular matrix and growth factors, is upregulated and serves as an identifying PC surface marker protein. In addition, the chemokine receptors CXCR5 and CCR7 are decreased on PCs, which impaires the migration to B and T cell zones. On the other hand, the chemokine receptor CXCR4 is highly expressed, which guides the PCs into CXCL12 expressing organs, including splenic red pulp, lymph nodes and bone marrow. However, it could be shown, that PCs in autoimmune mice not only migrate to the bone marrow, but also to the sites of inflammation. It has long been a matter of debate, whether the life span of PCs is intrinsically determined or dependent on several environmental factors. However, recent publications show that the longevity of the PC is influenced by a broad panel of stimuli, including cytokines like IL-5, IL-6, TNF-␣, granulocyte-macrophage colony stimulating factor, the chemokine CXCL12 and stromal cell-derived factor 1-alpha [37]. In addition, the contact with bone marrow stromal cells provides further adhesion-dependent signals that support PC longevity [38]. These signals are most probably mediated through VLA-4 and CD44. Interestingly, the affinity of serum antibodies increases long after the decline of the GC reaction, suggesting that either the longevity of the PC is determined during the GC reaction, or that there is an antigen-dependent selection of the PCs in a post-GC compartment as they home to the bone marrow [39]. The latter assumption would mean that the migrating PCs still rely on the expression of surface Ig for survival and selection and hence, it is possible that this cell population is specifically lost in transgenic mice that bear truncated surface Igs [4]. This assumption goes along with the finding that mice deficient for CD21 (a positive regulator of BCR signalling) appear to have PCs of reduced lifespan. This set of data suggests that PCs are still dependent on BCR expression. Summarizing, the existing data strongly support the concept that PC survival depends on niches, exhibiting a specific set of signals most of which remain yet to be identified. Most likely, these signals are redundant and act

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synergistically in order to provide PC longevity. Therefore, it is still matter of speculation whether the BCR on PCs is functional or not, and whether the PCs – like all cells of the B lineage – depend on the low-level expression of the BCR for survival and longevity. Taking these facts together it becomes clear that PCs are the limiting factor in the effectiveness of systemic IgE therapies.

Pros and Cons of Systemic IgE Therapies

Several strategies were followed to treat IgE-mediated allergic diseases by downregulating IgE levels [40]. Chimerized or humanized anti-IgE antibodies with a set of unique binding properties could be used for the isotype-specific control of IgE, and thus it would seem a logical therapeutic approach to treat IgE-mediated diseases. The anti-IgE antibody must have a high-affinity for IgE, should not bind IgE already bound by Fc␧RI on mast cells and basophils, nor to IgE bound by the low-affinity IgE Fc receptors (Fc␧RII, also known as CD23) on various other cell types and should bind to membrane-bound IgE (mIgE) on mIgE-expressing B cells. Summarizing, these antibodies are designed to neutralize free IgE and target IgE-expressing B cells. If these aims are achieved, the levels of IgE in blood and interstitial fluids for binding to Fc␧RI will be greatly reduced, and hence the sensitivity of mast cells and basophils to allergens should be gradually alleviated. On the other hand, as the anti-IgEs should not bind to IgE bound by Fc␧RI, they do not cross-link Fc␧RI-bound IgE and lack sensitization of mast cells and basophils [41]. Moreover, as the anti-IgEs do not bind to IgE bound by Fc␧RII – which is expressed broadly on lymphocytes, macrophages, platelets, and many other cell types – they are not expected to cause any adverse effects related with such binding. The anti-IgEs under clinical evaluation have an association constant, Ka, for soluble IgE of approximately 1010 M⫺1 [42], about the same as that of Fc␧RI for IgE. Thus, if anti-IgE is maintained at concentrations in excess of IgE in the body, it should effectively compete with Fc␧RI for IgE. The concentrations of IgE in the blood vary widely among allergic patients, ranging from 0.05 to 1 mg/ml. Taking this into account, anti-IgE given in excess to the basal concentration can bind most of IgE, leaving a minimum of free IgE for binding to IgE-Fc receptors. Probably the most readily appreciated pharmacological effect of anti-IgE therapy is its indirect effect on the downregulation of Fc␧RI on basophils. In earlier studies, the density of Fc␧RI on basophils was found to correlate strongly with the level of IgE in blood. In one of the clinical studies [43] and in additional in vitro studies, it was found that anti-IgE can also downregulate Fc␧RI in patients. The results show that the density of Fc␧RI on basophils had decreased by more than 95%, and in some cases to more than 99%, after

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anti-IgE was administered to patients for periods of five weeks to three months. The basophils isolated from patients after anti-IgE treatment were much less sensitive to allergen stimulation. In skin prick tests, it was also found that much larger amounts of allergens were required to elicit a positive reaction, indicating that mast cell function was also markedly decreased. Experimental evidence from in vitro and in vivo studies is generally supportive for the effectiveness of anti-IgE in targeting IgE-expressing B cells and in inhibiting the continuous production of IgE. The explanation for these effects is that anti-IgE binds to mIgE on IgE-expressing B cells, and as mIgE is a part of the B-cell receptor, anti-IgE may inhibit the B cells or even cause their lysis, like anti-IgM or anti-IgG [44]. However, IgE-secreting PCs do not, or only rarely express mIgE and presumably are not affected by anti-IgE. These cells reside in the bone marrow and probably have a life span of several months to years. Since new IgE-secreting PCs go through mIgE-expressing B-cell stages during differentiation, if their generation is abrogated by anti-IgE treatment, the existing PCs will die off in several weeks to several months, and thus the production of IgE will also gradually decline in similar periods. Furthermore, memory B cells may possibly be affected by anti-IgE. If this occurs, anti-IgE may have long-term effects on the fundamental disease process. The molecular mechanisms leading to the depletion of these cells can be explained by apoptosis. Transgenic animal studies confirmed that clonal deletion and clonal anergy are the principle mechanisms of self-tolerance. Clonal deletion is the physical elimination (death) of a B cell, while clonal anergy is characterized by the silencing of a living B cell suppressing Ig production and fast reactivation [45]. Both mechanisms and/or strategies naturally occur during B-cell development. Transferring these ideas to an affinity matured, class-switched IgE B-cell population, two scenarios of the effects of an anti-IgE antibody therapy would be possible. First, as shown by self reacting immature B cells, cross-linkage of the mIgE receptor without further T-cell support should directly induce apoptosis. Second, as normally shown during the induction of peripheral tolerance of mature B cells in answer to monovalent (self) antigen, receptor blockage of mIgE by a Fab-fragment should result in an anergic state of the mIgE population. In contrast to normal mature B cells, which have a half-life of 4–5 weeks, anergic B cells were found to last for only 3–4 days [46]. Nevertheless the fate of these anergic B cells finally turns out to be cell death [47].

Summary

Summarizing clinical applications and studies conducted so far, the most advanced systemic therapeutic approach for the treatment of allergic diseases is

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aiming to reduce increased IgE production in allergic condition. Despite optimized treatment regimens, including specific immunotherapy (SIT), antihistamines, corticosteroids and mast cell stabilizers, a subgroup of allergic patients have insufficient symptom control. Therefore, new treatment options, like antiIgE in combination with SIT are desirable to interment, more specifically and earlier, in the allergic cascade. Combination therapy may permit a broader use of SIT by reducing the risk of anaphylactic side effects after SIT injections. However, anti-IgE therapy has been shown to be effective not only for seasonal allergic diseases, but also for allergic bronchial asthma and food allergy. The optimal duration of anti-IgE therapy is unknown, but it is likely that anti-IgE has to be administered continuously in a dose-dependent fashion. The ultimate success of systemic anti-IgE therapy will, however, depends on the accessibility and sensitivity of long-lived PCs for the administered anti-IgE antibody.

Acknowledgements Experimental work was supported by the Austrian Science Foundation (S8809-MED and Hertha Firnberg Program T166-B12), and the Austrian National Bank (OENB grant: 9546). Work at SIAF was supported be the Swiss Federal Science Foundation Grant No. 3100–063381/2 and by the OPO foundation, Zürich. We are grateful to Prof. K. Blaser for his continuous support.

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Prof. Dr. Gernot Achatz Fachbereich für Molekulare Biologie Abteilung Allergologie und Immunologie, Hellbrunnerstrasse 34 AT–5020 Salzburg (Austria) Tel. ⫹43 662 8044 5764, Fax ⫹43 662 8044 183, E-Mail [email protected]

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Author Index

Aalberse, R.C. 134 Achatz, G. 204 Achatz-Straussberger, G. 204 Akdis, C.A. 159, 174, 195 Akdis, M. 159, 195 Behrendt, H. 1 Blaser, K. 159, 174, 195 Blümer, N. 30 Bonini, S. 110 Breuer, K. 76 Carballido, J.M. 147 Carballido-Perrig, N. 147 Crameri, R. 98, 121, 204

Erwin, E.A. 3 Flückiger, S. 121 Garn, H. 30 Glaser, A.G. 121 Heymann, P.W. 3 Jutel, M. 174 Kapp, A. 76 Kay, A.B. 59 Lamers, R. 204 Lametschwandtner, G. 147 Luger, E. 204 Platts-Mills, T.A.E. 3

Renz, H. 30 Rhyner, C. 121 Ring, J. 1 Santamaría-Babí, L.F. 87 Scheynius, A. 98 Schmid-Grendelmeier, P. 98 Schmidt-Weber, C.B. 188 Schultze-Werninghaus, G. 16 Schwärzler, C. 147 Sel, S. 30 Simon, H.-U. 49 Virna, S. 30 Weichel, M. 121 Werfel, T. 76 Woodfolk, J.A. 3

218

Subject Index

Air pollution, high-altitude climates 16, 17 Allergens, see also specific allergens cross-reactivity B cell activation 138 mast cell triggering 135–137 exposure high-altitude climates 17, 18 minimization 4, 12 fungus classification 129, 130 cross-reactivity 127, 129, 130 Malassezia sympodialis 101–103, 105, 106 pathogenesis of severe atopic diseases 130, 131 recombinant allergens 126, 127 modified Th2 response and allergenicity 139, 140, 144, 145 particle carriers 4 Allergic conjunctivitis classification 111 clinical features 110, 111, 114–116 diagnosis 116, 117 etiology and pathogenesis 111–114 prognosis 118, 119 treatment 117, 118 Allergic contact dermatitis, cutaneous lymphocyte-associated antigen positive T cells 92 Anergy interleukin-10 suppression of T cells 199, 200

tolerance comparison 159, 160 Antihistamines airway function effects 183 allergic conjunctivitis management 117 Apoptosis, eosinophils, see Eosinophil Aspergillus, allergen features 4, 5 Asthma fungal allergy association 125, 126 gene expression profiling 190–193 high-altitude climate response bronchial hyperreactivity 25–27 clinical studies 19–21 duration of benefits 26, 27 eosinophil inflammation markers 22 epidemiology 18 immunoglobulin E 22 inflammatory markers 22 lung function tests 25, 26 T cell function 22–24 immunoglobulin E response 8–11 inner-city children 7, 8, 11, 12 phenotypes 60, 61 T cell modulation, see T cell Atopic dermatitis (AD) cutaneous lymphocyte-associated antigen positive T cells 89–92 epidemiology 76, 77 food allergy relationship 78–80 histamine mediation 78 inhalant allergens 80 manganese superoxide dismutase role 130, 131

219

Atopic dermatitis (continued) Staphylococcus aureus as trigger factor 80–82 T cell response 77 treatment 92 Atopic eczema (AE) epidemiology 98 forms 99 fungal colonization 99, 100 Malassezia sympodialis allergens 101–103, 105, 106 intrinsic form and sensitization 103, 104 skin colonization 100, 101 management 104, 105 skin barrier dysfunction 99 Bacillus Calmette-Guérin (BCG), allergy protection 35, 36 B cell allergen cross-reactivity 138 antigen presentation effects on T helper cell balance 152, 153 nonmodified Th2 response 141–143 regulatory B cells 166 B cell receptor (BCR) internalization 206, 207 knockout mice 208 signaling 207, 208 structure 206, 207 Bcl-2 proteins, eosinophil apoptosis regulation 55, 56 Bee venom phospholipase A2 allergenicity 147, 148 structure 147 T helper balance studies 150–155 Blaser, Kurt, biography 1, 2 Caspases, eosinophil apoptosis regulation 53 Cat allergy allergen exposure immunoglobulin E titer response 6, 7 inhaled quantity 5 pet ownership and asthma relationship 31, 32 CD8⫹ T cell, asthma role 67

Subject Index

CD25, high-altitude climate effects on T cell expression 22, 23 CD30, eosinophil apoptosis signaling 52 CD45, eosinophil apoptosis signaling 52 CD69, eosinophil apoptosis signaling 52 CD137, eosinophil apoptosis signaling 52 Chemokines cutaneous lymphocyte-associated antigen positive T cell receptor expression 89 plasma cell receptors 212 T cell homing to airway in asthma 67–69 Cockroach allergens, asthma induction 8 Conjunctivitis, see Allergic conjunctivitis Corticosteroids atopic dermatitis management 83, 84 T cell suppression in asthma 69 CpG DNA, protection from asthma 34, 35, 42, 43 Cutaneous lymphocyte-associated antigen (CLA) adhesion molecule interactions 88, 89 interleukin-12 in induction 88 structure 87, 88 T cells allergic contact dermatitis 92 atopic dermatitis 89–92 chemokine receptor expression 89 drug allergy 92, 93 expression and distribution 87, 88 features in cutaneous allergic inflammation 93 Cyclosporin A allergic conjunctivitis management 118 asthma trials 69 Davos, health benefits 1 Dendritic cell (DC) asthma function 66, 67 histamine effects 179 T cell regulation 166, 167 T helper cell polarizing signals 150–152 DNA microarray allergy and asthma studies 190–193 principles 188, 189 Dog allergy allergen exposure 6, 7

220

pet ownership and asthma relationship 31, 32 Drug allergy, cutaneous lymphocyteassociated antigen positive T cells 92, 93 Dust mite allergens features 5, 6 purification 6 Eczema, see Atopic eczema Endotoxin cell wall 33 cytokine response 34 dose response in asthma and allergy 9, 10 farming environment protection from asthma 31–33 hygiene hypothesis testing 39, 40 structure 34 Eosinophil antigen presentation effects on T helper cell balance 153 apoptosis induction CD30 52 CD45 52 CD69 52 CD137 52 Fas system 51 overview 50, 51 Siglec-8 52 TRAIL 51 regulators Bcl-2 proteins 55, 56 caspases 53 IAP proteins 53–55 suppressors cytokines 52 tumor necrosis factor superfamily molecules 52 atopic dermatitis therapeutic targeting 84 bronchoalveolar lavage fluid cells in asthma 49, 50 Farming environment protection from asthma 31–35 Toll-like receptor-2 induction 34

Subject Index

Fas, eosinophil apoptosis signaling 51 Fetal immune response, allergen priming 37 Food allergy, atopic dermatitis relationship 78–80 Fungal allergy, see also Malassezia sympodialis allergens Aspergillus allergen features 4, 5 classification 129, 130 cross-reactivity 127, 129, 130 pathogenesis of severe atopic diseases 130, 131 recombinant allergens 126, 127 asthma association 125, 126 diagnosis extracts for testing 123, 124 skin prick test 124, 125 epidemiology 122, 123 fungus features 122 Gamadelta T cells, asthma role 67 GATA3 regulation of T cell cytokine production 64 T helper cell differentiation role 149, 150 Helminth infection, asthma protection 12 High-altitude climate air pollution 16, 17 allergen exposure 17, 18 asthma response bronchial hyperreactivity 25–27 clinical studies 19–21 duration of benefits 26, 27 eosinophil inflammation markers 22 epidemiology 18 immunoglobulin E 22 inflammatory markers 22 lung function tests 25, 26 T cell function 22–24 prospects for study 27 Histamine airway function effects 183 allergic conjunctivitis mediation 113 atopic dermatitis mediation 78

221

Histamine (continued) autoimmune response 184 cellular sources 175 chronic inflammatory response 182 history of study 174, 175 immune cell regulation dendritic cells 179 T cells 181, 182 malignancy effects 184 prospects for study 184, 185 receptors 176–179 synthesis and metabolism 175, 176 Hygiene hypothesis animal models 37–41 epidemiological and human exposure studies 30–37 mechanisms of allergy protection 41–45 origins 30, 31 prospects for study 45

effector cell suppression 198, 199 interleukin-10 suppression of T cells 199, 200 peripheral T cell tolerance allergen-specific immunotherapy 195–197 antibody isotype regulation 197, 198 Inner-city children asthma epidemiology 7, 8, 11, 12 hygiene hypothesis 32 Interleukin-9 (IL-9), asthma role 63 Interleukin-10 (IL-10), T cell suppression in immunotherapy 199, 200 Interleukin-12 (IL-12) asthma role 63 cutaneous lymphocyte-associated antigen induction on T cells 88 Interleukin-13 (IL-13), asthma role 63 Interleukin-16 (IL-16), asthma role 63 Interleukin-18 (IL-18), asthma role 63

IAP proteins, eosinophil apoptosis regulation 53–55 ICOS ligand (B7H), T helper balance role 152 Immunoglobulin E (IgE) animal allergy and titer response 6, 7 asthma prevalence and severity relationship 8–11 half-life 205 helminth infection response 204, 205 high-altitude climate effects 22 immunogenicity in allergy 135, 138, 139 membrane expression regulation overview 208, 209 polyadenylation of transcripts 209–211 plasma cells secretion 206 therapeutic targeting 211–213 receptors 205, 213 systemic inhibition 213–215 Immunoglobulin G (IgG), IgG4 in modified Th2 response 143, 144 Immunotherapy allergic conjunctivitis 118

Late-phase reaction, allergic conjunctivitis 112 Lipopolysaccharide, see Endotoxin Lodoxamide, allergic conjunctivitis management 118

Subject Index

Macrophilin-12, atopic dermatitis management 82, 83 Major histocompatibility complex (MHC), class II molecule-antigen affinity effects on T helper cell balance 153, 154 Malassezia sympodialis, atopic eczema role allergens 101–103, 105, 106 intrinsic disease and sensitization 103, 104 skin colonization 100, 101 Manganese superoxide dismutase (MnSOD), allergy role 130, 131 Mast cell allergen cross-reactivity 135–137 allergic conjunctivitis role 111, 112 Mepolizumab, atopic dermatitis management 84 Mountains, see Davos; High-altitude climate

222

Nerve growth factor (NGF), allergic conjunctivitis levels 113 Nonsteroidal anti-inflammatory drugs (NSAIDs), allergic conjunctivitis management 118 Obesity, indirect effects on asthma 12 Ovalbumin sensitization, hygiene hypothesis testing 38 OX40L, T helper balance role 151 Pimecrolimus atopic dermatitis management 83 atopic eczema management 104 Plasma cell chemokine receptors 212 immunoglobulin E secretion 206 life span 212 therapeutic targeting 211–213 Pollen grains, allergen features 4, 5 Programmed death-1 (PD-1), regulatory T cell function 168 Regulatory T cell (TReg) allergen response suppression 65 CD4⫹CD25⫹ cells 165 clinical relevance 169, 170 hygiene hypothesis 43–45 immunotherapy effects 199–201 peripheral tolerance role 170, 171 subsets 64, 162–166 suppression mechanisms 167–169 Th3 cells 163–165 therapeutic targeting in asthma 70, 71 Tr1 cells 162, 163 Rodent allergy, asthma association 8 SIAF, origins and functions 1, 2 Siglec-8, eosinophil apoptosis signaling 52 Staphylococcus aureus, atopic dermatitis triggering 80–82 STATs STAT6 regulation of T cell cytokine production 64 T helper cell differentiation role 148, 150

Subject Index

Tacrolimus atopic dermatitis management 83 atopic eczema management 104 T cell, see also specific cell types asthma CD8⫹ T cells 67 cell homing 67–69 cytokines 63, 64 dendritic cell function 66, 67 effector mechanisms 61, 62 gammadelta T cells 67 perpetuation 62 phenotypes 60, 61 provoked asthma studies 65, 66 regulatory T cells 64, 65 therapeutic targeting 69–71 atopic dermatitis response 77 high-altitude climate effects on function 22–24 histamine effects 181, 182 inflammatory response 160–162 T helper balance allergen response suppression by regulatory T cells 65 asthma 59, 60 bacillus Calmette-Guerin effects 36 B cell antigen presentation effects 152, 153 cytokine production 41 cytokine signaling in differentiation 148–150 dendritic cell polarizing signals 150–152 eosinophil antigen presentation effects 153 healthy antiallergen immune response and relation to modified Th2 response 139, 140 hygiene hypothesis 41, 44 inflammatory response 161, 162 major histocompatibility complex class II molecule-antigen affinity effects 153, 154 nonmodified Th2 response 141–143 TIM-4, T helper balance role 152 Tolerance anergy comparison 159, 160

223

Tolerance (continued) peripheral T cell tolerance immunotherapy allergen-specific immunotherapy 195–197 antibody isotype regulation 197, 198 regulatory T cells 170, 171 Toll-like receptors (TLRs) activation and allergy protection 42, 43

Subject Index

classification 42 farming environment TLR2 induction and asthma protection 34, 35 signaling 42 TRAIL, eosinophil apoptosis signaling 51 Tumor necrosis factor superfamily, eosinophil apoptosis signaling 52 Yogurt, allergy protection 36

224