Psychoneuroimmunology, Two-Volume Set, Volume 1-2, Fourth Edition

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Psychoneuroimmunology FOURTH EDITION Volume I

SECTION EDITORS

Robert Dantzer Department of Animal Sciences University of Illinois at Urbana-Champaign Urbana, Illinois Ronald Glaser Institute for Behavioral Medicine Research The Ohio State University College of Medicine Columbus, Ohio Cobi Heijnen Department of Psychoneuroimmunology University Medical Center Utrecht, The Netherlands Michael Irwin Cousins Center for Psychoneuroimmunology University of California Los Angeles, California David Padgett Institute for Behavioral Medicine Research The Ohio State University College of Dentistry Columbus, Ohio John Sheridan Institute for Behavioral Medicine Research The Ohio State University College of Dentistry Columbus, Ohio

Psychoneuroimmunology FOURTH EDITION Volume I

Edited by

Robert Ader Department of Psychiatry Center for Psychoneuroimmunology Research University of Rochester Medical Center Rochester, New York

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Elsevier Academic Press 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK This book is printed on acid-free paper. Copyright © 2007, 2001, 1991, 1981 by Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting “Customer Support” and then “Obtaining Permissions.”

Library of Congress Cataloging-in-Publication Data Psychoneuroimmunology / edited by Robert Ader.—4th ed. p. ; cm. Includes bibliographical references and indexes. ISBN-10: (invalid) 978012088576X (set : hardback : alk. paper) ISBN-10: 0-12-088576-X (set : hardback : alk. paper) ISBN-13: 978-0-12-088577-0 (v. 1 : hardback : alk. paper) ISBN-10: 0-12-088577-8 (v. 1 : hardback : alk. paper) [etc.] 1. Psychoneuroimmunology. I. Ader, Robert. [DNLM: 1. Psychoneuroimmunology. WL 103.7 P9734 2007] QP356.47.P79 2007 616.07′9—dc22 2006010164

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN ISBN ISBN ISBN ISBN ISBN

13: 10: 13: 10: 13: 10:

978-0-12-088576-8X (Two Volume Set) 0-12-088576-X (Two Volume Set) 978-0-12-088577-0 (Volume I) 0-12-088577-8 (Volume I) 978-0-12-088578-7 (Volume II) 0-12-088578-6 (Volume II)

For all information on all Elsevier Academic Press publications visit our Web site at www.books.elsevier.com

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Contents

7. Crosstalk between Insulin-like Growth Factors and Pro-inflammatory Cytokines . . . . . . . . . . 171 Robert H. McCusker, Klemen Strle, Suzanne R. Broussard, Robert Dantzer, Rose Marie Bluthé, and Keith W. Kelley

VOLUME I Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . ix Preface to the Fourth Edition . . . . . xv About the Editors . . . . . . . . . . . . . . . . . . . xix

8. The Neuroendocrine System and Rheumatoid Arthritis: Focus on the HypothalamicPituitary-Adrenal Axis . . . . . . . . . . . . . . . . . . . 193 Heather E. Gorby and Esther M. Sternberg

Prologue Exploring the Phylogenetic History of Neuralimmune System Interactions: An Update . . . . . 1 Nicholas Cohen and Kevin S. Kinney

9. Sex Steroids and Immunity . . . . . . . . . . . . . . . 207 Maurizio Cutolo and Alessandro Calvia 10. Emerging Concepts for the Pathogenesis of Chronic Disabling Inflammatory Diseases: Neuroendocrine-immune Interactions and Evolutionary Biology . . . . . . . . . . . . . . . . . . . . . 217 Rainer H. Straub, Adriana del Rey, and Hugo O. Besedovsky

I. Neural and Endocrine Effects on Immunity I. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 C. J. Heijnen

11. Neuroendocrine Regulation of Cancer Progression: I. Biological Mechanisms and Clinical Relevance . . . . . . . . . . . . . . . . . . . . . . . 233 Anil K. Sood, Susan K. Lutgendorf, and Steve Cole

1. Glucocorticoids and Immunity: Mechanisms of Regulation . . . . . . . . . . . . . . . . . 45 Onard J. L. M. Schoneveld and John A. Cidlowski 2. Adrenergic Regulation of Immunity . . . . . . . . 63 Virginia M. Sanders and Annemieke Kavelaars

12. Neuroendocrine Regulation of Cancer Progression: II. Immunological Mechanisms, Clinical Relevance, and Prophylactic Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Roi Avraham and Shamgar Ben-Eliyahu

3. Cholinergic Regulation of Inflammation . . . . . 85 Christopher J. Czura, Mauricio Rosas-Ballina, and Kevin J. Tracey 4. Significance of Sensory Neuropeptides and the Immune Response . . . . . . . . . . . . . . . . . . . . . 97 Hanneke P. M. van der Kleij and John Bienenstock

II. Immune System Effects on Neural and Endocrine Processes and Behavior

5. Vasoactive Intestinal Peptide: An Anti-inflammatory Neuropeptide . . . . . . 131 Doina Ganea and Mario Delgado

Robert Dantzer

6. Immune-derived Opioids: Production and Function in Inflammatory Pain . . . . . . . . . . . . 159 Halina Machelska and Christoph Stein

II. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Robert Dantzer

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13. Expression and Action of Cytokines in the Brain: Mechanisms and Pathophysiological Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Robert Dantzer 14. Cytokines, Sickness Behavior, and Depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Robert Dantzer, Rose-Marie Bluthé, Nathalie Castanon, Keith W. Kelley, Jan-Pieter Konsman, Sophie Laye, Jacques Lestage, and Patricia Parnet 15. The Differential Role of Prostaglandin E2 Receptors in the CNS Response to Systemic Immune Challenge . . . . . . . . . . . . . . . . . . . . . . 319 Michael Lazarus and Clifford B. Saper 16. The Role of Pro-inflammatory Cytokines in Memory Processes and Neural Plasticity . . . 337 Inbal Goshen and Raz Yirmiya 17. Aging, Neuroinflammation, and Behavior . . . 379 Rodney W. Johnson and Jonathan P. Godbout 18. Neuroimmune Interactions and Pain: The Role of Immune and Glial Cells . . . . . . . 393 Linda R. Watkins, Julie Wieseler-Frank, Mark R. Hutchinson, Annemarie Ledeboer, Leah Spataro, Erin D. Milligan, Evan M. Sloane, and Steven F. Maier

24. Psychoneuroimmunology of Depressive Disorder: Mechanisms and Clinical Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509 Lucile Capuron, Andrew Miller, and Michael R. Irwin 25. Immune and Neuroendocrine Alterations in Post-traumatic Stress Disorder . . . . . . . . . . . . 531 Gail Ironson, Dean Cruess, and Mahendra Kumar 26. Psychoneuroimmunologic Aspects of Alcohol and Substance Abuse . . . . . . . . . . . . . 549 Steven J. Schleifer 27. Schizophrenia and Immunity . . . . . . . . . . . . . 563 Matthias Rothermundt and Volker Arolt 28. Sleep and the Immune System . . . . . . . . . . . . 579 Mark R. Opp, Jan Born, and Michael R. Irwin 29. Emotions and the Immune System . . . . . . . . . 619 Margaret E. Kemeny 30. Behaviorally Conditioned Enhancement of Immune Responses . . . . . . . . . . . . . . . . . . . . . . 631 Gustavo Pacheco-López, Maj-Britt Niemi, Harald Engler, and Manfred Schedlowski 31. Exercise and Immunity: Clinical Studies . . . . 661 David C. Nieman

19. Cytokines and Non-immune Brain Injury . . . 415 Barry W. McColl, Chris J. Stock, and Nancy J. Rothwell

32. Behavioral Interventions: Immunologic Mediators and Disease Outcomes . . . . . . . . . . 675 Michael H. Antoni, Neil Schneiderman, and Frank Penedo

20. The Interaction between Brain Inflammation and Systemic Infection . . . . . . . . . . . . . . . . . . . 429 Leigh M. Felton and V. Hugh Perry

VOLUME II

III. Behavior and Immunity Michael R. Irwin III. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Michael Irwin 21. Mother-infant Interactions and the Development of Immunity from Conception through Weaning . . . . . . . . . . . . . . . . . . . . . . . . 455 Christopher L. Coe and Gabriele R. Lubach 22. Social Dominance and Immunity in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475 Mark L. Laudenslager and Sarah Kennedy 23. Social Context as an Individual Difference in Psychoneuroimmunology . . . . . . . . . . . . . . 497 Edith Chen and Gregory E. Miller

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . ix Preface to the Fourth Edition . . . . . xv About the Editors . . . . . . . . . . . . . . . . . . . xix IV. Stress and Immunity Ronald Glaser IV. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705 Ronald Glaser 33. Stress: A System of the Whole . . . . . . . . . . . . . 709 Bruce Rabin 34. Bi-directional Effects of Stress on Immune Function: Possible Explanations for Salubrious as Well as Harmful Effects . . . . . . 723 Firdaus S. Dhabhar and Bruce S. McEwen

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35. Positive Affect and Immune Function . . . . . . 761 Anna L. Marsland, Sarah Pressman, and Sheldon Cohen 36. Close Relationships and Immunity . . . . . . . . . 781 Jennifer E. Graham, Lisa M. Christian, and Janice K. Kiecolt-Glaser 37. Stress and Allergic Diseases . . . . . . . . . . . . . . . 799 Gailen D. Marshall and Sitesh R. Roy 38. Stress, Neuroendocrine Hormones, and Wound Healing: Human Models . . . . . . . . . . 825 Phillip T. Marucha and Christopher G. Engeland 39. Stress and Wound Healing: Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837 David A. Padgett, Phillip T. Marucha, and John F. Sheridan 40. Reactivation of Latent Herpes Viruses in Astronauts . . . . . . . . . . . . . . . . . . . . . . . . . . . 851 Duane L. Pierson, Satish K. Mehta, and Raymond P. Stowe 41. Psychosocial Influences in Oncology: An Expanded Model of Biobehavioral Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 Susan K. Lutgendorf, Erin S. Costanzo, and Scott D. Siegel 42. Stress-associated Immune Dysregulation Can Affect Antibody and T-cell Responses to Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 Mark A. Wetherell and Kavita Vedhara

V. Psychoneuroimmunology and Pathophysiology John Sheridan and David Padgett V. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917 John Sheridan and David Padgett 43. Psychoneuroimmunological Pathways Involved in Acute Coronary Syndromes . . . . 921 Willem J. Kop and Nicholas Cohen

44. Psychosocial Factors and Coronary Heart Disease: The Role of Psychoneuroimmunological Processes . . . . . . 945 Andrew Steptoe and Lena Brydon 45. Endocrine and Immune Responses to Stress in Chronic Inflammatory Skin Disorder (Atopic Dermatitis) . . . . . . . . . . . . . . . . . . . . . . 975 A. Buske-Kirschbaum 46. Obesity and Immunity . . . . . . . . . . . . . . . . . . . 993 Christopher B. Guest, Yan Gao, Jason C. O’Connor, and Gregory G. Freund 47. Endogenous Extracellular Hsp72 Release Is an Adaptive Feature of the Acute Stress Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1013 Monika Fleshner, Craig M. Sharkey, Molly Nickerson, and John D. Johnson 48. Cold-Restraint–induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1035 Rebecca T. Emeny and David A. Lawrence 49. Psychobiology of HIV Infection . . . . . . . . . . 1053 Erica Sloan, Alicia Collado-Hidalgo, and Steve Cole 50. Stress-induced Modulation of the Immune Response to Herpes Simplex Virus Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077 Robert H. Bonneau and John T. Hunzeker 51. Stress-induced Modulation of Innate Resistance and Adaptive Immunity to Influenza Viral Infection . . . . . . . . . . . . . . . . . 1097 Michael T. Bailey, David A. Padgett, and John F. Sheridan 52. Social Stress Alters the Severity of a Virally Initiated Model of Multiple Sclerosis . . . . . . 1107 Mary W. Meagher, Robin Johnson, Elisabeth Good, and C. Jane Welsh

Author Index Subject Index

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1125 1249


Contributors

Numbers in parentheses indicate the volume number and the chapter(s) where the authors’ contribution(s) begin.

Lena Brydon (2: Ch. 44) Psychobiology Group, Department of Epidemiology and Public Health, University College London, London WC1E 6BT, UK A. Buske-Kirschbaum (2: Ch. 45) Department of Biopsychology, Technical University of Dresden, Dresden D-01362 Germany Alessandro Calvia (1: Ch. 9) Research Laboratory and Division of Rheumatology, Department Internal Medicine, University of Genova, Viale Benedetto XV, 6 16132 Genova, Italy Lucile Capuron (1: Ch. 24) Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia 30322 Nathalie Castanon (2: Ch. 14) Neurobiologie Intégrative, UMR INRA Université de Bordeaux 2, FRE CNRS, Rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France Edith Chen (1: Ch. 23) Department of Psychology, University of British Columbia, Vancouver BC V6T 1Z4 Lisa M. Christian (2: Ch. 36) Department of Psychology, The Ohio State University, Columbus, Ohio 43210 John A. Cidlowski (1: Ch. 1) Laboratory of Signal Transduction, Molecular Endocrinology Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 Christopher L. Coe (1: Ch. 21) Harlow Center for Biological Psychology, University of Wisconsin, Madison, Wisconsin 53715 Nicholas Cohen (1: Prologue; 2: Ch. 43) Department of Microbiology and Immunology and the Center for Mind-Body Research, The University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Michael H. Antoni (1: Ch. 32) Department of Behavioral Medicine, University of Miami, Coral Gables, Florida 33146 Volker Arolt (1: Ch. 27) Department of Psychiatry, University of Muenster, 48129, Muenster, Germany Roi Avraham (1: Ch. 12) Department of Psychology, Tel Aviv University, Tel Aviv 69978 Israel Michael T. Bailey (2: Ch. 51) Section of Oral Biology, College of Dentistry, The Ohio State University, Columbus, Ohio 43218 Shamgar Ben-Eliyahu (1: Ch. 12) Department of Psychology, Tel Aviv University, Tel Aviv 69978 Israel Hugo O. Besedovsky (1: Ch. 10) Institute of Normal and Pathological Physiology, University of Marburg, 35033 Marburg, Germany John Bienenstock (1: Ch. 4) Brain-Body Institute and Department of Pathology and Molecular Medicine, St. Joseph’s Healthcare Hamilton and McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Rose-Marie Bluthé (1: Ch. 7, Ch. 14) Neurobiologie Intégrative, UMR INRA Université de Bordeaux 2, FRE CNRS, Rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France Robert H. Bonneau (2: Ch. 50) Department of Microbiology and Immunology, College of Medicine, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033 Jan Born (1: Ch. 28) Department of Neuroendocrinology, University of Lübeck, Lübeck, Germany Suzanne R. Broussard (1: Ch. 7) Integrated Immunology and Behavior Program, Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

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Contributors

Sheldon Cohen (2: Ch. 35) Department of Psychology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15238 Steve Cole (1: Ch. 11; 2: Ch. 49) Division of Hematology-Oncology and Department of Psychiatry and Biobehavioral Sciences, and the Norman Cousins Center for Psychoneuroimmunology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095 Alicia Collado-Hidalgo (2: Ch. 49) Cousins Center for Psychoneuroimmunology, UCLA Semel Institute for Neuroscience and Human Behavior, Los Angeles, California 90095 Erin S. Costanzo (2: Ch. 41) Department of Psychology, University of Iowa, Iowa City, Iowa 52242 Dean Cruess (1: Ch. 25) Department of Psychology, University of Connecticut, Storrs, Connecticut 06269 Maurizio Cutolo (1: Ch. 9) Research Laboratory and Division of Rheumatology, Department Internal Medicine, University of Genova, Viale Benedetto XV, 6 16132 Genova, Italy Christopher J. Czura (1: Ch. 3) Laboratory of Biomedical Science, Center for Inflammation and Immunity, Feinstein Institute for Medical Research, Manhasset, New York 11030 Robert Dantzer (1: Ch7, Ch. 13, Ch. 14) Integrative Immunology and Behavior, Department of Animal Sciences, University of Illinois at UrbanaChampaign, Urbana, Illinois 61801 Mario Delgado (1: Ch. 5) Instituto de Parasitología y Biomedicina, Granada, Spain Adriana del Rey (1: Ch. 10) Institute of Normal and Pathological Physiology, University of Marburg, 35033 Marburg, Germany Firdaus S. Dhabhar (2: Ch. 34) Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305 Rebecca T. Emeny (2: Ch. 48) Laboratory of Clinical and Experimental Endocrinology & Immunology, Wadsworth Center, New York State Department of Health, Albany, New York 12201 Christopher G. Engeland (2: Ch. 38) Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 Harald Engler (1: Ch. 30) Division of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland Leigh M. Felton (1: Ch. 20) CNS Inflammation Group, School of Biological Sciences, University of Southampton, Southampton, S016 7PX, UK

Monika Fleshner (2: Ch. 47) Department of Integrative Physiology and the Center for Neuroscience, University of Colorado, Boulder, Colorado 80309 Gregory G. Freund (2: Ch. 46) Integrated Immunology and Behavior, Department of Pathology Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801 Doina Ganea (1: Ch. 5) Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Yan Gao (2: Ch. 46) Integrated Immunology and behavior, Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801 Jonathan P. Godbout (1: Ch. 17) The Ohio State University, Department of Molecular Virology, Immunology, and Medical Genetics and the Institute for Behavioral Medicine Research, Columbus, Ohio 43210 Elisabeth Good (2: Ch. 52) Department of Psychology, College of Liberal Arts, Texas A&M University, College Station, Texas 77843 Heather E. Gorby (1: Ch. 8) Section on Neuroendocrine Immunology and Behavior, Integrative Neural Immune Program, National Institute of Mental Health, National Institutes of Health, Rockville, MD 20852 Inbal Goshen (1: Ch. 16) Department of Psychology, The Hebrew University of Jerusalem, Mount Scopus, Jerusalem 91905, Israel Jennifer E. Graham (2: Ch. 36) Institute for Behavioral Medicine Research, The Ohio State University College of Medicine, Columbus, Ohio 43210 Christopher B. Guest (2: Ch. 46) Integrative Immunology and Behavior, College of Medicine, Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801 John T. Hunzeker (2: Ch. 50) Department of Microbiology and Immunology, College of Medicine, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033 Mark R. Hutchinson (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 Gail Ironson (1: Ch. 25) Department of Psychology and Psychiatry, University of Miami, Miami, Florida 33124 Michael R. Irwin (1: Ch. 24, Ch. 28) Cousins Center for Psychoneuroimmunology, University of California, Los Angeles, Neuropsychiatric Institute, Los Angeles, California 90095

Contributors

John D. Johnson (2: Ch. 47) Department of Integrative Physiology and the Center for Neuroscience, University of Colorado, Boulder, Colorado 80309 Robin Johnson (2: Ch. 52) Department of Psychology, College of Liberal Arts, Texas A&M University, College Station, Texas 77843 Rodney W. Johnson (1: Ch. 17) Integrative Immunology and Behavior, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Annemieke Kavelaars (1: Ch. 2) Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, Utrecht, The Netherlands Keith W. Kelley (1: Ch. 7, Ch. 14) Integrated Immunology and Behavior Program, Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Margaret E. Kemeny (1: Ch. 29) Health Psychology Program, University of California, San Francisco, California 94143 Sarah Kennedy (1: Ch. 22) Department of Integrative Physiology, University of Colorado, Boulder, Colorado 80309 Janice K. Kiecolt-Glaser (2: Ch. 36) Department of Psychiatry and Institute for Behavioral Medicine Research, The Ohio State University College of Medicine, Columbus, Ohio 43210 Kevin S. Kinney (1: Prologue) Department of Biology, DePauw University, Greencastle, Indiana 46135 Jan-Pieter Konsman (1: Ch. 14) Neurobiologie Intégrative, UMR INRA Université de Bordeaux 2, FRE CNRS, Rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France Willem J. Kop (2: Ch. 43) Department of Medical and Clinical Psychology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 Mahendra Kumar (1: Ch. 25) University of Miami, Miami, Florida 33124 Mark L. Laudenslager (1: Ch. 22) Department of Psychiatry, University of Colorado Denver & Health Sciences Center, Denver, Colorado 80220 David A. Lawrence (2: Ch. 48) Laboratory of Clinical and Experimental Endocrinology & Immunology, Wadsworth Center, New York State Department of Health, Albany, New York 12201 Sophie Laye (1: Ch. 14) Neurobiologie Intégrative, UMR INRA Université de Bordeaux 2, FRE CNRS, Rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France

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Michael Lazarus (1: Ch. 15) Department of Neurology, Beth Israel Deaconess Medical Center, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115 Annemarie Ledeboer (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 Jacques Lestage (1: Ch. 14) Neurobiologie Intégrative, UMR INRA Université de Bordeaux 2, FRE CNRS, Rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France Gabriele R. Lubach (1: Ch. 21) Harlow Center for Biological Psychology, University of Wisconsin, Madison, Wisconsin 53715 Susan K. Lutgendorf (1: Ch. 11; 2: Ch. 41) Departments of Psychology and Obstetrics and Gynecology, Holden Comprehensive Cancer Center, University of Iowa, Iowa City, Iowa 52242 Halina Machelska (1: Ch. 6) Klinik für Anaesthesiologie und operative Intensivmedizin, CharitéUniversitätsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany Steven F. Maier (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 Gailen D. Marshall (2: Ch. 37) Division of Clinical Immunology and Allergy, Department of Medicine The University of Mississippi Medical Center, Jackson, Mississippi 39216 Anna L. Marsland (2: Ch. 35) Department of Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Philip T. Marucha (2: Ch. 38, Ch. 39) Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 Barry W. McColl (1: Ch. 19) Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK Robert H. McCusker (1: Ch. 7) Integrated Immunology and Behavior Program, Laboratory of Immunophysiology, Department of Animal Sciences, College of Agriculture, University of Illinois at UrbanaChampaign, Urbana, Illinois 61801 Bruce S. McEwen (2: Ch. 34) Laboratory of Neuroendocrinology, The Rockefeller University, New York, New York 10021 Mary W. Meagher (2: Ch. 52) Department of Psychology, College of Liberal Arts, Texas A&M University, College Station, Texas 77843

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Contributors

Satish K. Mehta (2: Ch. 40) National Aeronautics and Space Administration/Johnson Space Center, Enterprise Advisory Services, Inc. Houston, Texas 77058 Andrew Miller (1: Ch. 24) Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia 30322 Gregory E. Miller (1: Ch. 23) Department of Psychology, University of British Columbia, Vancouver BC V6T 1Z4 Erin D. Milligan (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 Molly Nickerson (2: Ch. 47) Department of Integrative Physiology and the Center for Neuroscience, University of Colorado, Boulder, Colorado 80309 David C. Nieman (1: Ch. 31) Department of Health and Exercise Science, Appalachian State University, Boone, North Carolina 28608 Maj-Britt Niemi (1: Ch. 30) Division of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland Jason C. O’Connor (2: Ch. 46) Integrative Immunology and Behavior, Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801 Mark R. Opp (1: Ch. 28) Department of Anesthesiology, University of Mechigan Medical School, Ann Arbor, Michigan 48109 Gustavo Pacheco-López (1: Ch. 30) Division of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland David A. Padgett (2: Ch. 39, Ch. 51) Section of Oral Biology, College of Dentistry, Institute for Behavioral Medicine Research, Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43218 Patricia Parnet (1: Ch. 14) Neurobiologie Intégrative, UMR INRA Université de Bordeaux 2, FRE CNRS, Rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France Frank Penedo (1: Ch. 32) Department of Behavioral Medicine, University of Miami, Coral Gables, Florida 33146 V. Hugh Perry (1: Ch. 20) CNS Inflammation Group School of Biological Sciences, University of Southampton, Southampton, S016 7PX, UK Duane L. Pierson (2: Ch. 40) National Aeronautics and Space Administration/Johnson Space Center, Houston, Texas 77058

Sarah Pressman (2: Ch. 35) Department of Psychology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15238 Bruce Rabin (2: Ch. 33) University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213 Mauricio Rosas-Ballina (1: Ch. 3) Laboratory of Biomedical Science, Center for Inflammation and Immunity, The Feinstein Institute for Medical Research, Manhasset, New York 11030 Matthias Rothermundt (1: Ch. 27) Department of Psychiatry, University of Muenster, 48129 Muenster, Germany Nancy J. Rothwell (1: Ch. 19) Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK Sitesh R. Roy (2: Ch. 37) Division of Allergy and Immunology, Department of Pediatrics, The University of Mississippi Medical Center, Jackson, Mississippi 39216 Virginia M. Sanders (1: Ch. 2) Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University School of Medicine and Public Health, Columbus, Ohio 43210 Clifford B. Saper (1: Ch. 15) Department of Neurology, Beth Israel Deaconess Medical Center, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115 Manfred Schedlowski (1: Ch. 30) Division of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland Steven J. Schleifer (1: Ch. 26) Department of Psychiatry, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103 Neil Schneiderman (1: Ch. 32) Department of Behavioral Medicine, University of Miami, Coral Gables, Florida 33146 Onard J. L. M. Schoneveld (1: Ch. 1) Laboratory of Signal Transduction, Molecular Endocrinology Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 Craig M. Sharkey (2: Ch. 47) Department of Integrative Physiology and the Center for Neuroscience, University of Colorado, Boulder, Colorado 80309 John F. Sheridan (2: Ch. 39, Ch. 51) Section of Oral Biology, College of Dentistry, Institute for Behavioral Medicine Research, Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43218

Contributors

Scott D. Siegel (2: Ch. 41) Department of Psychology, University of Miami, Miami, Florida 33124 Erica Sloan (2: Ch. 49) Division of HematologyOncology and Department of Psychiatry and Biobehavioral Sciences, and the Norman Cousins Center for Psychoneuroimmunology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095 Evan M. Sloane (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 Anil K. Sood (1: Ch. 11) Departments of Gynecologic Oncology and Cancer Biology, University of Texas M. D. Anderson Cancer Center, Unit 1362, Houston, Texas 77230 Leah Spataro (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 Christoph Stein (1: Ch. 6) Klinik für Anaesthesiologie und operative Intensivmedizin, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany D-12200 Andrew Steptoe (2: Ch. 44) Psychobiology Group, Department of Epidemiology and Public Health, University College London, London WC1E 6BT, UK Esther M. Sternberg (1: Ch. 8) Section on Neuroendocrine Immunology and Behavior, Integrative Neural Immune Program, National Institute of Mental Health, National Institutes of Health, Rockville, MD 20852 Chris J. Stock (1: Ch. 19) Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK Raymond P. Stowe (2: Ch. 40) Microgen Laboratories, La Marqué, Texas 77568

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Rainer H. Straub (1: Ch. 10) Department of Internal Medicine I, University Hospital Regensburg, 93042 Regensburg, Germany Klemen Strle (1: Ch. 7) Integrated Immunology and Behavior Program, Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Kevin J. Tracey (1: Ch. 3) Laboratory of Biomedical Science, Center for Patient Oriented Research, General Clinical Research Center, The Feinstein Institute for Medical Research, Manhasset, New York 11030 Hanneke P.M. van der Kleij (1: Ch. 4) Brain-Body Institute and Department of Pathology and Molecular Medicine, St. Joseph’s Healthcare Hamilton and McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Kavita Vedhara (2: Ch. 42) MRC Health Services Research Collaboration, University of Bristol, Clifton, Bristol, BS8 2PR, UK Linda R. Watkins (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 C. Jane Welsh (2: Ch. 52) Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843 Mark A. Wetherell (2: Ch. 42) MRC Health Services Research Collaboration, University of Bristol, Clifton, Bristol, BS8 2PR, UK Julie Wieseler-Frank (1: Ch. 18) Department of Psychology and the Center for Neuroscience, University of Colorado at Boulder, Boulder, Colorado 80309 Raz Yirmiya (1: Ch. 16) Department of Psychology, The Hebrew University of Jerusalem, Mount Scopus, Jerusalem 91905, Israel


Preface to the Fourth Edition

Psychoneuroimmunology is a convergence of disciplines—namely, the behavioral sciences, the neurosciences, endocrinology, and immunology—intended to achieve a more complete understanding of the way the interactions among these systems serve homeostatic ends and influence health and disease. As I have previously remarked, psychoneuroimmunology is an interdisciplinary field that has developed and now prospers by exploring and tilling fertile territories secreted by the arbitrary and illusory boundaries of the biomedical sciences. Disciplinary boundaries, codified by bureaucracies, are historical fictions that can restrict the imagination and impede the transfer and application of technologies and lend credence to Werner Heisenberg’s assertion that “What we observe is not nature itself, but nature exposed to our method of questioning.” Today, still, the business of science, by which I mean the training of scientists, the formulation of questions and hypotheses, the implementation of research, the communication among scientists, and even the funding of research, takes place within disciplinary boundaries that represent the disassembled parts of natural phenomena. This is not a representation of nature. It is an expedience that reflects our own intellectual limitations. This theme of disciplinary integration is now being endorsed by the National Institutes of Health (NIH). The NIH Roadmap, a description of new trans-NIH initiatives, argues that, “Biomedical research traditionally has been organized . . . into broad areas of scientific interest and then [grouped] into distinct, departmentally based specialties. But, as science has advanced over the past decade . . . , two fundamental themes are apparent: the study of human biology and behavior is a wonderfully dynamic process, and the traditional divisions within biomedical research may in some instances impede the pace of scientific discovery. To lower these artificial organizational barriers, the

NIH will implement several initiatives designed to facilitate interdisciplinary research collaborations and research training and, ultimately, lead to the development of new hybrid disciplines that will provide a more complete understanding of psychological, social and biological interactions in health and disease.” Psychoneuroimmunology is one such hybrid discipline that should prosper under these NIH initiatives. This is the temporal context in which I proposed a fourth edition of Psychoneuroimmunology. Like previous editions, Psychoneuroimmunology-IV is addressed to a broad audience in a continuing effort to draw attention to the field and promote interdisciplinary research in the laboratory and in the clinic. The primary targets would be laboratory and clinical investigators in endocrinology, immunology, neurochemistry, neurophysiology, pharmacology, psychiatry, psychology, virology, and medical specialists in allergic and infectious diseases, oncology, rheumatology, etc. In contrast to previous editions, the editorial responsibilities for Psychoneuroimmunology-IV were shared among a board of Associate Editors. These individuals, Robert Dantzer, Ronald Glaser, Cobi Heijnen, Michael Irwin, David Padgett, and John Sheridan, are well known and respected investigators who did a superb job in selecting and editing the chapters in their respective parts. Having established that (a) lymphocytes bear receptors for neurotransmitters, neuropeptides, and hormones and that activated lymphocytes can produce these same neuroendocrine signals, and that (b) neurons bear receptors for messenger molecules produced by cells of the immune system and that these cytokines can be released by cells of the neuroendocrine system, current research on the mechanisms involved in this neuroendocrineimmunecircuitry are presented in Part I of this volume. Emphasizing either shared mediators or shared recep-

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tors, these several chapters describe functional neuroendocrine-immune system regulatory pathways at the intercellular, interorgan, and whole body level— and how these circuits might influence health and disease. Continuing and reinforcing this discussion of shared mediators and receptors, Part II illustrates the diversity of effects and the complexity of the circuitry involved that has made research on cytokines and neuroinflammatory processes a rapidly expanding interdisciplinary area of research in the neurosciences, including behavior. Part III details the influence of behaviors, disordered behavioral states, and behavioral interventions, including classical conditioning, on immune functions that bear on health. The premise underlying this research is that psychosocial and stressful life events induce emotional responses that are associated with neuroendocrine and autonomic nervous system changes capable of modulating immune function and, thus, the susceptibility and/or progression of disease. It is further hypothesized that interventions calculated to elicit positive emotions and behaviors might, through alterations in immune states, ameliorate the effects of stressful life experiences on disease. The final two Parts most directly address the perennial issue of whether stress-induced changes in immune function are sufficiently large (and/or of sufficient duration) to influence the health of the organism. Within the context of behavioral medicine or psychosomatic medicine, could changes in immune function serve as mediators of the effects of psychosocial factors, including stressful life experiences, on health and disease? The several chapters in Part IV on stress and immunity affirm, within the immune system, an existing literature indicating that different stressors elicit different physiological responses and, therefore, would have different effects on different disease processes. This part also reviews the effects of positive affect and social relationships on immune function and their possible health consequences; the data on the role of stressful life experiences and associated changes in immune function in allergic diseases, wound healing, reactivation of latent viruses, the progression of malignant disease, and how stressful life experiences are capable of altering the individual’s response to vaccines. Part V provides further examples of how the immune system plays a role in mediating the effects of psychosocial factors on specific disease processes. These chapters review the provocative new studies of immune system involvement in cardiovascular disease, skin disorders, obesity, infectious and autoimmune diseases—and the psychoneuroimmunologic mechanisms that may play a role in host resistance.

Although the nervous, endocrine, and immune systems evolved to serve specialized functions, the premise underlying psychoneuroimmunology, confirmed by the extraordinary amount of research that has been conducted in the past couple of decades, is that each of these “systems” is also capable of responding to information derived from the other. Together, behavioral, neural, endocrine, and immune processes of adaptation constitute an integrated network of defenses and, insofar as immunoregulatory processes are concerned, the assumption of an autonomous immune system is no longer tenable. It is not possible to obtain a full understanding of immunoregulatory processes without considering the organism and the internal and external environment in which immune responses take place. The first edition of Psychoneuroimmunology was published 25 years ago. It described the relatively meager amount of research being conducted on the relationship between the brain and the immune system. However, it was, as one reviewer prophesied, the signature volume of a new field of research. That new field of research has since come of age and is being described as a paradigm shift in the way we view the multi-tiered organization of adaptive processes. Certainly, that is not a universally accepted proposition. Psychoneuroimmunology has been and, in reduced volume, continues to be assailed by gratuitous and uninformed commentaries. That is not altogether surprising. Any number of writers has commented that the everyday business of most scientists does not concern itself with the development or implementation of new ideas and theories—and is often intolerant of such developments by others. “Normal science,” as Thomas Kuhn refers to it in The Structure of Scientific Revolutions (University of Chicago Press, 1970), is concerned with an elaboration of the phenomena that the prevailing paradigm already supplies. And, in accord with the prevailing paradigm, we refer to connections between the CNS and immune system, which is not an especially satisfactory or accurate expression. It does not adequately describe the essence of the integration that the term psychoneuroimmunology is meant to convey. The phrase enables one to retain the historical fiction that these are separate and distinct systems; it is a two-dimensional expression for a threedimensional concept. Psychoneuroimmunology does magnify the complexity of already complex fields of study. That, however, is a small price to pay for a more complete understanding of biological processes that constitute the foundation of the study of health and illness. As we learn more about how behavioral, neural, and endocrine states influence immunologically based dis-


eases and how immune processes affect behavior and what are presumed to be endocrine or neurologic disorders, there may be a need to redefine the nature of some diseases and, thus, the strategies for therapeutic interventions. Accepting the notion that there is a single, integrated system of adaptive processes pre-

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sents new challenges and new opportunities for approaching the study of health and the treatment and prevention of disease. The authors of Psychoneuroimmunology-IV invite your participation in this venture. Robert Ader


About the Editors

of Psychoneuroimmunology at the Department of Pathology and Department of Animal Sciences of the University of Illinois at Urbana-Champaign, where he coordinates a program in Integrative Immunology and Behavior. Robert Dantzer has carried out research for many years on the psychobiology of stress, the influence of neuropeptides on behavior, and the interactions between the immune system and the brain. His current research aims at understanding the mechanisms of cytokine-induced sickness behavior and the possible involvement of cytokines in symptoms of depression. He has authored and co-authored 350 original research papers and 100 book chapters on stress, anxiety, neuropeptides, and psychoneuroimmunology. He is also the author or the editor of several books on stress in intensive husbandry, emotions, psychosomatics, and neurobiology of cytokines. He is the Editor-in-Chief of Psychoneuroendocrinology (Elsevier), Associate Editor of Brain, Behavior and Immunity (Elsevier), and was President of the Psychoneuroimmunology Research Society from 2002 to 2003.

Robert Ader, Ph.D., M.D. hc Dr. Robert Ader is Distinguished University Professor at the University of Rochester School of Medicine and Dentistry. He received his Ph.D. in psychology from Cornell University in 1957 and immediately thereafter joined the Rochester faculty, becoming Professor of Psychiatry and Psychology in 1968. He was awarded an honorary M.D. from Trondheim University in Norway in 1992 and an honorary D.Sc. from Tulane University in 2002. Dr. Ader was Visiting Professor at the Rudolf Magnus Institute for Pharmacology in Utrecht, The Netherlands (1970–71), and a Fellow at the Center for Advanced Study in the Behavioral Sciences at Stanford (1992–93). He serves on several Editorial Boards and was Editor-in-Chief of Brain, Behavior and Immunity from 1986 through 2002. Dr. Ader is a past President of the American Psychosomatic Society, the International Society for Developmental Psychobiology, the Academy of Behavioral Medicine Research, and was Founding President of the Psychoneuroimmunology Research Society. Robert Dantzer, D.V.M., Ph.D. Robert Dantzer was born in Saint-Etienne, France. He received his Doctorate in Veterinary Medicine in 1968, his Ph.D in 1972, and his Doctorate es-Sciences in 1977. From 1993 to 2006 he was Director of Research at the French National Institute for Agronomic Research and the Director of the Laboratory of Integrative Neurobiology at the University of Bordeaux, France, an operation funded by the French Scientific Research Council, the French National Institute of Agronomic Research, and the University of Bordeaux 2. He worked there with several senior scientists and technicians on various aspects of the expression and action of cytokines in the brain and their pathological consequences. Robert Dantzer is currently professor

Ronald Glaser, Ph.D. Ronald Glaser is Professor of Molecular Virology, Immunology, and Medical Genetics and Director of the Institute for Behavioral Medicine Research at The Ohio State University College of Medicine. He has published over 282 articles and chapters in the area of viral oncology and in the area of stress and immune function. He is an American Association for the Advancement of Science (AAAS) fellow, a fellow of the Academy of Behavioral Medicine Research, was named Distinguished Scholar by The Ohio State University, and holds the Gilbert and Kathryn Mitchell Endowed Chair in Medicine.

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About the Editors

Cobi Heijnen, Ph.D. Dr. Cobi Heijnen trained in medical biology and basic immunology and defended her thesis on regulation of antibody production by T-cell subsets at the Sorbonne University Marie Curie in Paris in 1982. She began her research in psychoneuroimmunology as a post-doctoral fellow in Utrecht. In 1997, she became a full professor in psychoneuroimmunology. This PNI chair was the first in Europe. In 1998, Dr. Heijnen received the Norman Cousins Award from the PsychoNeuroImmunology Research Society (PNIRS) and, in 2005–06, she served as President of the PNIRS. Michael Irwin, M.D. Michael Irwin, M.D., is Norman Cousins Professor of Psychiatry and Biobehavioral Sciences and Director of the Cousins Center for Psychoneuroimmunology and the UCLA Training Program in Psychoneuroimmunology. His primary research interests focus on the bi-directional relationships between behavioral processes (e.g., depression, disordered sleep) and autonomic and immunological function and their consequences for infectious disease risk and inflammation in humans. Current studies are also examining the neural, autonomic, and immunological mechanisms that underlie the action of mind-body interventions to promote health in older adults and in patients with inflammatory disorders. Dr. Irwin serves on the Advisory Council of the National Center for Complementary and Alternative Medicine, is past President of the Academy of Behavioral Medicine Research and of the Psychoneuroimmunology Research Society, Associate Editor of Brain, Behavior and Immunity, and recipient of numerous awards from the National Institutes of Health. For over a decade, Dr. Irwin has demonstrated leadership in the training of post-doctoral research fellows as Training Director of the UCLA Psychoneuroimmunology Post-Graduate Training

Program at UCLA as well as the Psychoneuroimmunology Research Society Scholars Program that sponsors training in psychoneuroimmunology at the national level. David Padgett, Ph.D. David Padgett received his Ph.D. in 1994 from the Medical College of Virginia and then joined the psychoneuroimmunology research group at The Ohio State University as a post-doctoral fellow. In the decade that he has been at Ohio State, he has risen to Associate Professor and is a member of the Institute for Behavioral Medicine Research. Dr. Padgett’s laboratory embraces the overarching concept that hormonal modulation of transcriptional activators that control gene expression is responsible for modulating innate immune responses to viral infection, the inflammatory phase of wound repair, and the induction of programmed cell death. John F. Sheridan, Ph.D. John F. Sheridan is an Associate Dean for Research, Professor of Oral Biology in the College of Dentistry, and Director of the Comprehensive Training in Oral and Craniofacial Biology program at the Ohio State University. He also holds appointments in the Departments of Molecular Virology, Immunology and Medical Genetics, and the Department of Psychology. He currently holds the George C. Paffenbarger Distinguished Alumni Endowed Chair in Dental Research, and he is an Associate Director of the Institute for Behavioral Medicine Research at the Ohio State University. Dr. Sheridan’s research is devoted to understanding the cellular and molecular mechanisms underlying mind body interactions as they relate to host defense and resistance to infectious disease. Dr. Sheridan is a past President of the PsychoNeuroImmunology Research Society.

PROLOGUE Exploring the Phylogenetic History of Neural-immune System Interactions: An Update NICHOLAS COHEN AND KEVIN S. KINNEY

I. INTRODUCTION 1 II. NEURAL-DEFENSE SYSTEM INTERACTIONS IN INVERTEBRATES 2 III. NEURAL-IMMUNE INTERACTIONS IN TELEOST FISH 6 IV. NEURAL-IMMUNE INTERACTIONS IN AMPHIBIANS 12 V. NEURAL-IMMUNE INTERACTIONS IN REPTILES 20 VI. NEURAL-IMMUNE SYSTEM INTERACTIONS IN BIRDS 22 VII. CONCLUDING REMARKS AND FUTURE RESEARCH DIRECTIONS 25

vertebrate species are serving, or have served, as living tools to probe the evolutionary origins of neuralimmune system interactions. In 2001, we published a comprehensive review of the research from these laboratories (Cohen and Kinney, 2001); the present review updates that information. The reader should bear in mind that the descriptive comparative approach we have taken here allows us only to make educated guesses about the true evolutionary history of the integration of two complex physiological systems. To avoid redundancy with what is presented in the rest of this 4th edition of Psychoneuroimmunology, we will not summarize the voluminous data from research with mammals as we did in our earlier review (Cohen and Kinney, 2001). The main phenomenology emerging from the literature that deals with neural-immune system interactions in rodents and primates, however, still serves as the gold standard for the questions asked by investigators using non-mammalian model systems. Thus, for comparative purposes, the mammalian reference points include the following facts: (1) mammalian lymphoid tissues are richly innervated (Felten et al., 1992; Felten et al., 2003); (2) cells of the mammalian immune system express receptors for neuropeptides, neurotransmitters, and hormones (Sanders et al., 1997, 2001); (3) activation of these receptors by their appropriate ligands affects functional behavior of the cells (Sanders and Straub, 2002; Sanders et al., 2001); (4) the SNS exerts a tonic regulatory role over the immune system as revealed, for example, by experiments

I. INTRODUCTION Psychoneuroimmunology, the study of behaviorally associated immunological changes and immunologically associated behavioral changes that result from reciprocal interactions among the nervous, endocrine, and immune systems, has emerged as a new field of scientific inquiry within the past 2 decades (Ader, 1981; Ader et al., 1991, 2001). It is a field that has been defined phenomenologically and is currently being explored mechanistically, primarily by studying rodents and primates. Although hundreds of investigators are using these and other mammalian species to address basic and clinical facets of psychoneuroimmunology, we are aware of only a handful of laboratories in which invertebrates, avian, and ectothermic (cold-blooded) PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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Copyright © 2007 by Elsevier, Inc. All rights of reproduction in any form reserved.

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Prologue

involving sympathectomy (Kruszewska et al., 1995, 1998); (5) cells of the immune system themselves produce, as well as respond to, neuropeptides and hormones (Blalock, 2005; Smith, 2003); (6) cytokines (e.g., IL-1, IL-6, TNF-α) produced by cells of both systems act as signal molecules in the bi-directional dialogue between the nervous and immune systems (Danzer et al., 2002; Goehler, et al., 1997; Maier, 2003; Maier and Watkins, 1998); and (7) behavioral responses to diverse stimuli (stressors) can trigger central neuroendocrine and peripheral autonomic responses that can alter immune parameters and, thereby, under certain conditions, affect the health of the organism (Glaser et al., 1999).

II. NEURAL-DEFENSE SYSTEM INTERACTIONS IN INVERTEBRATES By all current definitions, invertebrates display features of innate immunity in the complete absence of adaptive immunity (e.g., major histocompatibility complex [MHC], immunoglobulin genes, re-arranging T-cell receptor genes, immunological specificity, and memory). In the past 15 years, two major research groups, one in Italy and the other in New York state, have explored the possibility that communication between the neuroendocrine and innate defense systems exists in invertebrates as well as vertebrates. These investigators have used four basic interrelated approaches to address this phylogenetically critical issue. In the following review of their work and the more recent work of others, the generic term hemocyte refers to the invertebrate equivalent of vertebrate blood leukocyte.

A. Synthesis of Neuroendocrine and Neurotransmitter Molecules by Invertebrate Hemocytes The first approach focused on whether endogenous neuroendocrine and/or neurotransmitter substances are synthesized by, and can affect the behavior of, invertebrate blood cells. Hemocytes from several molluscan species (Planorbarius corneus, Lymnaea stagnalis, Mytilus edulis) exhibit immunoreactivity for several vertebrate neuropeptides including met-enkephalin, oxytocin, somatostatin, vasoactive intestinal peptide (VIP), substance P (SP) (Ottaviani and Cossarizza, 1990), ACTH (Ottaviani et al., 1991; Ottaviani et al., 1992a; Smith et al., 1991), and β-endorphin (Ottaviani et al., 1990). ACTH- and TNF-α–like molecules are also found in some types of leukocytes residing in the hemolymph of the dipteran Calliphora vomitoria; staining for both ACTH and TNF-α of the mitotically active

plasmacytes was related to their activated state during the formation of capsules to wall off foreign substances (Franchini et al., 1996b). Immunoreactive met-enkephalin has also been detected in the coelomic fluid of earthworms, and treatment of earthworm coelomocytes with DAMA stimulates coelomocyte migration, much as is seen in human granulocytes and molluscan hemocytes (Cooper et al., 1993). Eleven years ago, Ottaviani et al. (1995a) reported that hemocytes from freshwater snails, Planorbarius corneus and Viviparus ater, express pro-opiomelanocortin (POMC) mRNA as assessed by in situ hybridization with a digoxigenin-labeled human DNA probe. Interestingly, this probe did not detect POMC mRNA in another morphologically distinct hemocyte from these species, a hemocyte that Ottaviani (1992) believes has features of the vertebrate T-cell. This is the same probe mentioned subsequently in connection with detection of POMC mRNA in phagocytic leukocytes from both the edible frog and goldfish, and in lymphocytes from frogs but not fish (Ottaviani et al., 1995a). It also appears that “stress” stimulates invertebrate hemocytes to produce endogenous neural-immune mediators. For example, Stefano et al. (1989b) found elevated levels of endogenous morphine-like material in the hemolymph of Mytilus that had been subjected to electrical shock combined with mechanically preventing closure of their shells. Concurrent with this rise was a substantial increase in the proportion of activated (ameboid, as opposed to rounded or resting) hemocytes (Stefano et al., 1993). With respect to stressors and immunological changes in invertebrates, it is worth noting that Malham et al. (2003) reported that abalone subjected to 15 minutes of mechanical disturbance (i.e., shaking in a rotating box) resulted in an elevation of norepinephrine (NE) and epinephrine (EPI) levels. Whether this increase was causally responsible for the accompanying reduction of numbers of circulating hemocytes, their migratory and phagocytic activity, and respiratory burst is possible, but this component of the regulatory pathway has yet to be studied. Interestingly, these changes in immune parameters were short lived in that the values returned to baseline levels (with the exception of superoxide anion production) 100–480 minutes after stress exposure. In a related study, Malham and co-workers (2002) “stressed” octopuses (Eledone cirrhosa) by handling them and exposing them to air for a few minutes. They found that both NE and EPI were released into hemolymph but that the increased levels returned to basal levels within 30–60 minutes after stressor exposure. They also saw a decrease in the numbers of circulating hemocytes that was followed by a rebound effect (i.e., greater numbers than controls within an hour). In contrast to

Prologue

their observations with abalone, the stressor in the octopus effected an increase in hemocyte activity (measured by phagocytosis of heat-killed Vibrio) that peaked at 1 hour and returned to baseline by 2 hours. The production of superoxide anions peaked as early as 5 minutes post stressor exposure and remained elevated for upwards of 2 hours. The norendocrine effectors responsible for these transitory changes are unknown, as is whether such short-lived changes have repercussions with respect to the health of these invertebrate species. There is, however, a very recent study that addresses the long-term health consequences of a stressor exposure in another invertebrate, the fresh water clam Anodonta piscinalis (Saarinen and Taskinen, 2005). These investigators explored the susceptibility of Anodonta to the ergasilid copepod, Paraergasilus rylovi. Clams were field collected from two populations in late summer. They were then transported to the laboratory and marked. The stressed clams were subjected to low oxygen for 25 days, whereas the unstressed control clams were housed in their lakes of origin for the same period. Eleven months after exposure to the stressor, the stressed clams were more intensively parasitized than controls. They also showed lower growth, lower reproduction, and poorer survival than the “unstressed” control clams. Thus, this model suggests that even in an invertebrate, a stressor may evoke long-lasting effects on susceptibility of natural populations to parasitism.

B. Effects of Mammalian Neuroendocrine and Neurotransmitter Molecules on Invertebrate Hemocytes Several investigators have examined the possible influence of exogenous neuroendocrine, neuropeptide, and/or neurotransmitter messenger molecules on the behavior of invertebrate hemocytes. Recent research on crustaceans by Cheng and colleagues (2005) revealed that 4 hours after white shrimp received 10−7 M dopamine, there was a 25% decrease in total hemocyte count, a 15% decrease in phenoloxidase activity, a 21% decrease in respiratory burst, and a 50% decrease in superoxide dismutase activity. Further, the phagocytic activity and clearance of Vibrio also diminished significantly, and bacterial challenge resulted in a higher mortality. Li et al. (2005) reported a similar response to dopamine (except for the change in numbers of circulating hemocytes) in a different host (giant prawn) and parasite combination. Catecholamines have been shown to affect the behavior of hemocytes from bivalves. Specifically, Lacoste et al. (2001) reported that 0.1 μM NE (and higher) inhibited phagocytosis in oysters, an effect that

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was mimicked by isoproterenol, but not by the alpha agonist, phenylephrine. The antagonist, propranolol (a beta-blocker), blocked the NE effect, but an alphablocker did not. Further experiments indicated that the reported effect was mediated via a cAMP-protein kinase A-dependent signal. In the 1990s, Franchini and Ottaviani (1994) and Ottaviani et al. (1992d) published that ACTH induces cytoskeletal and motility changes of phagocytic hemocytes from snails. Genedani et al. (1994) basically confirmed these studies for CRF. They also reported that ACTH fragments (1–24), (1–4), (4–9), (1–13), (1–17), and (11–24) stimulate molluscan hemocyte migration, whereas the entire sequence (1–39) and the fragment (4–11) have an inhibitory effect. Differences between species were noted with respect to the response to individual fragments. Additionally, the (4–11) fragment could antagonize some of the stimulatory fragments (4–9) as well as TNF-α–induced chemotaxis. More recently, Malagoli et al. (2000) again confirmed (in the mollusc Mytilus galloprovincialis) that exogenous CRH provokes changes in the cellular shape of immunocytes and that this response is dependent on extracellular Ca++. By using various inhibitors of transduction signaling pathways, they could completely or partially inhibit these changes. These findings are consistent with the proposition that PKA, PKC, and PKB/Akt are involved in CRH-induced cell shape changes in immunocytes and that the cellular effect of CRH needs the synergistic action of the two second messengers, cAMP and IP(3). In this study, Malagoli and colleagues also reported that immunocytes from the mussel express mRNAs for the CRH receptors, CRH-R1 and CRH-R2. ACTH also causes molluscan hemocytes to release biogenic amines (NE, EPI, and dopamine) that influence chemotactic and phagocytic activities of hemocytes (Franchini and Ottaviani, 1994; Ottaviani et al., 1992d). The greatest release occurred after 15 minutes, but after 45 minutes, the values were similar to those of the controls. Culturing hemocytes with CRF also provoked release of biogenic amines, suggesting that endogenous ACTH mediates this release. These experiments also suggest that molluscan hemocytes have the capacity to bind and respond to CRH in a manner reminiscent of the way in which mammalian leukocytes respond to this releasing factor (Ottaviani et al., 1993a). These authors further demonstrated immunoreactive tyrosine hydroxylase and dopamine betahydroxylase (enzymes involved in biogenic amine biosynthesis) in these hemocytes. Ottaviani et al. (1994) found a similar but less significant catecholamine response when mammalian interleukin-2 (IL-2) rather than ACTH was added to cultures of hemocytes. Inter-

4

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estingly, pre-incubation of hemocytes with IL-2 or with anti–IL-2 monoclonal antibody significantly reduced or completely eliminated the CRF-induced release of biogenic amines. Further direct evidence of competition between CRF and IL-2 was revealed by immunocytochemical and cytofluorimetric analysis. One explanation favored by these investigators (at least at that time) was the presence of a unique (ancestral?) receptor on molluscan hemocytes that is capable of binding both CRF and IL-2. If this is indeed the case, it would have significant implications for understanding the evolution of neural-immune system interactions. At the very least, these and other observations suggest that in terms of catechol biosynthesis, the invertebrate hemocyte may be a major player in an ancestral stress response that is associated with the HPA axis in mammals. Stefano and colleagues (Dureus et al., 1993) also found that administration of mammalian neuropeptide Y (NPY) to either molluscan hemocytes or to human granulocytes inhibited both spontaneous activation and chemotaxis in response to the chemoattractant synthetic peptide, N-formyl-methionyl-leucylphenylalanine. In a somewhat more recent investigation, Sassi et al. (1998) confirmed that ACTH (1–24) induces cell shape changes in the immunocytes of the mollusc, Mytilus galloprovincialis. Using computer-assisted microscopic image analysis, they reported that a G protein antagonist (suramin sodium), an adenylate cyclase inhibitor (2′,5′-dideoxyadenosine), and a protein kinase inhibitor (staurosporine) inhibited this effect. The highly specific inhibitors H-89 (for protein kinase A) and calphostin C (for protein kinase C) only partially inhibited the morphological alterations, whereas the simultaneous action of H-89 and calphostin C completely blocked them. Thus, mammalian ACTHinduced changes in cell shape appear to involve the adenylate cyclase/cAMP/protein kinase A pathway, as well as the activation of protein kinase C. In a related paper (Ottaviani et al., 1998b), ACTH receptor-like messenger RNA was detected in molluscan hemocytes (and, as a control, in human blood mononuclear cells) using a digoxigenin-labeled bovine cDNA probe. These findings imply that the ACTH receptor gene has been highly conserved during evolution and, according to these investigators, support their hypothesis that there is a phylogenetic relationship between the immune and neuroendocrine systems in invertebrates. Stefano et al. (1989a) reported that opioids can also affect the behavior of hemocytes of the mussel, Mytilus edulis. Specifically, they found that the synthetic enkephalin analogue, DAMA (D-Ala2, met5enkephalinamide), modulated locomotion, adherence,

and conformation of a subset of hemocytes that resulted in their assuming a flattened and elongated conformation with extended pseudopodia. These morphological characteristics of hemocyte activation are similar to those seen following similar treatment of human granulocytes (Hughes et al., 1991b; Stefano et al., 1989a; Stefano et al., 1989b; Stefano et al., 1991a).

C. Effects of Mammalian Cytokines on Invertebrate Hemocytes The third approach to studying invertebrate neuralinnate immune system interactions has involved exploring the impact of molecules, purported to be homologues of mammalian pro-inflammatory cytokines, on hemocyte locomotion and phagocytosis, and on the production of nitric oxide synthase (NOS) and biogenic amines (Ottaviani et al., 1995c; Ottaviani et al., 1997). Some of these studies provide suggestive evidence that the cytokines tested can bind to, and compete with, CRF for the same membrane receptor (Ottaviani and Franchini 1995). However, given the known lack of cross-reactivity of most mammalian cytokines with cells from different mammalian species (Haynes and Cohen, 1991), these results with mammalian cytokines and invertebrate blood cells must remain more provocative than definitive.

D. Production of Cytokine-Like Molecules by Invertebrate Hemocytes The final approach taken by these investigators addresses the production of cytokine-like molecules by invertebrate hemocytes in response to signals that clearly elicit cytokine production by mammalian leukocytes. Like human granulocytes, molluscan hemocytes respond to lipopolysaccharide (LPS) stimulation by assuming the active conformation changes described above (Hughes, et al., 1990; Hughes et al., 1991a; Hughes et al., 1991c). Similar LPS-induced changes of hemocytes from the insect Leucophaea maderae have also been published (Ottaviani et al., 1995e). At least for molluscs, this effect could be blocked by anti-mammalian TNF-α and/or anti–IL-1 antibodies. DAMA also is able to induce molluscan hemocytes to produce immunoreactive (ir)IL-1 (Stefano et al., 1991b). Administration of naloxone blocked the DAMAinduced conformational change by hemocytes, but these cells could still be activated by administration of recombinant human (rh)IL-1α, suggesting that opioid activation may be triggered by an IL-1–like molecule (Stefano et al., 1991b). As mentioned earlier, molluscan hemocytes release biogenic amines when they are cultured with CRF, a

Prologue

phenomenon that Ottaviani and co-workers (1991) described as a prototypic stress response. This response is significantly reduced when hemocytes are preincubated with IL-1α, IL-1β, TNF-α, or TNF-β prior to adding CRF to the incubation mixture (Ottaviani et al., 1995b). Ottaviani and Franchini (1995) and Franchini et al. (1996a) used immunocytochemistry to detect immunoreactive platelet-derived growth factor α and β (PDGFα/β) and transforming growth factor (TGF)-β in phagocytic invertebrate leukocytes. The presence of PDGF-α/β–like receptors and TGF-β receptor (type II)-like molecules on the plasma membranes of the immunocytes of the mollusc Mytilus galloprovincialis was also suggested by immunocytochemistry (Kletsas et al., 1998). This latter study also revealed that PDGFα/β and TGFβ1 provoke changes in the shape of the molluscan hemocytes following interactions of these mammalian ligands with their putative receptors and that these extracellular signals are transduced along the phosphoinositide-signaling pathway. Ottaviani et al. (1998a) suggest that in the mussel, the major pathway followed by PDGFα/β and TGFβ in provoking the release of NE, EPI, and dopamine into cell-free hemolymph is mediated by a CRH-ACTH biogenic amine axis. In mammals, microglial cells, like macrophages, are phagocytic and synthesize pro-inflammatory cytokines. Sonetti et al. (1997) argue that the snail, Planorbius corneus, also has a class of glial cells that resemble vertebrate microglia. Interestingly, these cells can be identified by their immunopositivity to anti–POMCderived peptide antibodies. As in the vertebrates, snail microglia exhibit macrophage-like mobility, and when exposed in vitro to LPS or bacteria, they underwent conformational and mobility changes and also became phagocytic. Moreover, when activated, they also expressed TNF-α–like molecules and increased production of NOS, as shown immunocytochemically. Morphine (which appears to bind these cells via a μ3 receptor) inhibited this mobility and phagocytic activity of invertebrate microglia, suggesting to these investigators that opioid-like compounds may influence invertebrate microglia as well as hemocytes. Similar microglial-like cells have also been described in the mussel and the insect Leucophaea maderae (Sonetti et al., 1994). Excitability of a population of nociceptive sensory neurons in Aplysia were influenced by neighboring hemocytes (Clatworthy, 1998); Clatworthy and Grose (1999) suggest that in vitro activation of these hemocytes by LPS causes them to produce cytokinelike factors which modulate expression of injuryinduced sensory nerve hyperexcitability. The aforementioned studies with invertebrates are clearly provocative in terms of their suggesting a

5

common evolutionary origin of the immune and neuroendocrine systems with their attendant inflammatory and stress responses (Ottaviani and Franchini, 1995; Ottaviani and Franceschi, 1996, 1997, 1998). However, before accepting the validity of this hypothesis, or even some of the data that led to its formulation, we must emphasize the importance of characterizing all the immunoreactive molecules and their receptors described in the previous paragraphs at the structural and genomic levels to determine if they are true homologues of their mammalian counterparts rather than, for example, an artifact of the detection methods used to identify them (Hahn et al., 1996). A few intriguing studies we encountered in our review of the literature do not fit into the outline we’ve followed for this section on invertebrates. Indeed, they so beautifully reveal the evolutionary conservation of the links between behavior and its neuroimmune consequences that they merit their own paragraph. Mallon et al. (2003) and Riddell and Mallon (2006) presented behavioral evidence indicating a link between the immune system and the nervous system in insects. In brief, bumblebees that were injected with LPS to incite an anti-bacterial response in their hemolymph (Moret and Schmid-Hempel, 2000) have reduced abilities to learn (or recall memory) in a classical conditioning paradigm. Their study further points out that this associative learning deficit occurs only after bees are deprived of pollen (their only protein sources). As will be discussed later in the section on birds, some evolutionary biologists have become interested in the immune system in general (and psychoneuroimmunology in particular) because of an interest in energy trade-offs between immune processes and various behaviors (e.g., reproduction, foraging for food, nest building, sexual displays). With respect to invertebrates, Fedorka et al. (2004) tested the hypothesis that “immune suppression” mediates a phenotypic trade-off between reproduction and immunity by manipulating reproductive effort and measuring immune function and mortality rates in the striped ground cricket, Allonemobius socius. In this species, male crickets provide females with a hemolymphbased “nuptial gift” during copulation. Based on their knowledge that hemolymph contains many immune mediators, these investigators predicted that sexual selection might differentially affect how disease resistance evolves in males and females. Indeed, they found that for both sexes, an increased mating effort resulted in a reduced immune ability. In their words, immune suppression appears to be a link between reproductive effort and cost in this system. Also of note are their observations that males and females differentially invest in several aspects of immunity prior to mating:

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Prologue

Males exhibit a higher concentration of circulating hemocytes and a superior bacterial defense capability than females. In a related study from this group, Zuk et al. (2004) reported that the ability of seasonal breeding crickets (both in the field and in the lab) to encapsulate foreign material was better in males than females. No sex dimorphism was noted in an aseasonal breeding species; however, when food was restricted, the males again did better. Although an interaction between the endocrine and defense systems are suspect in the results described in this and the preceding paragraph, no mechanisms have been explored formally. No doubt many experimental questions of potential psychoneuroimmunological interest raised by this intriguing line of research will keep these and other investigators gainfully occupied for several years.

III. NEURAL-IMMUNE INTERACTIONS IN TELEOST FISH Given the phylogenetic success of the teleosts (Hickman et al., 1993), it would not be unreasonable to propose that fish, like all other ectotherms discussed in the following sections, display coordinated and integrated immune and neuroendocrine responses to environmental challenges. The information reviewed in this section and elsewhere (Weyts et al., 1999; Yada and Nakanishi, 2002) clearly support this hypothesis.

A. Innervation of Lymphoid Organs and Response of Leukocytes to Biogenic Amines Autonomic innervation in the spleens of cod (Nilsson and Grove, 1974), coho salmon (Flory, 1989), and rainbow trout (Flory, 1990) has been demonstrated. At least for coho salmon, however, splenic innervation appears to be largely associated with the vasculature, with some branching out into the parenchyma (Flory, 1989). Chemical sympathectomy (SyX) of salmon with 6-hydroxydopamine (6-OHDA) depletes noradrenergic (NA) innervation as measured either by HPLC for NE, or by the absence of fluorescent NA nerve fibers (Flory, 1989) using sucrose-potassium phosphateglyoxylic acid-induced (SPG) histofluorescence for catecholamines (de la Torre, 1980). Chemical SyX has also been reported to increase the number and percentage of splenic anti-sheep red blood cell (SRBC) plaqueforming cells (PFCs) in fish denervated prior to immunization; no effect was seen if immunization preceded SyX. These data are consistent with the augmented antibody response seen following SyX of neonatal rats

(Besedovsky et al., 1979) and adult mice (Kruszewska et al., 1995, 1998), but contrast with the decreased antibody and cell-mediated responses seen after SyX in adult mice (Livnat et al., 1985; Madden et al., 1989a; Madden et al., 1989b; Sanders and Straub, 2002). Flory (1990) also demonstrated for rainbow trout that adrenergic and cholinergic agents can alter in vitro antibody response to TNP-LPS. Specifically, the in vitro induction of a primary anti–TNP-LPS PFC response was suppressed by the β-adrenergic agonist, isoproterenol (10−4–10−7 M), whereas it was enhanced by the αadrenergic agonist, phenylephrine. The β-agonist effect could be blocked by propranolol, consistent with receptor mediation, and the α-agonist effect was blocked by yohimbine but not phentolamine. This suggestion of an α–2 adrenoreceptor was confirmed by the demonstration that clonidine (10−7–10−11 M), an α–2 specific agonist, enhanced antibody responses. A cholinergic agonist also enhanced PFC responses over a dose range of 10−5–10−11 M; this was blockable by the muscarinic antagonist, atropine. Subsequent studies have revealed an influence of adrenergic and cholinergic agents on the chemiluminescent and mitogenic responses of trout leukocytes (Bayne and Levy, 1991; Flory and Bayne, 1991). Plytycz and co-workers (Józefowski and Plytycz, 1998; Józefowski et al., 1995) extended Flory’s studies by demonstrating first that there are adrenergic and cholinergic receptors on head kidney leukocytes of the goldfish, Carassius auratus; and second, that high concentrations of the βadrenergic agonist, isoproteronol (10−4 M), and the cholinergic agonist, carbachol (10−5 M), enhanced phorbol myristate acetate (PMA)-induced oxidative burst of goldfish macrophages, effects that could be blocked by equimolar concentrations of propranolol and atropine, respectively. Both EPI and NE enhance the respiratory burst activity of carp anterior kidney macrophages and neutrophils (Verburg-van Kemenade, personal communication). Finally, Narnaware and colleagues (Narnaware and Baker, 1996; Narnaware et al., 1994) observed that both α- and β-adrenergic agonists depress in vitro phagocytosis of yeast by rainbow trout macrophages and that injection of the adrenergic blocker phentolamine can prevent the depressive effects of “stress” on the phagocytic index of cells from the same species. Serotonin (5-HT) is also immunomodulatory in fish. According to a set of detailed experiments (Ferriere et al., 1996), 5-HT suppressed LPS- and PHA-induced proliferation of trout PBLs. This inhibitory effect could be mimicked by an agonist of 5-HT1A receptors (8-OH-DPAT) and was reversed by an antagonist of 5-HT1A and 5-HT1B receptors (spiperone). Scatchard plot analyses confirmed the existence of specific sero-

Prologue

tonin receptors on lymphocytes. In a competition study, serotonin inhibited the binding of 3H-5HT to receptors in both resting and mitogen-stimulated lymphocytes. However, the agonists (8-OH-DPAT and buspirone) and antagonist (NAN-190) of the 5-HT1A receptor subtype failed to displace 3H-5HT binding to receptor sites in resting cells, but they did inhibit 3H5HT binding in LPS- and PHA-stimulated lymphocytes. Based on these observations, the authors propose that 5-HT1A receptors are expressed on activated lymphocytes only after mitogenic stimulation. An agonist of 5-HT1B receptors (CGS-12066B) failed to affect 3H5HT binding on either resting or mitogen-stimulated lymphocytes, suggesting that this 5-HT receptor subtype is absent on lymphocytes. A subsequent pharmacological study from the same group (Meyniel et al., 1997), in which additional antagonists of mammalian 5-HT receptors (ICS-205–930 and metoclopramide) were used, suggests that fish 5-HT3 lymphocyte receptors may differ pharmacologically from mammalian receptors.

B. Neuropeptide Production by Cells of the Teleost Immune System Recently, investigators have begun to explore whether fish leukocytes, like mammalian lymphocytes (Blalock, 2005), synthesize hormones typically associated with the hypothalamo-pituitary-interrenal (HPI) gland axis.1 POMC-derived peptides (ACTH, α-MSH, and β-endorphin) have been detected immunocytochemically in goldfish thymic epithelial cells (Ottaviani et al., 1995d), and constitutive and mitogenstimulated production of immunoreactive POMC products by catfish lymphocytes has also been reported (Arnold and Rice, 1997). Since both studies were based on antibody detection of antigenic cross-reactivities, their interpretation may be questioned. However, Ottaviani et al. (1995a) have also reported that goldfish (C. auratus) phagocytic leukocytes express POMC mRNA as determined by in situ hybridization with a digoxigenin-labeled human DNA probe. The same probe also detected POMC mRNA in phagocytic leukocytes and peripheral blood lymphocytes from the frog, Rana esculenta, but lymphocytes from goldfish did not express this gene. A study of different teleost and amphibian species, however, seems necessary before endorsing these authors’ suggestion that expres-

1 Since in fish, the interrenal glands serve the function provided by the adrenal cortex in mammals (Chester Jones et al., 1980), the hypothalamo-pituitary-adrenal axis and the hypothalamo-pituitaryinterrenal axis are functionally equivalent.

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sion of this gene in vertebrate lymphocytes first occurred in the Amphibia. A major contribution to psychoneuroimmunology in the past decade has been research revealing that in mammals communication between the neuroendocrine and immune systems is mediated, at least in part, by pro-inflammatory cytokines (e.g., IL-1, IL-6, TNFα). For example, in rodents, IL-1 can act on the hypothalamus and pituitary to elicit CRH and ACTH, respectively (Parsadaniantz et al., 1994, 1997). Thanks largely to the work of Secombes and colleagues in Scotland, several cytokine genes that include IL-1 (Pleguezuelos et al., 2000; Secombes et al., 1998); IL-8 (Laing et al., 2002); IL-10 (Zou et al., 2003); TNF-α (Zou et al., 2002, 2003); IFN-γ (Zou et al., 2005); IL-6 (Bird et al., 2005); lymphotoxin b (Kono et al., 2006), IL-11 (Wang et al., 2005); and IL-18 (Zou 2004) have been cloned in different species (e.g., salmon, rainbow trout) and, at least for some genes, their products expressed as recombinant proteins (reviewed in Secombes et al., 2001; Secombes and Cunningham, 2004). This major sustained effort has permitted research on the role of cytokines as mediators of neuroendocrine-immune interactions in teleosts. Indeed, there is now solid evidence that in fish, IL-1β is an effector molecule that activates the HPI axis as evidenced by a strikingly elevated level of plasma cortisol in trout receiving an i.p. injection of 0.1–0.6 nmol/kg of the recombinant protein (Engelsma et al., 2002; Holland et al., 2002). Holland et al. (2002) also reported that trout IL-1β peptides (P1 and P3), which are homologous to receptor-binding sequences of human IL-1β, failed to influence the prevailing cortisol concentration even though an equivalent dose was immunostimulatory in vivo. In this study, blockade of endogenous ACTH release by administration of the synthetic glucocorticoid dexamethasone (DEX) prevented the rIL-1β–mediated elevation of plasma cortisol. Inhibition studies with the cloned fish IL-1 receptor-associated protein and IL-1 receptor (Stansberg et al., 2005) have yet to be carried out to further probe the role of this cytokine in the neuroimmune circuitry. Nevertheless, the important phylogenetic take-home message from these studies is that IL-1 signaling between the immune and neuroendocrine systems in mammals is conserved in lower vertebrates. Just how early this neuroimmune regulatory pathway evolved (i.e., is it present in elasmobranchs and agthanans?) remains unresolved. So too does the question of whether, like mammals, other pro-inflammatory cytokines play a signaling role at the teleost level of phylogeny. LPS is known to activate the fish HPI axis (Balm, 1997). Since increased cortisol levels could be induced by either an i.p. injection of LPS or rIL-1β, it is reason-

8

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able, by extrapolating from the mammalian literature, to conclude that in fish LPS induces an increase in IL-1 that, in turn, effects increased levels of cortisol via activation of the HPI axis. An early literature points out that direct exposure of pituitary tissue to LPS in vitro blunts ACTH and α-MSH release (Balm et al., 1995). Although it is once again reasonable to assume that these changes result from an increase in cytokine secretion following LPS treatment, it should be noted that LPS can also affect endocrine tissues directly (Brunetti et al., 1994; Milton et al., 1993). One of the most important reasons for conducting studies using a homologous system (i.e., trout rIL-1b injected into trout) is that it obviates problems inherent in using mammalian cytokines in fish. Earlier studies like one in which murine IL-1α was reported to inhibit α-MSH release by the HPI axis (Balm et al., 1993) or studies pointing out that mammalian IL-1 has no effect on teleost lymphocytes (see review by Haynes and Cohen 1991; see also Ellsaesser and Clem, 1994; Verburg-van Kemenade et al., 1995) need to be repeated in the homologous system, especially since mammalian and fish IL-1 have minimal identity at the DNA and amino acid sequence levels (Secombes et al., 1998).

C. Glucocorticoids, Neuropeptides, and Stressor Effects on Immunity The neuroendocrine stress response of fish (Wendelaar Bonga, 1997) is quite similar to that of mammals (Chrousos and Gold, 1992) in that it consists, in part, of a stressor-sensitive HPI axis. Cortisol, the major glucocorticoid in fish, is produced by the interrenal gland. Primary mediators of cortisol in teleosts are ACTH (apparently for acute stress situations) and α-MSH (in more chronic situations) (Donaldson, 1981; Lamers et al., 1994; Sumpter et al., 1994). These hormones, in turn, are under hypothalamic control via CRF for ACTH (Olivereau and Olivereau, 1991) or via both CRF and TRH α-MSH (Lamers et al., 1994). Parenthetically, in an in vitro study, Harris and Bird (1998) demonstrated that a 1-hour exposure to α-MSH increases the phagocytic ability of head kidney macrophages and neutrophils from rainbow trout. Although the neuroendocrinology of the stress response in teleosts has been well studied (Huising et al., 2004, 2005; Rotlant et al., 2003; van den Burgh et al., 2005), information that causally relates, in a stepwise and mechanistic fashion, stressor-associated neuroendocrine levels and pathways to changes in immune system parameters and susceptibility to pathogens is relatively fragmentary. This is not to say that there is a dearth of studies that explore the effects of stressors

on selected aspects of immune function and on mortality in agriculturally important teleost species. Quite the contrary (Barton and Iwama, 1991; Ellis, 1981; Ndoye et al., 1991; Pickering, 1981; Wendelaar Bonga, 1997). For example, a 30-second dip net removal of Chinook salmon from the water temporarily elevated plasma cortisol, increased leukocyte numbers in the thymus and anterior kidney, decreased blood and spleen leukocytes, and altered resistance to the fish pathogen Vibrio anguillarum (Maule et al., 1989). Altered resistance was manifested by an increased mortality and decreased time-to-death in salmon exposed to Vibrio 4 hours after stressor exposure and by a decreased mortality (relative to controls) and longer survival times in fish exposed 1 day after this acute stressor (Maule and Schreck, 1990a; Maule et al., 1989). Altered immune function was also reflected by a decreased in vitro anti-TNP antibody production (relative to unstressed fish) by anterior kidney leukocytes 4 hours and 7 days after stressor exposure. At this later time-point, plasma cortisol levels had returned to normal (Maule et al., 1989), indicating that the effects of the stressor persist beyond the time of cortisol elevation. A similar observation was made by Betoulle et al. (1995), who subjected trout to hyperosmotic shock for 7 days or 30 days and measured cortisol, PRL, and anti-Yersinia ruckeri antibodies in the serum. Relatively short-term “stress” was associated with a correlation between high levels of both stress hormones and a delayed production and lower titers of antibody. More chronically exposed animals had no increase in stress hormones but still had low antibody titers. These two reports, among others, are consistent with the idea that the putative immunomodulatory effects of stress hormones impact the earlier phases of antibody production. A recent study (Binuramesh et al., 2006) has convincingly demonstrated that the social environment of fish plays an important role in their adaptive and innate immune responses to pathogens. This research examined the effects of sex ratio of a tilapia (Oreochromis mossambicus) housed for 4 weeks either as same sex or mixed (1:1) sex cohorts on antibody responses to Aeromonas hydrophila; serum lysozyme activity; production of intracellular reactive oxygen species (ROS); on reactive nitrogen species (RNS) by peripheral blood leukocytes; and on disease resistance against live, virulent Aeromonas hydrophila. Their data convincingly showed an enhanced antibody response and increased number of antibody-producing cells in the mixed-sex cohorts relative to fish in monosex ratio groups. Similar enhancement was also observed in non-specific immune parameters such as serum lysozyme level, ROS, and RNS production. The host resistance test

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revealed that enhanced immunity in the equal male and female sex ratio group was protective against Aeromonas hydrophila infection. Hence, natural sex ratios may enhance disease resistance to pathogens in this species; just how it does so is an important but unresolved issue. As indicated in the aforementioned Binuramesh study, stressors can modify various aspects of the innate as well as adaptive immune system of teleosts. In another example from other species (e.g., trout), in vitro respiratory burst activity of fish anterior kidney phagocytes was diminished following handling and exposure to anoxic shock, as well as by crowding (Angelidis et al., 1987; Pulsford et al., 1994; Yin et al., 1995), but it was increased in trout following transfer from fresh to sea water (Marc et al., 1995), indicating a variance across species and/or stress modalities. A social stress paradigm in rainbow trout led to increased in vivo phagocytosis of bacteria by peripheral blood phagocytes (Peters et al., 1991). Transition from fresh to seawater, however, had no effect on activity of natural cytotoxic cells (NCC) isolated from brown trout (Marc et al., 1995). Although decreasing the water temperature in which carp were held enhanced their NCC activity (Le Morvan-Rocher et al., 1995), the in vitro assays were all performed at 28°C, which could have different consequences for cells isolated from fish adapted to different temperatures (Clem et al., 1984, 1991). A social stress paradigm in aggressive fish (Tilapia) resulted in depressed NCC activity and mitogenic responses in the subordinate fish (Ghoneum et al., 1988). As determined by blocking studies with naltrexone, this effect seems to be mediated, at least in part, by endogenous opioids (Faisal et al., 1989). A group of Polish investigators led by Plytycz has demonstrated that endogenous opioids (i.e., morphine) also appear to be involved in reducing numbers, but increasing respiratory burst activity, of thioglycollate-elicited inflammatory cells in peritoneal exudates from goldfish (Chadzinska et al., 1997; Gruca et al., 1996) and salmon (Chadzinska et al., 1999). This morphine-induced increase in respiratory burst activity did not occur if the exudate cells were harvested from stressed salmon (Plytycz et al., 1996). As would be predicted by the results from these studies involving the parenteral administration of morphine and naltrexone, opioid receptors have been identified on teleost head kidney leukocytes cells and characterized by radiolabeled ligand binding (Józefowski and Plytycz, 1997). As in mammals, lysozyme plays a non-specific antibacterial defense role in fish. Plasma lysozyme levels (as well as plasma cortisol and epinephrine) in rainbow trout increased following 30 seconds of handling (Demers and Bayne, 1997). Lysozyme levels were also

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increased in brown trout following their transition from fresh to seawater (Marc et al., 1995) but were unaffected by parr-smolt transformation (see below) in Atlantic salmon (Olsen et al., 1993) and were actually decreased in carp following 30 days of crowding (Yin et al., 1995). These differences may well relate to the duration and severity of the stressor, as was shown by Möck and Peters (1990), who observed that 30 minutes of handling increased lysozyme levels in rainbow trout, whereas a 2-hour transport stressor decreased their levels. It seems clear that many of the effects of stressors on non-specific and specific defense modalities in fish involve the HPI axis and glucocorticoids. Fish leukocytes possess receptors for corticosteroids (Maule and Schreck, 1990a, 1990b; Verburg-van Kemenade et al., 1999; Weyts et al., 1998a). In coho salmon, receptor-like binding of a synthetic corticosteroid analogue (triamcinolone acetonide) to cells isolated from spleen and head kidney was reported (Maule and Schreck, 1990b). Carp peripheral blood cells also express cortisol receptors with a high binding affinity (Kd 3.8 nM). Neutrophilic granulocytes isolated from the carp head kidney contain cortisol-binding sites with the same characteristics (Weyts et al., 1998a, 1998c), suggesting that both PBL and head kidney neutrophils express the same glucocorticoid receptor. Basal receptor densities in both cell types are approximately 500 per cell. Following cortisol treatment in vivo, receptor numbers in carp PBL decrease (Weyts et al., 1997), whereas numbers of corticosteroid receptors in coho salmon spleen and head kidney leukocytes increase following exposure to an acute or chronic stressor or by cortisol treatment in vivo (Maule and Schreck, 1991). These changes in receptor densities have been explained by a stress- or cortisol-induced trafficking of receptor-rich leukocyte subtypes from the circulation into lymphoid organs. However, since corticosteroid receptors in coho salmon head kidney leukocytes are also increased following an in vitro exposure to cortisol (Maule and Schreck, 1991), an actual upregulation of receptor numbers resulting from the cortisol treatment also seems reasonable. The immunosuppressive effects of glucocorticoids have been demonstrated in several studies with teleosts. Anderson et al. (1982) injected rainbow trout with a synthetic glucocorticoid 24 hours after immunizing them with the O-antigen of Yersinia ruckeri and observed depressed in vitro and in vivo antibody production and a reduced number of splenic lymphocytes. A similar reduction in numbers of antibody-producing cells has been described in flounder (Carlson et al., 1993). Ellsaesser and Clem (1987) injected channel catfish i.v. with cortisol (6.7 μg/kg body weight),

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which resulted in a plasma level of cortisol 30 minutes following injection equivalent to that seen 30 minutes following “transport stress” in this species. This increase correlated with decreased numbers of circulating leukocytes, increased neutrophils, and decreased LPS- and Con A-induced lympho-proliferation. This last observation has also been made for salmon by Espelid et al. (1996). Since these Norwegian investigators noted that the addition of physiologic levels of cortisol to normal fish leukocytes in vitro did not alter mitogen responses, they suggested that an indirect mechanism was involved in the observed effects. However, Tripp and colleagues (1987) found that physiological concentrations of cortisol in vitro did, in fact, depress both LPS-induced mitogenesis and the primary anti–TNP-LPS antibody responses of splenic and head kidney lymphocytes from coho salmon. In this paradigm, pronephric lymphocytes were sensitive early in the antibody response, whereas splenic lymphocytes were sensitive throughout the culture period. Although it is unknown whether the aforementioned differences in the in vitro effects of cortisol on salmon and catfish lymphocyte mitogenesis are related to the species used or to methodological considerations, others have also shown that vitro, cortisol inhibits teleost lymphocyte proliferation (Grimm, 1985; Pulsford et al., 1995; Tripp et al., 1987) and reduces antibody production (Tripp et al., 1987; Wechsler et al., 1986). It has been suggested that cortisol may act on fish lymphocytes by inhibiting cytokine production as it does in mammalian cells (Kaattari and Tripp, 1987; Tripp et al., 1987). On the other hand, the observation that in vivo cortisol treatment resulting in plasma concentrations of 400 ng/ml enhances the numbers of apoptotic lymphocytes in the skin of rainbow trout (Iger et al., 1995) suggests that apoptosis may be regulated by cortisol. Indeed, apoptosis appears to have been conserved as an immune regulatory mechanism in fish as well as other ectothermic vertebrates including frogs (Haberfeld et al., 1999; Rollins-Smith, 1998; Rollins-Smith and Blair, 1993; Rollins-Smith et al., 1997a; Ruben et al., 1994) and salamanders (Ducoroy et al., 1999). Cortisol-induced apoptosis of fish leukocytes is mediated by a glucocorticosteroid receptor since the glucocorticoid receptor antagonist RU486 (Weyts et al., 1998a, 1998b, 1998c) could block apoptosis. The low concentration of cortisol (0.1 μM) that was effective in inducing B-cell apoptosis contrasts with the lack of effects of cortisol’s natural conversion product, cortisone. Since the conversion of cortisol to cortisone in fish is highly preferred over the reverse reaction (Donaldson and Fagerlund, 1972), this conversion may

provide the fish with a mechanism to regulate the effects of corticosteroids on cells of the immune system. The lack of apoptosis induction by cortisone correlates with the low affinity of the glucocorticosteroid receptor in carp PBL for cortisone (250 times lower than that for cortisol) (Weyts et al., 1998c). Effects of cortisol on leukocyte viability are cell type-specific. For example, B-cells from carp are especially sensitive to cortisol, whereas thrombocytes and T-cells are insensitive. Induction of apoptosis depends on the developmental state and/or activation state of the lymphocyte. In the periphery, only activated Bcells appear sensitive, whereas in head kidney and spleen apoptosis induction in B-cells is independent of the activation state (Verburg-van Kemenade et al., 1999). Interestingly, in vitro apoptosis of carp head kidney neutrophils was reduced when cells were cultured with cortisol, and this effect of cortisol was also mediated by a glucocorticoid receptor (Weyts et al., 1998b). Analysis of the glucocorticoid receptors in these cells revealed that they may be the same as those detected in PBLs since both have the same affinity and specificity (Weyts et al., 1998c). The inhibition of neutrophil apoptosis by cortisol, combined with the observation that neutrophil respiratory burst activity was not affected by cortisol, would augment the supply of functional neutrophils in stressful conditions. Taking into account that neutrophils, together with macrophages, form the first line of defense against invading microorganisms (Dalmo et al., 1997), mobilization of these cells under stressful conditions may be important for survival. Although cortisol can trigger apoptosis of leukocytes, and stressors elevate this steroid in fish, Alford et al. (1994) observed that confinement stress of channel catfish was associated with a decrease in apoptotic PBLs, and in vitro culture of lymphoid cells with cortisol failed to induce apoptosis. This apparent discrepancy with some of the previously cited literature may relate to the fact these investigators used unstimulated cells in their experiments and, at least in carp, only mitogen-stimulated PBLs are sensitive to cortisolinduced apoptosis (Weyts et al., 1998b). Plasma cortisol concentrations in stressed salmonids and cyprinids range between 100 and 500 ng/ml (Barton and Iwama, 1991), of which approximately 25–125 ng/ml is present in an unbound configuration (Caldwell et al., 1991; Flik and Perry, 1989). Therefore, concentrations in the micromolar range or higher may not be physiological. It appears that in vitro, cortisol does not affect phagocytosis or respiratory burst activity (Narnaware et al., 1994; Weyts et al., 1998b) unless

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supraphysiological concentrations in the micromolar range (or higher) are used (Ainsworth et al., 1991; Pulsford et al., 1995; Stave and Roberson, 1985). Accordingly, respiratory burst activity of a goldfish macrophage cell line was unaffected by up to 10 μM cortisol (Wang and Belosevic, 1995). The inhibition of phagocytosis of SRBC that was described in the same study was again only detected at relatively high (1 μM) cortisol concentrations. Studies of cellular immune functions associated with either stressor administration or in vivo cortisol treatment often fail to consider leukocyte trafficking and redistribution as an explanation of the apparent immunosuppression observed in vitro. A wealth of information reveals that many stressors (e.g., transport, anoxia, social conflict, handling, injection) in several fish species are associated with decreased numbers of circulating B-lymphocytes and increased numbers of circulating neutrophils (Ainsworth et al., 1991; Angelidis et al., 1987; Bly et al., 1990; Ellsaesser and Clem, 1987; Espelid et al., 1996; Faisal et al., 1989; Pulsford et al., 1994; Salonius and Iwama, 1993). These effects are mimicked by in vivo corticosteroid treatment (Ainsworth et al., 1991; Ellsaesser and Clem, 1987; Espelid et al., 1996; Weyts et al., 1997). Increased infiltration of leukocytes into the thymus, head kidney, skin, and gill (Balm and Pottinger, 1993; Iger et al., 1995; Maule and Schreck, 1990a; Peters et al., 1991) has also been observed following either stress or in vivo cortisol administration. Thus, interpretation of data obtained from in vitro functional analysis of leukocytes from stressed fish or from fish injected with cortisol needs to take into consideration the possible (dis)appearance of cell populations rather than simply changes in cell activity. Although there are more data supporting a neuroendocrine-immune link in fish than there are for any other non-mammalian vertebrate, studies to date have used many different species and many different types of stressors. Thus, no “best use” model has emerged to allow an in-depth study of the effects of various sorts of stressors on several immune parameters of a single species. Effects are leukocyte type dependent, and the final outcome may depend on the severity and duration of the stressor, as it does in mammals (Moynihan et al., 1994). The cortisol-mediated rescue of neutrophils from apoptosis shows that cortisol does not suppress all aspects of the fish defense system. Rather, cortisol acts as a regulator, inhibiting some parts of the (specific) immune response and enhancing other (nonspecific) components that may be functional in stressful situations. Stimulation of an innate immune response may be part of an adaptive response neces-

sary to combat potential pathogens under stressful conditions (Weyts et al., 1999).

D. Seasonal Influences on Immunity Smolting, a series of profound physiological changes that prepare juvenile freshwater salmon for entry into salt water, is characterized by increases in plasma thyroxine and cortisol levels (Maule et al., 1987). These hormonal changes correlate with decreased numbers of splenic PFCs in salmon immunized with the Oantigen from V. anguillarum and also with decreased numbers of PBL (although there was an increase in the proportion of small lymphocytes) relative to either erythrocytes or fish body weight. Such changes, together with increased mortality to Vibrio infection, have also been seen following implantation of cortisolcontaining pellets (Maule et al., 1987). Like reptiles and amphibians (discussed in following sections), immune reactivities and lymphoid tissues of teleosts undergo seasonal changes that are unrelated to smolting. For example, Yamaguchi et al. (1981) found that the agglutinating and cytotoxic antibody responses of trout immunized in the spring with the pathogen Aeromonas salmonicida were higher and increased more rapidly than those of fish immunized in the winter, even though animals were held at a constant temperature of 18°C. Seasonal modulation in antibody production in relation to the state of lymphoid tissue development has also been studied in the ovoviviparous marine fish, Sebastiscus marmoratus (Nakanishi, 1986). Fish immunized with SRBC in summer after having been acclimated for at least 2 weeks to 23°C, had higher antibody titers than fish immunized in winter, even when the environmental temperature of acclimation and immunization was constant. A sexual dimorphism was noted in that antiSRBC antibody titers of mature females were lower than that of either males or immature females in the winter spawning season. In addition, the thymus of pregnant and especially post-spawning females was entirely involuted, showing a marked decrease in the number of lymphocytes in both the cortex and medulla. The neuroendocrine regulation of such dramatic changes seems well worth further study. Circadian rhythm has been shown to influence immune responses in fish. The gulf killifish, Fundulus grandis, for example, exhibits a circadian variation in immune reactivity during scale allograft rejection. Specifically, a two- to three-fold higher level of immune activity and cellular destruction occurred during the dark period, resulting in a longer survival time for grafts transplanted at light onset than for those grafted

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at lights off (Nevid and Meier, 1993, 1994). Phase relationships between two circadian neuroendocrine oscillations (daily photoperiod and non-photic daily stimuli) appear to be involved (Nevid and Meier, 1995a), as do levels of hormones and neuropeptides/ neurotransmitters (Nevid and Meier, 1995b). For example, daily rhythms of alloimmune reactivity could be abrogated by treating fish with naloxone or propranolol at light offset only, GH or atropine at light onset only, or PRL at either light onset or light offset. Timed treatments with PRL or GH reduced the length of time needed to completely destroy scale grafts, whereas timed treatments with propranolol or naloxone prolonged graft survival (Nevid and Meier, 1995b).

IV. NEURAL-IMMUNE INTERACTIONS IN AMPHIBIANS Based on a variety of morphological and physiological characteristics that distinguish Amphibia from Teleosteii and Reptilia, this class of vertebrates is generally thought of as a phylogenetically pivotal group. As such, the immune systems of a few (one hopes representative) amphibian species have been exhaustively studied. In the last decade, a few “amphibian immunologists” have broadened their research focus to include neural-immune interactions in both frogs and salamanders. Areas being investigated include: (a) innervation of lymphoid tissues; (b) neuropeptide/ neurotransmitter regulation of immunity; (c) seasonal/ neuroendocrine effects on immunity in adults; (d) neuroendocrine regulation of immunity during metamorphosis; and (e) the impact of environmental stressors on anti-microbial immunity and its implications for the worldwide decline of amphibians.

A. Innervation of Lymphoid Organs Several lines of evidence point to catecholamines as “neuroimmune transmitters” in amphibians. Prior to the published observations in mammals by the Feltens in the 1980s (Felten and Olschowka, 1987; Felten et al., 1987), Nilsson (1978) had used fluorescence histochemistry to reveal sympathetic innervation of the spleen of the cane toad, Bufo marinus, and Zapata et al. (1982) published electron microscopic evidence of direct contacts between nerve endings and lymphoid cells in the jugular body of the leopard frog, Rana pipiens (Manning and Horton, 1982). More recently, Kinney et al. (1994b) described noradrenergic and peptidergic innervation of the spleen of the adult South African clawed frog, Xenopus laevis, using: SPG histofluorescence (de la

Torre, 1980) for catecholamines and immunocytochemistry for tyrosine hydroxylase (TH), the ratelimiting enzyme in the synthesis of catecholamines (Felten and Olschowka, 1987); PGP 9.5 (a general neuronal antigen); and NPY. Spleens of this species, like those of other anurans, have a clearly defined red and white pulp (Manning, 1991; Manning and Horton, 1982). Noradrenergic fibers are almost exclusively restricted to the white pulp in association with the central artery. These fibers have occasional varicosities in the parenchyma, and additional fibers are present in the boundary cell/perifollicular areas where they may come into contact with B-cells and macrophages of the white pulp; non-lymphoid dendritic cells involved in trapping and retention of soluble antigen (Manning, 1991); and possibly the T-cells at the extreme boundary of the white pulp. In some instances, fibers were also noted in the splenic capsule. In short, this innervation pattern in Xenopus is similar to that described for the murine spleen (Felten et al., 1987). The profile of fine varicose nerve fibers staining for NPY in the white pulp of the Xenopus spleen was similar to, but less abundant than, TH+ fibers, an observation that may reflect a difference in sensitivity of the antibody used rather than an actual difference in amount of neurotransmitter present. Substance P-staining fibers were also found around vessels in the splenic white pulp (Kinney et al., 1994b). Although frogs, toads (Anura), and salamanders (Urodela) are all amphibians, the two taxonomic orders differ immunologically in several interesting ways, such as the delayed kinetics of antibody production and allograft rejection characteristic of urodeles relative to anurans (Cohen and Koniski, 1994). In addition, and in sharp contrast to the mammalian-type pattern of innervation that characterizes the compartmentalized Xenopus spleen, SPG histofluorescence analysis of the non-compartmentalized spleens of the salamanders Taricha torosa, Notophthalmus viridescens, and Ambystoma mexicanum revealed a diffuse pattern of innervation associated with the reticular network (Kinney et al., 1994b). The third order of amphibians, the Gymniophiona (Apoda or caecelians), has received only limited attention from immunologists, no doubt because they are difficult to obtain for laboratory research. The spleen of one such apodan, Typhlonectes sp., is elongate like that of salamanders. Unlike salamander spleens, however, the Typhlonectes spleen exhibits some aggregation of lymphocytes into white pulp-like regions that are less organized than the white pulps of the anuran or mammalian spleen (Manning, 1991). The spleen of this species is also characterized by less abundant innervation than the urodele spleen, both in

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immediate proximity to blood vessels and also in areas removed from blood vessels, as shown by SPG histofluorescence. PGP 9.5 staining of the Typhlonectes spleen revealed occasional individual fibers and fiber bundles in a pattern similar to that seen with histofluorescence (Kinney et al., 1994b). The ontogeny of splenic innervation during the larval life of Xenopus has also received some attention. Clothier et al. (1991) reported changes in splenic innervation during the period shortly before metamorphic climax. Specifically, they described a drop in the levels of splenic NE, as assessed by HPLC and SPG histofluorescence at Nieuwkoop and Faber (1967) larval stage 58, a time when lymphocyte mitogen responsiveness is also significantly decreased (Rollins-Smith et al., 1984). Unfortunately, this reported developmental loss of splenic NE was not accompanied by micrographic documentation of a loss of sympathetic nerve fibers in the spleen. The lack of such documentation becomes important in view of our observations (Kinney, 1996) that the larval Xenopus spleen is innervated earlier than stage 58 (i.e., from stage 54 onward), and that the appearance of innervation is very sensitive to the environmental conditions (e.g., temperature, animal density) under which the larvae are reared. We have also reported that chemical SyX during larval life prior to the appearance of splenic compartmentation does not affect the subsequent development of the demarcation into a red and white pulp (Kinney, 1996), and that early larval thymectomy that renders animals T-cell deficient does not influence the normal development of innervation (Kinney et al., 1993; Rollins-Smith and Cohen, 1995).

B. Sympathetic and Neuroendocrine Regulation of Immunity SyX (using 6-OHDA) of adult Xenopus is associated with a significant increase in the in vitro proliferative response by splenocytes cultured with the mitogens LPS, Con A, and PMA (Kinney, 1995; Kinney and Cohen, 2005). A similar increase in in vitro proliferation was noted when splenocytes from frogs that had been parenterally immunized with keyhole limpet hemocyanin (KLH) 2 days after SyX were cultured with KLH (Kinney, 1995). This SyX-associated enhancement of polyclonal- and antigen-driven proliferation of Xenopus splenocytes is similar to observations in mice (Kruszewska et al., 1995, 1998). Unlike these murine studies, our initial (Kinney, 1995) SyX experiments did not reveal any alteration in the primary serum anti-KLH IgM antibody response (assayed 1–55 days post-immunization) in frogs immunized 2 days after 6-OHDA treatment. This apparent lack of effect

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was recently reproduced (Kinney, unpublished). However, when such primed frogs were again treated with 6-OHDA 47 days after the initial SyX and injected with a second dose of KLH 2 days later, the secondary IgY anti-KLH response was increased relative to the appropriate controls. To the best of our knowledge, the impact of SyX on secondary antibody responses in other species has never been examined. SyX does not appear to affect the time-course of skin allograft rejection (Kinney, 1995; Kinney et al., 1994a), a T-dependent immune process in Xenopus (Manning et al., 1976). Specifically, injection of 6-OHDA 2 days before transplantation, and repeated weekly during the course of the experiment, did not affect skin graft survival, regardless of whether donor and hosts differed by MHC plus minor histocompatibility (H) locus antigens or by minor H-antigens only. There was also no effect of SyX on the accelerated second-set rejection of minor H locus-disparate grafts. In confirmation of these data, Józefowski et al. (1996) reported that chronic in vivo administration of β-adrenergic (propranolol) or muscarinic (atropine) antagonists had no effect on skin allograft survival in R. esculenta and R. temporaria. Morphine, too, was without effect. The picture is slightly different, however, when the fate of xenografts rather than allografts was investigated using R. esculenta as hosts. Specifically, repeated injections of propranolol increased the survival time of xenogeneic skin grafts from R. temporaria and B. bomina, injections of atropine significantly accelerated rejection of skin from B. bomina but not R. temporaria, and injections of morphine had no effects regardless of the donor species used. Interestingly, binding of radiolabeled ligands to muscarinic and adrenergic receptors on PBLs was increased significantly in xenografted animals but not on cells from recipients of allografts. It is also noteworthy that unlike classic T-cell–mediated rejection of allografts, xenograft rejection in anurans is thought to primarily involve innate and antibody-mediated immunity (Horton et al., 1992; Józkowicz., 1995). In Xenopus, immunological tolerance characterizes the alloimmune response of perimetamorphic animals to skin grafts from adult donors that differ from the hosts only by minor H-antigens (DiMarzo and Cohen, 1982). SyX of newly metamorphosed recipients of such grafts had no effect on either the induction or maintenance of this non-deletional form of tolerance (Kinney, 1995). As in so-called higher vertebrates, immunological effects of SyX suggest that cells involved in amphibian immunity express receptors for noradrenergic ligands. Indeed, in the late 1970s, an English group (Hodgson et al., 1978, 1979) reported that antigen-binding splenocytes from several species of SRBC-immunized sala-

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manders (T. cristatus, T. alpestris, Cynops hongkongensis, C. pyrrhogaster, N. viridescens) were decreased following their in vitro exposure to both α- and β-adrenergic receptor (AR) stimulation2. In immunized frogs (R. temporaria, X. laevis, R. esculenta, Bufo bufo), α-AR stimulation decreased, whereas β-AR stimulation increased the number of antigen-binding splenocytes. In these early studies, it was erroneously assumed that all antigen-binding cells were antibody-producing cells. Subsequently, this group (Clothier et al., 1989, 1992) used an ELISA to examine the effects of in vivo NE administration on in vitro antibody responses to T-dependent and T-independent antigens. They reported differential effects based on the thymus dependency of the antigen and the timing of immunization relative to NE administration. Implantation of an NE-containing pellet prior to priming with a Tdependent antigen resulted in increased antibody production, whereas pellet implantation at some (unstated) time after priming was without effect. The influence of NE on antibody responses to T-independent antigens was studied by injecting NE rather than implanting an NE-containing pellet. A single injection of an (unspecified) amount of NE at the time of immunization with TNP-LPS effected a reduction of the splenic anti-TNP antibody response. Unfortunately, the difference in routes of administration of the NE between the two experiments makes a general conclusion difficult. Addition of NE (10−6–10−8 M) to cultures of splenocytes from animals primed in vivo with TNP-LPS also reduced the in vitro anti-TNP antibody response. Interestingly, an increase was seen if the cells were exposed to 10−12 M NE, a finding apparent from the data but not pointed out in the text. A low concentration (10−12– 10−15 M) of the β-AR agonist, isoproterenol, also enhanced in vitro antibody production when it was added on day 7 in culture. In contrast, the α2-agonist, clonidine (10−9–10−15 M) resulted in a reduced response, an effect that was blocked by the α-antagonist, yohimbine. β-AR stimulation with 10−10–10−12 M isoproterenol was also reported to reduce PHA-stimulated T-cell mitogenesis, a finding which we were unable to replicate using physiological or even subpharmacological (i.e., less than 10−4 M) concentrations of isoproterenol (Cohen and Kinney, unpublished). Although Clothier and colleagues (1992) also claimed that immunization resulted in a depletion of NE in the spleen, an unex-

To stimulate α receptors, either the specific α-agonist, phenylephrine, or a combination of epinephrine plus the β–receptor antagonist, timolol, were used; to stimulate β–receptors, either the specific β agonist, isoproterenol, or a combination of epinephrine plus the α–receptor antagonist, thymoxamine, was used. 2

plained decrease in splenic NE was also seen in animals injected only with phosphate buffered saline (PBS). In these same studies, Clothier et al. (1989, 1992) suggested reciprocal interactions between splenic NE content and processes involved in antibody production. Treatment with 6-OHDA reduced primary antihapten antibody responses to TNP-SRBC. When TNP was coupled to a T-independent carrier (LPS or Ficoll), the primary anti-hapten response was increased. Most recently, Haberfeld et al. (1999) reported that adrenoceptor agonists modulate in vitro apoptosis of lymphocytes. Although α-2 and β-2 receptor agonists themselves did not induce apoptosis of Xenopus splenocytes cultured for 4 or 20 hours, they did modulate apoptosis of Xenopus splenocytes that were cultured with a calcium ionophore. More specifically, clonidine (α-2 agonist) enhanced ionophore-induced apoptosis of lymphocytes cultured for 4 but not for 20 hours, whereas isoproterenol (β-2 agonist) decreased apoptosis in 4-hour cultures but enhanced this programmed cell death when lymphocytes were cultured for the longer period. By itself, the synthetic glucocorticoid, DEX (10−4–10−6 M), also induced apoptosis of frog lymphocytes cultured for 4 or 20 hours. Whereas clonidine did not affect cell death caused by this synthetic steroid at either time-point, isoproterenol enhanced apoptosis after 4 but not after 20 hours of co-culture. Haberfeld et al. (1999) suggested that their data might reflect a different apoptotic pathway induced by the ionophore and the synthetic steroid. All the aforementioned experiments dealing with noradrenergic modulation of immunity in amphibians were based on the assumption that the agonists and antagonists used were actually binding to bona fide β-AR receptors. Józefowski and Plytycz (1998) used radiolabeled ligand binding to actually characterize β-AR receptors on splenocytes and activated peritoneal leukocytes from frogs (R. temporaria and B. bufo) and splenocytes from the salamander (Salamandra salamandra). Saturation, competition, and kinetic studies revealed a single site with a similar binding capacity. These investigators estimated that there were about 4,000 and 14,000 receptors per frog splenocyte and peritoneal cell, respectively (the difference probably reflects the larger number of phagocytic adherent cells in the peritoneal exudate) and as many as 183,000 such receptors on salamander splenocytes (a number consistent with their greater cell size). Although leukocytes from amphibians and goldfish (see previous section) all have β-AR receptors with similar affinities for the ligands [3H]CGP-12177 and [3H]DHA, competition experiments (Józefowski and Plytycz, 1998) suggest that there may be taxa (class)-specific differences not only for cells of the amphibians and teleosts

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studied, but also relative to birds and mammals. Radiolabeled ligand-binding studies have also revealed cholinergic muscarinic receptors on elicited peritoneal leukocytes from two species of anurans (Józefowski et al., 1996). As discussed in great detail in the first section, which deals with invertebrates, a series of studies published throughout the 1990s by Ottaviani and coworkers used immunocytochemical procedures to investigate whether neuropeptides, neurohormones, and cytokines are produced by vertebrate and invertebrate cells and tissues involved in adaptive and innate immunity. With respect to the Amphibia, POMC-derived ACTH, β-endorphin, and αmelanocyte–stimulating hormone (α-MSH), as well as molecules that show antigenic cross-reactivity with antibodies directed against mammalian IL-1α, IL-1β, IL-2, IL-6, and TNF-α were revealed in PAS-positive epithelial cells of the thymus of the anuran amphibian R. esculenta. Three groups of PAS-positive epithelial cells were identified in subcapsular cortex, inner cortex, and medulla. The cells containing ACTH-, α-MSHand cytokine-like molecules were distributed in the cortex, whereas those containing β-endorphin–like molecules were found in the medulla and inner cortex. Thymic lymphocytes were always negative for POMCderived peptides and “cytokines.” This does not appear to be the case for peripheral lymphocytes, however, since immunocytochemistry and cytofluometry (Ottaviani et al., 1992c, 1995a) detected POMC (or POMC mRNA) in peripheral blood lymphocytes as well as in phagocytic leukocytes from this same species. Ottaviani et al. (1998) also detected CRH and cortisol-like molecules immunocytochemically in the epithelial cells, interdigitating cells and macrophages, but not lymphocytes, in thymuses from fish, frog, chicken, and rat. These data suggest that throughout vertebrate evolution, the thymus can function as a neuroendocrine organ that is fully capable of displaying characteristics of a “stress response.” In a 1992 study, the presence of immunoreactive ACTH and βendorphin molecules in phagocytic basophils and neutrophils of salamanders (S. salamandra, T. carnifex, Speleomantes imperialis) has been established (Ottaviani et al., 1992d); ACTH increased phagocytic activity of these cells. Morphine-induced inhibition of zymosan-induced peritoneal inflammation consistently recorded in several mouse strains and in fish (salmon and goldfish) was not observed in the anuran amphibians R. temporaria, R. esculenta, and Bombina bombina (Kolaczkowska et al., 2000). Given the number of parameters involved in this type of study, however, a negative result cannot be considered as definitive.

In a more recent study, Chadzinka and Plytycz (2004) pre-incubated leukocytes from mice, goldfish, and frogs with agonists of mu, delta, or kappa opioid receptors (morphine, deltorphine, or U-50,488H, respectively), and then recorded their in vitro migration after culturing them in either medium (M), control serum (S), or serum from zymosan-treated animals (SZAS). In all species, migration of control leukocytes was in the order SZAS > S > M. Also for all species, pre-treatment of leukocytes with a mu or delta opioid receptor agonist, but not a kappa agonist, enhanced their migration when they were cultured with either medium or control serum. When the leukocytes were harvested from animals treated in vivo with zymosan to activate them, the migration of mouse and fish leukocytes, but not frog leukocytes, was inhibited. This inhibition could be reversed by specific antagonists of mu and delta opioid receptors (CTOP and naltrindole, respectively). According to the authors, these results imply that the final effects of opioids on cell migration are “dependent on a species-specific balance between up- and down-regulation of leukocyte migration that results from an interplay between receptors for opioids and chemotactic factors.”

C. Seasonal Influences on Immunity The amphibian immune system, like that of reptiles (see next major section), is influenced by seasonal variations (Plytycz and Seljelid, 1996, 1997). Several examples are worth noting: (1) The inguinal lymphoid bodies of toads undergo seasonal cyclic changes in morphology (Plytycz and Szarski, 1987). (2) Gutassociated lymphoid tissues of some anuran species also undergo seasonal changes (Saad and Plytycz, 1994). For example, Wojtowicz and Plytycz (1997) found that the number of lymphoid nodules in the gut was negligible in field-collected B. bufo that were emerging from hibernation. This number increased in spring, reached its highest level in the summer, and declined in autumn. (3) The magnitude of the antiSRBC response and the number of splenic lymphocytes in the toad B. regularis are high in spring, low in summer, high in autumn, and low again in winter (Hussein et al., 1984; Saad and Ali, 1992). (4) The percentage of lymphocytes in peripheral blood and hematopoiesis in the perihepatic subcapsular tissue is strikingly reduced in winter relative to summer in two species of newts: T. carnifex and T. alpestris (Barni et al., 1993). (5) The thymus of the frog R. temporaria undergoes cyclical changes with maximal development occurring during the summer and involution in the winter (Bigaj and Plytycz, 1984; Miodonski et al., 1996; Plytycz, et al., 1991).

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Surprisingly, only a few investigators have examined seasonal effects on the amphibian immune system by modeling the normal winter hibernation of amphibians in the laboratory. In an early study of this kind, Green (Donnelly) and Cohen (1977) found that complement CH50 titers of Rana pipiens kept at 4°C for several months in a hibernaculum decreased to undetectable levels. This finding was confirmed and extended to other components of the immune system by Maniero and Carey (1997). Cooper et al. (1992) performed a more comprehensive study of hibernationassociated changes in the anuran immune system. Leopard frogs (acclimated to 22°C) were placed in a hibernaculum (in the dark) in late autumn. Animals, sampled at days 45 and 90 after initiation of hibernation, underwent a progressive 9- to 10-fold loss of leukocytes in the blood, thymus, spleen, jugular bodies and, to a much lesser extent (1- to 2-fold), in the hematopoietic bone marrow. These changes were reflected by marked aplastic changes in these organs. By the end of the experimental hibernation period (day 135), however, numbers of leukocytes in these organs had begun to increase, even though the photoperiod was unchanged and the temperature remained at 4°C. At day 30 after the frogs had been returned to room temperature, cell numbers had returned to, or were greater than, pre-hibernation values, and the architecture of the organs again appeared normal. Despite the clear importance of temperature effects rather than season on graft rejection discussed below, at least in the case of the R. temporaria thymus, manipulation of the environmental temperature during the period of winter involution and summer growth did not override the endogenous seasonal rhythms (Bigaj and Plytycz, 1984; Zapata et al., 1992). These morphological and functional changes may result from seasonal changes in levels of circulating hormones such as corticosteroids, sex steroids, GH, PRL, and/or thyroxine (Mosconi et al., 1994; Plytycz et al., 1993; Saad and Ali, 1992; Zapata et al., 1992). Indeed, nearly 60 years ago, Holzapfel (1937) reported changes in the gross and histological changes in the adrenals, pituitary, gonads, pancreas, and thyroid during winter hibernation. This yearly pattern of thymic involution and loss of lymphocytes followed by expansion of thymocytes and peripheral lymphocytes in the spring may offer a unique model for studying repertoire development and self-tolerance in the context of neuroendocrine regulation. Regardless of whether cold and/or neuroendocrine changes are responsible for the striking winter-associated depression of lymphoid tissues and specific immune responses (Plytycz and Seljelid, 1997), immunologists still have to contend with the issue of

how such immunosuppressed amphibians survive during such periods. Several possibilities come to mind. First, at least some animals might not survive. Second, at least for amphibians living in temperate climates, low temperature may also suppress the growth of potential pathogens. Third, immunological memory (i.e., memory cells) to pathogens to which amphibians were exposed during the spring, summer, and fall may persist during the winter (Cooper et al., 1992). Finally, components of the non-specific innate immune system may not be depressed during the winter or may recover more rapidly after hibernation than specific adaptive immune responses. With respect to this last possibility, Maniero and Carey (1997) noted that complement activity increased within 2 days after R. pipiens were brought from artificial hibernation to room temperature. Moreover, complement levels continued to increase until, in some instances, they were significantly greater than those of controls held at room temperature. Similarly, Plytycz and Józkowicz (1994) reported that endocytosis of peritoneal macrophages from fish as well as anuran and urodele amphibians was effective in vitro over a wide temperature range and was actually enhanced in cells obtained from cold-acclimated animals. Although the aforementioned studies reveal important seasonal (endocrine) effects on parameters of the amphibian immune system, it is well known that immune responses in this vertebrate class are, like those in other ectotherms, regulated by temperature. In this regard, it is noteworthy that the timing of skin graft survival by anurans grafted during different seasons appears reflective of temperature rather than neuroendocrine changes (Józkowicz and Plytycz, 1998; Saad and Plytycz, 1994). In the Józkowicz and Plytycz (1998) study, the effects of ambient temperature (22°C vs. 10°C) and season (summer vs. winter) on anuran skin allograft rejection were examined in frogs (R. temporaria and R. esculenta) and toads (B. bufo, Bombina variegate, and B. bombina). Mean graft survival times were significantly prolonged at the low temperature regardless of the season. Interestingly, R. esculenta was the most sensitive to the temperature effect, whereas B. bufo was relatively resistant. Rejection of second-set grafts in R. esculenta was accelerated at both 22°C and 10°C, but second-set grafts were less temperature-sensitive than the initial sensitizing ones. In both summer and in winter, R. esculenta rejected allografts promptly at 22°C but slowly at 10°C. In both seasons, B. variegate kept at 22°C rejected allografts chronically. This indicates that amphibian transplantation immunity depends on the donor-host genetic disparity and ambient temperature but is independent of season.

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D. Glucocorticoid Effects on Amphibian Immunity Exogenous administration of a single injection of DEX in Rana perezi caused thymic involution and massive destruction of cortical lymphocytes, diminution in white pulp, and lymphocyte redistribution to the bone marrow from blood and spleen (Garrido et al., 1987). The nature and extent of these changes depended on whether DEX was administered in autumn or winter (Garrido et al., 1989). Pharmacologic concentrations of hydrocortisone suppress antibody responses in Xenopus and cause massive cell death in vivo and in vitro in the thymuses and jugular bodies, but not the spleen, of R. temporaria (Plytycz et al., 1993). In the Plytycz et al. (1993) study, the number of viable cells in the thymus and jugular body returned to normal within 1 week after a single injection of hydrocortisone3. Rollins-Smith and Blair (1993) also reported that physiological (10−5 M–10−9 M) rather than pharmacological concentrations of corticosterone inhibited PHA-induced proliferation of adult Xenopus splenocytes.

E. Neuroendocrine-immune System Interactions During Metamorphosis Thyroxine (T4) and triiodothyronine (T3) play a major role in driving amphibian metamorphosis (Kikuyama et al., 1993; Leloup and Buscaglia, 1977; White and Nicoll, 1981). Circulating levels of these hormones are low at pre-metamorphic stages, increase during pro-metamorphosis, peak during climax, and decline at the end of metamorphosis. Metamorphosis is also accompanied by increases in circulating levels of corticosteroids (Jaudet and Hatey, 1984; Kikuyama et al., 1986), PRL, and GH (Buckbinder and Brown, 1993; Clemons and Nicoll, 1977; White and Nicoll, 1981). Each of these hormones has immunomodulatory effects in mammalian species (Madden and Felten, 1995), and manipulation of at least some of these hormones also influences immune function in amphibians (Rollins-Smith, 1998; Rollins-Smith and Cohen, 1995, 2003). Thyroid hormones: Since metamorphosis is strictly regulated by the availability of thyroid hormones, manipulating their levels makes it possible to inhibit or accelerate metamorphosis and thereby determine the extent to which development of an adult parameter of immunity in frogs requires a normal metamorphic transition. Xenopus larvae reared in the water contain3 It should also be noted that thymic and splenic morphology may be affected by laboratory and/or husbandry conditions regardless of season (Dulak and Plytycz, 1989).

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ing the goitrogen, sodium perchlorate, to inhibit iodine uptake by thyroid follicle cells (Capen, 1994) undergo arrested development and do not metamorphose. Such blocked animals reveal that development of some structural and functional components of the adult Xenopus immune system are dependent on thyroid hormones and a normal metamorphic transition, whereas others are metamorphosis-independent. Specifically, perchlorate-blocked larvae lack the adult pattern of antibody responses to a specific hapten (Hsu and Du Pasquier, 1984), and adult-type MHC class II+ T-lymphocytes do not appear in the periphery (RollinsSmith and Blair, 1990b). Moreover, the thymus does not assume an adult-type morphology (Clothier and Balls, 1985); lymphocytes in the thymus and spleen do not achieve the expanded cell numbers characteristic of post-metamorphic adults (Rollins-Smith and Blair, 1990a, 1990b); adult-type skin allograft rejection does not replace the typical tolerogenic responses of larvae (Cohen and Crosby, unpublished); and, as pointed out previously, splenic innervation and compartmentation of the spleen into a clearly defined red and white pulp is dramatically delayed (Kinney, 1996). Immune system changes that occur in perchlorateblocked tadpoles (i.e., are independent of thyroxine levels) include the expression of MHC class I antigens (Rollins-Smith, 1998; Rollins-Smith et al., 1994), the immigration and expansion of T-cell precursors in the thymus (Rollins-Smith et al., 1992), and the development of high titer IgY antibody production (Hsu and Du Pasquier, 1984). Just how thyroid hormones are related to the maturation of some facets of the Xenopus immune system is unknown. Thyroxine appears to drive the terminal maturation of larval erythrocytes (Galton and St. Germain, 1985) and the expansion of a separate adult erythrocyte population (Flajnik and Du Pasquier, 1988). However, since thyroid hormones have no deleterious effects on lymphocyte viability and inconsistent effects on proliferation (Rollins-Smith and Blair, 1993), the direct action of thyroid hormones does not appear to be responsible for the dramatic loss of larval lymphocytes at metamorphosis. In this regard, it is noteworthy that thyroid hormone deprivation initiated after metamorphosis does not appear to affect lymphocyte populations of post-metamorphic animals or the ability of T-cells from such animals to respond to mitogens (Rollins-Smith et al., 1993). Corticosteroid hormones (CH): In short-term in vitro studies, both adult and larval lymphocytes are killed by concentrations of corticosterone that are comparable to those occurring at metamorphosis (RollinsSmith, 1998; Rollins-Smith and Blair, 1993; Rollins-Smith et al., 1997a). Corticosterone and aldosterone signifi-

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cantly inhibit PHA-induced proliferation of larval and adult spleen cells (Marx et al., 1987; Rollins-Smith and Blair, 1993; Rollins-Smith and Cohen, 1995), and corticosterone induces apoptotic death of larval splenocytes at concentrations as low as 1–10 nM (Rollins-Smith, 1998; Rollins-Smith et al., 1997a). Adult lymphocytes may be protected in vivo from the destructive effects of corticosterone since there is less total corticosterone in the circulation after metamorphosis, and most is in a bound state (Jolivet-Jaudet and Leloup-Hatey, 1986). Since aldosterone levels do not appear to be affected by specific serum-binding factors, the observed elevated levels of aldosterone in plasma of metamorphosing and adult frogs reflect the level of freely available aldosterone. Because larval lymphocyte function is affected by aldosterone at these physiologically relevant concentrations (Rollins-Smith and Cohen, 1995), aldosterone could play a role in the loss of larval lymphocytes. Based on in vitro sensitivities of Xenopus lymphocytes to corticosterone and aldosterone, it is reasonable to propose that the naturally increasing concentrations of these hormones during metamorphosis are causally related to the death of significant numbers of larval splenic lymphocytes (Rollins-Smith and Cohen, 1995). This hypothesis is supported by evidence that the in vitro inhibition of PHA-stimulated splenocyte proliferation by CH can be blocked by the CH-receptor antagonist, RU486 (Rollins-Smith, 1998; Rollins-Smith and Cohen, 1995; Rollins-Smith et al., 1997a), and that in vivo treatment of stage 57–58 larvae with RU486 reveals a dose-dependent inhibition of the loss of splenocytes that normally occurs during metamorphosis (Rollins-Smith, 1998). Thus, corticosterone and/or aldosterone appear to be important regulators of peripheral splenocyte populations at metamorphosis in Xenopus. The putative hormonally induced loss of splenic lymphocytes during metamorphosis is quite important, for if these immunocompetent larval cells were not eliminated, they could potentially be activated by newly emerging adult-specific self-molecules. The role of increasing levels of CH on the viability and development of thymocytes is more difficult to evaluate. Viability of larval thymocytes cultured in 1– 10 μg/ml corticosterone is significantly reduced (Rollins-Smith and Blair, 1993); this reduction can be inhibited by RU486 in vitro (Rollins-Smith, 1998; Rollins-Smith et al., 1997a). However, other studies have reported significant apoptosis of cultured thymocytes from all larval and metamorphic that does not appear to be increased by culture with DEX (Ruben et al., 1994). Regardless of their outcome, these in vitro

studies are difficult to interpret because thymocyte development is critically dependent on influences of the stromal/epithelial microenvironmental. For example, in mammalian systems, thymic epithelial components can synthesize corticosteroids (Vacchio et al., 1994). Thus, thymocytes may constantly receive local corticosteroid signals that influence their viability. Growth hormone (GH) and prolactin (PRL): Although a role for GH and PRL in mammalian immunity is now well established (Dorshkind and Horseman, 2000; Kelly et al., 1992), very little is known about the involvement of these hormones in the development and function of cells of the amphibian immune system. Hypophysectomized tadpoles continue to grow, can usually reject skin or organ allografts differing by presumed or defined MHC antigens (Rollins and Cohen, 1980; Rollins-Smith and Cohen, 1982; Maéno, and Katagiri, 1984) and, like control tadpoles, do not reject minor H-locus disparate grafts or grafts expressing organ-specific antigens (Maéno and Katagiri, 1984; Rollins-Smith and Cohen, 1982). To study the possible role of pituitary-derived hormones in the development of the amphibian immune system, Rollins-Smith and colleagues (2000) hypophysectomized (hypx) early larval stage Xenopus and examined the pattern of immigration of host-derived T-cell precursors into an implanted thymus using a ploidy marker (Rollins-Smith et al., 1992; Turpen and Smith, 1989). There were significantly decreased numbers of lymphocytes in the spleen and thymuses of hypx frogs in comparison with intact controls. However, no apparent delay or inhibition of the repopulation of the triploid donor thymus implanted into a diploid tadpole host was observed (Rollins-Smith et al., 2000). These results suggest that during the larval period, pituitary hormones are not required for either maintenance of the extra-thymic stem cell compartment, for maintenance of the attractiveness of the thymus for precursors, or for the process of thymocyte immigration. Analysis of cell division in the developing thymus populations revealed an inhibition of proliferation in hypx hosts. Thus, pituitary hormones appear to be required for the normal expansion of Tcell precursors after they colonize the thymus. In unpublished studies, we and Rollins-Smith have independently observed that chicken anti-PRL antibodies suppress proliferation of mitogen-stimulated (LPS, PHA, Con A) larval and adult splenic lymphocytes. Thus, PRL may be an essential lymphocyte growth factor that developed early in vertebrate evolution (see discussion in Kelly et al., 1992). To produce sufficient Xenopus PRL to investigate the role of PRL in normal lymphocyte function, Rollins-Smith and

Prologue

colleagues (personal communication) have inserted cDNA corresponding to the known sequence for a Xenopus PRL into a bacterial expression vector. The resulting expression products (single peptides of about 26 kDa (including 21 additional amino acids on the amino terminal end) stain with anti-PRL antibodies by immunoblotting. These expressed proteins can enhance PHA-induced lymphocyte proliferation driven by the T-cell mitogen, PHA (Rollins Smith and Reinhart, personal communication). Hypx frogs exhibit a larval-type pattern of expression of MHC class I and class II antigens (Rollins-Smith et al., 1997b). That is, surface expression of MHC class I on spleen cells and erythrocytes is low in comparison with age-matched intact post-metamorphic controls at 1 year of age. The monoclonal anti-T cell antibody XT1 (Nagata, 1988) recognizes determinants that are predominantly on MHC class II negative cells in hypx frogs, whereas the XT-1+ population in age-matched post-metamorphic adults is class II positive. These experiments suggest that the pituitary-derived hormones that orchestrate normal metamorphosis are required for a post-metamorphic T-cell expansion and for development of the adult pattern of MHC expression on cells of the immune system.

F. The Impact of Environmental Stressors on Anti-microbial Immunity and Its Implications for the Worldwide Decline of Amphibians An ecological problem area that is being studied deliberately from a psychoneuroimmunological perspective is the worldwide decline of populations and species of frogs and salamanders (Collins et al., 2005). One cause of these declines and extinctions is clearly the destruction of habitat where amphibians breed and grow. A second cause stems from the introduction of superior predators or competitors that displace native species. At face value, these two explanations would not appear to involve psychoneuroimmunological considerations although they do involve what might be considered environmental stressors. However, we also know that two pathogens, a Chytrid fungus (Batrachochytrium dendrobatidis) and iridoviruses (ranavirus, Ambystoma tigrinum virus), are causally involved (Carey et al., 1999; Rollins-Smith et al., 2002; Woodhams et al., 2006) in the decline; so too are anthropogenic environmental changes or “ecological stressors” that include toxic chemicals (e.g., agricultural pesticides), UV radiation, and/or global climate change. Some pesticides are immunomodulatory in amphibians. For example, immunotoxicological studies have revealed the immunosuppressive capacity of various

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single and combinations of pesticides (e.g., atrazine, metribuzine, endosulfan, lindane, aldicarb, DDT, malathione, and dieldrin) in Xenopus and Rana (Christin et al., 2004; Gilberton et al., 2003). These agents could be immunotoxic by acting directly on cells of the immune system. Alternatively, and from a psychoneuroimmunological perspective, they could be immunosuppressive owing to their action on the neuroendocrine system that, in turn, negatively signals the immune system and increases susceptibility to fungal and/or viral pathogenicity, eventuating in a species decline (Rollins-Smith and Cohen, 2003). Evidence for this latter possibility is scanty since studies of immunity to chytrids and ranaviruses are only in their infancy. Nevertheless, we do know that naturally produced anti-microbial skin peptides of frogs are capable of inhibiting growth of zoospores and mature fungal cells (Rollins-Smith et al., 2002). Secretion of these peptides from amphibian skin glands is regulated by the sympathetic nervous system (i.e., EPI and NE) (Benson and Hadley, 1969; Dockray and Hopkins, 1975; Holmes and Balls, 1978). Could environmental “stressors” (e.g., pesticides) be affecting peptide production/ secretion via their effects on the sympathetics? Although this possibility has yet to be tested in a laboratory environment, it is known that chlorotriazine herbicides decrease the intracellular content and release of DA and NE from PC12 cells, a cell line with neuronal characteristics (Das et al., 2000). Also, a dithiocarbamate compound similar to agents used as pesticides inhibits NE synthesis by suppressing the activity of dopamine-beta-hydroxylase (Goldman et al., 1994). In other words, this possibility may not be so far-fetched. With respect to iridoviruses, we now know that in addition to anti-microbial peptides (Chinchar et al., 2001) and NK cells (Horton et al., 2003), Xenopus uses CD8 T-cells, NK/T-cells, and antibody to deal with laboratory-induced infections with frog virus 3 (FV3) (Gantress et al., 2003; Robert et al., 2005). We also know that x-irradiation increases mortality associated with FV3 infection (Robert et al., 2005). Metamorphosing frogs have a naturally downregulated immune system4, and larval and metamorphosing frogs are much more susceptible to infection with this iridovirus than are adults (Robert et al., 2005). Finally, we know that in amphibians glucocorticoids are highly immunomodulatory, especially during metamorphosis (RollinsSmith et al., 1997a). In this regard, xenobiotic chemicals in the environment can induce increases in circulating 4 It is thought that this downregulation evolved to protect animals from immune responses to adult-specific antigens that are emerging during the metamorphic transition from immunocompetent larva to immunocompetent adult.

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corticosteroids (Gendron et al., 1997; Hopkins et al., 1999). Although we’ve not directly tested this possibility in Xenopus, we do know that elevated levels of corticosteroids have been detected in toads (Bufo terrestris) collected from areas polluted by coal ash (Hopkins et al., 1999) and in mudpuppies (Necturus maculosus) that have been exposed to organochlorine (Gendron et al., 1997) in a setting in which maximal concentrations approximate those associated with the natural glucocorticoid-associated immunosuppression in Xenopus that occurs during metamorphosis (Barker et al., 1997; Rollins-Smith and Blair, 1993).

V. NEURAL-IMMUNE INTERACTIONS IN REPTILES Our very limited knowledge of the interactions of the neuroendocrine and immune systems in reptiles is derived primarily from the following three lines of research by Egyptian and Spanish groups of investigators: (1) lymphoid tissue innervation, (2) seasonal influences on several immunological parameters, and (3) effects of steroids on immunity. Much of what has been accomplished following the last two approaches is the focus of reviews by Zapata et al. (1992) and Saad and Plytycz (1994).

A. Innervation of Lymphoid Organs Saad (1993) described, in turtles, the presence of nerve endings in close proximity to lymphocytes in the spleen, as well as changes in brain catecholamine levels during the course of an immune response. Brain EPI levels were increased 10 hours after immunization with SRBC; they then decreased to below-control levels at 48 hours, after which time they returned to baseline. DA was elevated at 4 hours, decreased at 2–4 days, and then returned to normal values by day 10 (Saad, 1993). Clearly, more details are needed about innervation of lymphoid organs in representatives of various reptilian orders. It is also unknown whether the seasonal waxing and waning of immune reactivity in reptiles described below is associated with seasonal changes in the pattern of innervation of lymphoid tissues.

B. Seasonal Influences on Immunity In the snake (Natrix natrix), a seasonal cyclicity in circulating leukocytes has been reported, with females showing lymphocytopenia and neutrophilic granulocytosis during the time of egg laying, and males also showing similar, but less marked, white cell count cycling (Wojtaszek, 1992). Thymic involution in the

turtle, Mauremys caspica, occurs in April–May, as do diminished antibody responses and delayed graft rejection (Zapata and Leceta, 1986; Zapata et al., 1992). Other investigators (Muñoz and De La Fuente, 2001, 2004), however, reported for the same species that spring is associated with a higher splenic mitogen response and summer with enhanced chemotaxis and cytotoxicity; spring-summer is associated with a higher proportion of splenic and thymic lymphocytes as well as better thymocyte proliferation. Since there is disagreement in these results from the same chelonian species, resolution of the differences awaits further study, as does an understanding of the mechanisms responsible for such seasonal changes. In the lizards Eumeces schneideri and Chalcides ocellatus (Saad et al., 1993), the numbers of lymphocytes in the peripheral blood, bone marrow, thymus, and spleen are lower during the fall and winter than in spring and summer. There also appears to be a trend toward increased humoral immunity (both in vivo and in vitro) in the summer relative to winter (Saad et al., 1993; Zapata et al., 1992). Lizards have also been reported to show seasonal variability in their ability to reject skin allografts in that animals grafted in winter months did not reject until the warmer spring months (Afifi et al., 1993). These winter-spring responses, however, were still slower than those seen in warmer summer months. Reptiles are ectotherms, and high and low environmental temperatures are immunomodulatory in such species (Clem et al., 1984, 1991). Therefore, interpretation of data describing seasonal effects on the immune system is confounded by the possibility that variation in ambient temperature, rather than (or in addition to) neuroendocrine changes associated with seasonality and altered photoperiod, is the important variable. The fact that antibody titers in the turtle Mauremys capsica are higher in autumn than summer in turtles kept at 27°C and at a constant photoperiod (Zapata and Laceta, 1986) argues against this possible confound. Comparable data presented below for a ranid frog (Bigaj and Plytycz, 1984) and for fish (Nakanishi, 1986; Yamaguchi et al., 1981) also support some sort of seasonal cue (e.g., photoperiod?) other than temperature as an important component of this cyclicity of immune responses and lymphoid system changes. The impact of different photoperiods at a constant temperature on immune system parameters still needs to be determined at all seasons.

C. Effects of Steroids on Immunity Some of the aforementioned seasonal changes may be related to seasonal changes in circulating levels of

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corticosteroids and/or gonadal steroids. In this regard, some correlational data on seasonal levels of testosterone, glucocorticoids, and changes in the reptilian immune system are worth highlighting (Zapata et al., 1982; Zapata et al., 1992). Based on observations of a sexual dimorphism in immunity in a snake (Saad and Shoukrey, 1988) and of an increase in testosterone levels in the blood of Chalcides ocellatus during the spring, Saad et al. (1990, 1992) investigated the impact of adult orchiectomy and testosterone treatment on antibody production in Chalcides. Five injections of testosterone propionate (each separated by 5 days) during the summer (animals maintained at ambient and, therefore, fluctuating, temperatures), followed by immunization with SRBC, did not affect in vitro PFC responses but were associated with a significantly diminished serum antibody at some, but not all times, post-immunization. Orchiectomy during the spring effected a marked and long-lived reduction in serum levels of testosterone and was associated with an increased primary PFC and serum antibody response to SRBC. Although antibody responses in orchiectomized lizards that received a course of testosterone did not differ from responses of orchiectomized animals, this reconstitution experiment would have been strengthened had the investigators confirmed the efficacy of the orchiectomy itself by including a group of unmanipulated animals in their protocol. A single injection of testosterone proprionate also resulted in thymic involution and depletion of splenic and PBLs in the turtle Mauremys caspica (Saad et al., 1991a, 1991b; Varas et al., 1992). The immunocompetence handicap hypothesis (ICH) was developed to explain the maintenance of exaggerated male traits under sexual selection in birds. The hypothesis requires that such traits (e.g., bright plumage in birds) (1) are preferred by females; (2) are promoted by testosterone (T); and (3) are maintained as “honest signals” by the presumed immunosuppressive action of testosterone (i.e., only a high-quality male can afford to be so immunosuppressed) (Fostad and Karter, 1992). As might be expected given their close phylogenetic apposition to birds, reptiles have also been the subject of speculations concerning the ICH. Oppliger et al. (2004) found decreased skin response to PHA in testosterone-implanted male lizards (Podarcis muralis) but did not find any effect on the presence of blood or ectoparasites, a common functional measure of immunity in both birds and reptiles. Berger et al. (2005) examined Galapagos marine iguanas during the breeding season and found no overall differences between dominant and subordinate males, but that restraint stress or corticosteroid injection elicited a depressed PHA response. The authors

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argue that this is evidence that corticosteroids, not gonadal steroids, are key immunosuppressive agents during the breeding season and that the ICH needs to be revised to account for this. In an earlier series of studies, the Egyptian group studied the histological, cytological, and immunological consequences of exogenously administered pharmacological concentrations of hydrocortisone acetate in Chalcides during the summer. As would be predicted from studies with mammals, exogenous hydrocortisone acetate caused profound, but seemingly selective, loss of both T- and B-lymphocytes in the thymus, peripheral blood, and spleen. Physiological concentrations of 10−3 M hydrocortisone also led to in vitro destruction of thymocytes, PBLs and, to a lesser extent, splenic lymphocytes (Saad and Bassiouni, 1991; Saad et al., 1984a, 1984b). Long-term corticosteroid administration has also been associated with decreases in lymphocytes and heterophils in juvenile alligators (Morici et al., 1997). More recently, exogenous glucocorticoids have been associated with apoptosis of thymocytes in the wall lizard, Hemidactylus flaviviridis (Hareramadas et al., 2004). In Chalcides, a single injection of 1.0 mg/g body weight of hydrocortisone acetate “caused” a 3-week-long physiological increase in serum corticosterone (peak 2 × 10−7 M) and cortisol (peak 1 × 10−6 M). This was associated with a suppression of the primary antibody response to rat RBC in vivo and in vitro, a delay in allograft rejection, and a reduction in proliferation in mixed leukocyte culture (Saad et al., 1986). An obvious confound in this study is the extent to which the impaired immune response reflects the exogenously administered steroid rather than, or in addition to, endogenously synthesized glucocorticoid. Also, an increase in endogenous steroids in response to exogenous steroids is puzzling if the HPA axis of turtles, like that of mammals, has a characteristic feedback loop. In a somewhat more naturalistic study, environmental stress (density, presence of new conspecifics, altered feeding protocol) was associated with an increase in blood parasite infections in the lizard Lacerta vivipara, a finding which could have implications for understanding the effects of habitat loss (Oppliger et al., 1998) on the survival of lizard populations. Infection with blood-borne parasites has also been associated with a drop in corticosteroid levels (Hanley and Stamps, 2002). An important caveat in interpreting studies in which a correlation is being made between seasonally associated alterations in immune function and in levels of endogenous hormones is whether levels of a given hormone have been determined sequentially during a 24-hour period in each season. In instances of the

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single time-of-day measurements that appear typical of the immunological studies in reptiles, amphibians, and teleosts reviewed in this chapter, a possible seasonal phase shift in the diurnal rhythm associated with a given hormone (Spieler, 1979) could significantly alter interpretation of the published data regarding mechanisms responsible for seasonal changes in immune function. Regardless of the underlying mechanisms, however, seasonal and endocrine-associated changes in lymphoid tissue architecture and cellularity remain real events. In contrast to mammals (and to a lesser extent, amphibians and teleosts), the role of catecholamines in immunity in reptiles has received minimal attention. One recent study, however, indicates that in the lizard Hemidactylus flaviviridis, high doses of catecholamines (as well as 1-hour restraint stress) inhibited phagocytic activity of splenic macrophages, whereas very low concentrations enhanced the response (Roi and Rai, 2004). Clearly, much remains to be examined in this area.

VI. NEURAL-IMMUNE SYSTEM INTERACTIONS IN BIRDS The study of neural-immune system interactions in birds, like the study of avian immunity itself (Sharma, 1991), has lagged behind comparable studies in mammals. Nevertheless, several lines of evidence (Marsh and Scanes, 1994) are totally consistent with neuroendocrine regulation of immune processes in the few commercially important avian species that have been studied.

A. Innervation of Lymphoid Organs and Immunomodulation by Neuropeptides The earliest indication that the bursa of Fabricius, the primary lymphoid organ involved in Blymphocyte ontogeny in birds, is innervated was provided by observations of Pintea et al. (1967), Cordier (1969), and Inue (1971). More recently, Zentel et al. (1991) described peptidergic innervation of the chicken bursa. Specifically, immunoreactivity for tachykinins, calcitonin gene-related peptide (CGRP), and NPY was described in proximity to bursal vasculature and specific cell populations, primarily B-cells. Even though some T-lymphocytes are present in the bursa, these investigators did not find peptidergic fibers near labeled T-cells. Although Zentel and Weihe (1991) were unable to directly demonstrate sympathetic innervation of the bursa, they associated the NPY staining with sympathetic innervation since, at least in

mammals, NPY and NE are co-localized in sympathetic fibers (Bellinger et al., 1990). More recently, Ciriaco et al. (1995) investigated innervation of the pigeon bursa of Fabricius from hatching to 4 months of age, by histochemically staining for noradrenergic- and acetylcholinesterase (AChE)-reactive nerve fibers. NA nerve fibers were largely perivascular, but a small number were also seen in interfollicular connective tissue. In bursas from 30–75-day-old animals, fibers were occasionally observed beneath the bursal epithelium, in the cortex, and at the cortico-medullary border, but not the medullary portion, of the lymphoid follicles. Although no age-associated changes in the density of NA innervation were observed in the vascular areas, there was a progressive increase in the density of non-vascular NA nerve fibers for the first 30 days of life, no variation from 30–75 days, and an apparent regional increase during bursal involution. The distribution and density of perivascular AChE-positive nerve fibers were similar to that for NA fibers. AChE-reactive fibers were also observed in the capsule of the bursa and in the interfollicular septa, and AChE-reactive dendritic-like cells and cell processes were seen subepithelially, in the cortex, and at the corticomedullary border of the lymphoid follicles. No age-dependent changes in AChE staining were noted. Finally, in 1999, Franchini and Ottaviani described the presence of immunoreactive pro-opiomelanocortin derivatives (ACTH, α-MSH, β-endorphin) in both the thymus and bursa of chickens. In the thymus, immunoreactive staining was associated with the thymic epithelium and a few interdigitating cells. In the bursa, staining was associated with cells of the follicle. Immunoreactivity was seen as early as day 4 and increased with age. Given the change with development and the pattern of association, the authors suggest a physiological role in the development and/or involution of the organs. Suggestive evidence for sympathetic modulation of immunity in chickens has been provided by BrownBorg and Edens (1992). These investigators administered 6-OHDA to chicks in ovo on day 18 post-fertilization, immunized the birds with SRBC 25 days post-hatching, and observed a small but statistically significant increase in hemagglutinating antibody titers (relative to vehicle-injected controls) at 6, 8, and 10 days post-immunization. No change, however, was observed in a cutaneous cell-mediated response to PHA measured at the same times as anti-SRBC antibody responses. This suggests that their manipulation of the sympathetic nervous system may differentially affect different arms of the immune system. SyX of adult mice causes an increase in glucocorticoids (Callahan et al., 1998). Although this does not appear

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to be responsible for the SyX-associated immune changes seen in such mice (Kruszewska et al., 1998), this may not be the case in birds. There is also evidence that innervation of primary lymphoid organs plays a role in development of avian immune responses (Franchini et al., 1995). Administration of 6-OHDA to chicks in ovo was associated with reduced expression of IL-2 mRNA and reduced in vitro proliferative response to pokeweed mitogen, as well as alterations in lymphocyte expression of β-AR mRNA expression and response to NE administration (Motobu et al., 2003). That young chickens exhibit a corticosterone-unresponsive phase during the first 2–3 weeks post-hatching (Guellati et al., 1991) also needs to be considered in the context of differential effects of SyX on cellular and humoral immunity described by Brown-Borg and Edens (1992). In rodents, neonatal SyX results in a profound and long-lasting drop in splenic NE (Ackerman et al., 1991a) that is accompanied by increased antibody response to T-dependent antigens (Ackerman et al., 1991a; Besedovsky et al., 1979) and changes in a variety of other in vivo and in vitro immune system parameters (Ackerman et al., 1991b; Chelmicka-Schorr et al., 1992a, 1992b; Madden et al., 1993). Although in chickens, the injection of 6-OHDA in ovo causes central as well as peripheral denervation (Spooner and Winters, 1966), Brown-Borg and Edens (1992) did not directly demonstrate denervation in their 6-OHDA study. Indeed, they could not detect NE (the principal sympathetic neurotransmitter in the chick) (Rome and Bell, 1983) in the thymus or bursa at the time of hatching, and NE was detected only in the brain stem and spleens of fewer than 30% of the control and experimental chicks examined. Moreover, similar levels of EPI and 5HT were found in the brain stem, thymus, bursa, and spleen of both control and 6-OHDA–treated animals. Although the levels of splenic NE increased during the course of the antibody response, at the time of each antibody titration, no differences among levels of splenic NE, DA, and 5HT in controls and experimental birds were seen. The significance of their observation that the peak splenic NE and DA response in 6-OHDA– treated chicks lagged a few days behind that of vehicleinjected controls is unclear. Further studies are needed to clarify the developmental time course of innervation of lymphoid tissues in chicks as well as to provide definitive HPLC, immunohistochemical, and immunocytochemical evidence of peripheral denervation by 6-OHDA treatment in ovo. In chickens, suppression of a PHA-induced cellmediated immune reaction in the wattle (Kelsius et al., 1977) could be effected by a single injection of NE, 5HT, and DA (Lukas et al., 1987) or by continuous DA

administration alone (McCorkle and Taylor, 1994). In vivo DA and 5HT administration also suppressed PFC responses (McCorkle et al., 1986); enhanced migration of PBLs was noted following in vitro exposure of chicken leukocytes to each of these monoamines (McCorkle and Taylor, 1994; McCorkle et al., 1990). In a somewhat more recent study, the same group (Gray et al., 1991) found that parenteral 5-HT and DA increased and decreased plaque formation, respectively; only the DAassociated effect could be mimicked by in vitro administration. No effect of neurotransmitters on IgG PFC formation was seen in either case, but in a related study, Denno et al. (1994) demonstrated a suppression of IgG PFCs by in vivo and in vitro administration of EPI, but an enhancement of IgM PFCs in response to in vitro EPI. In vitro administration of catecholamines has also been demonstrated to alter macrophage functions. For example, an increase in phagocytosis and the percentage of cells expressing FcR was seen following incubation with NE, EPI, or dopamine for 1 hour, whereas a longer exposure (3 hours) to DA only reduced SRBC phagocytosis (Ali et al., 1994). Taken together, these studies indicate a substantial, albeit not easily interpretable, influence on the sympathetic nervous system on immunity in birds.

B. Neuroendocrine Regulation of Immunity In addition to modulation of immunity by peripheral nerves and neurotransmitters, hormones (e.g., corticosterone, thyroid hormone, growth hormone, PRL, and gonadal steroids) all affect in vivo and in vitro immune function in fowl (Bachman and Mashaly, 1986, 1987; Glick, 1984; Haddad and Mashaly, 1990, 1991; Marsh and Scanes, 1994; Mashaly et al., 1998; Skwarlo-Sonta, 1992; Trout and Mashaly, 1995). A few examples should suffice: (1) CRF stimulates chicken macrophages, and to a lesser extent lymphocytes, to secrete ACTH, and concentrations of circulating corticosterone increase following antigen challenge (Hendricks and Mashaly, 1998; Hendricks et al., 1991; Mashaly et al., 1993). The increased corticosterone causes a redistribution of different lymphocyte subpopulations from the blood to spleen (Mashaly et al., 1993, 1998). (2) In mallards, the size of the thymus and bursa are reduced following corticosteroid treatment or blockade of thyroid hormone production (Glick, 1984). In vivo treatment of mallards with DEX suppresses primary antibody responses to SRBC. At the same concentration of DEX, NK cell activity is increased, apparently resulting from the decreased production of PGE-2 by macrophages included in the cultures (Fowles et al., 1993). (3) Castrated chickens, or

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chickens with early lesions of the anterior pituitary, display decreased T-cell–mediated immunity such as GVH in vivo and PHA-induced lympho-proliferation in vitro (Glick, 1984; Mashaly, 1984). (4) Chickens with either a genetic thyroid hormone deficiency (SLD dwarf chickens) or a thyroid hormone deficiency induced by synthesis blockade exhibit depressed antiSRBC antibody responses (Glick, 1984). The genetic deficiency and its associated immune dysfunction can be reversed by administration of thyroxine and growth hormone (Marsh et al., 1984). (5) PRL has a stimulatory effect on avian immunity in that it is associated with increased bursal mitotic activity in leghorns (Glick, 1984), increased bursa weight in hypophysectomized pigeons, enhanced chicken anti-SRBC responses, and enhanced mitogenic activity of chicken thymocytes and splenocytes (Skwarlo-Sonta, 1992). (6) Feeding thyrotropin-releasing hormone (TRH) or triiodothyronine (T3) to 1-day-old chicks for 8 weeks is associated with an increase in bursal weight; T3 but not TRH also increases splenic weight and numbers of white blood cells (Haddad and Mashaly, 1990); T3 treatment also increases the number of resting splenocytes expressing IL-2 receptors as well as the number of CD3-positive cells (Chandratilleke et al., 1996). (7) The bursa of Fabricius, like the thymus, has an endocrine as well as a critical immunological function. Surgical bursectomy (BsX) of 1-day-old chickens reduces the subsequent in vitro corticosterone response of adrenal glands exposed to ACTH (el Far et al., 1994). BsX is also associated with a reduction in testosterone production by Leydig cells (el Far et al., 1994). (8) Humoral immunity to a Tindependent antigen (LPS) has also been associated temporally with a drop in triiodothyronine levels and a rise in CORT levels in immature male chickens (Gehad et al., 2002). One of the most thoroughly examined roles of neuroendocrine regulation of immunity in birds comes from investigations by the group of Georg Wick (Hu et al., 1993), who studied the Obese Strain (OS) of chicken. This strain is characterized by the spontaneous onset of an autoimmune thyroiditis and hypothyroidism (Wick et al., 1990). OS birds exhibit increased corticosteroid-binding globulin, decreased free corticosteroid, and an impaired or even absent increase in plasma glucocorticoids in response to either the intravenous (i.v.) administration of antigen, IL-1, or a conditioned medium from Con-A–stimulated splenocytes that is known to contain factors that increase glucocorticoid levels (GIFs) in birds (i.e., IL-1 but not IL-6 or TNF). This impaired glucocorticoid response is not seen in either normal birds or in the UCD 200 strain of chickens that spontaneously develops a different autoimmune pathology from OS chickens. Given that there

is no strain-associated difference in the ability of mitogen-stimulated splenocytes from the OS and UCD 200 strain to produce conditioned medium with GIF activity (Schauenstein et al., 1986), the reduced glucocorticoid synthesis in OS chickens may well be referable to a neuroendocrine immunoregulatory defect in the HPA axis (Brezinschek et al., 1990; Wick et al., 1990) such as that associated with induced or spontaneous autoimmune diseases in certain strains of rats and mice (Wick et al., 1993). The situation for OS birds, however, is not as clear-cut as it might seem since hypothyroidism itself causes blunted HPA axis responses (Kamilaris et al., 1991). Given their economic importance and public concerns about battery-raised chickens, it is somewhat surprising that relatively little has been published on the effects of stressors on immunity in domestic fowl. What is known, however, holds no surprises (Cunnick et al., 1994). For example, social isolation as a stressor in young domestic fowl led to an increase in the number of leukocytes in the blood, but not in the spleen. This same study reported a time-dependent decrease in T-cell proliferation and an increase in production of an IL-1–like factor by adherent cells in the spleen (Cunnick et al., 1994). Increasing housing density decreased bursal weight in broilers but did not affect anti-SRBC production, cell proliferation, or total white blood cell count (Heckert et al., 2002). In a study examining free-living Adelie penguins, repeated handling had no effect on heterophil/lymphocyte ratios, but nesting status did. The ratio was lowest during the stage of chick care, a change which apparently preceded the rise in CORT associated with fasting at this time (Vleck et al., 2000). Perhaps the most fascinating area of investigation involving neural-immune interactions in birds involves the fields of ecology and evolutionary biology, where a hypothesis has emerged that actually assumes a neuroendocrine-immune system link. The immunocompetence handicap hypothesis (ICH), described in the previous section on reptiles, requires that certain traits (e.g., bright plumage in birds) that are preferred by females are promoted by the immunosuppressive action of testosterone (Fostad and Karter, 1992). Since the ICH was proposed, many studies have focused on demonstrating the immunomodulatory action of testosterone. For example, male house finches implanted with capsules to artificially elevate testosterone prior to immunization with SRBC exhibited a diminished hemagglutinating antibody response 2 weeks following immunization. A diminished cell-mediated response, assessed by a local inflammatory response to PHA, was also seen (Deviche and Cortez, 2005). A similar suppressive response was seen in song spar-

Prologue

rows (Owen-Ashley et al., 2004). Since these latter authors also reported that dehydroepiandrosterone did not affect immune responses, a direct effect of androgens is questionable. Further, these authors’ demonstration that CORT levels were elevated in testosterone pellet-implanted birds also argues that this effect is not directly due to the action of testosterone on the immune system. Artificial elevation of testosterone has also been demonstrated to depress immune parameters in dark-eyed juncos (Casto et al., 2001) and European starlings (Duffy et al., 2001). Not all experiments testing the ICH have demonstrated an immunosuppressive action of testosterone. Experiments employing free-living versus captive superb fairy-wrens (Peters, 2000) or examining resistance to viral infection in greenfinches (Lindstrom et al., 2001) have indicated that, at least in some instances, testosterone may be immunoenhancing. At least one other study examining antibody responses to SRBC failed to find any immunological effect of testosterone (Hasselquist et al., 1999). Thus, validation of ICH, and on the immunosuppressive effect of testosterone under naturalistic conditions, requires further investigation.

VII. CONCLUDING REMARKS AND FUTURE RESEARCH DIRECTIONS Although there have been only limited analyses of components of the neural-immune system communication network in the few favorite non-mammalian species, enough data exist to support the proposition that the phylogenetic emergence of a link between these two systems was not a recent event. To find the earliest origins of the integration of these systems and to determine whether the neuroendocrine and immune systems evolved from a “common ancestor” (Ottaviani and Franceschi, 1997) prior to the appearance of a blood-brain barrier (Cserr and Bundgaard, 1984), studies are needed that exploit the most primitive extant vertebrates: the Agnatha (hagfish and lampreys) that lack MHC, Ig, and TCR genes; the elasmobranchs (sharks and rays), in which these critical hallmarks of adaptive immunity emerge for the first time (Du Pasquier and Flajnik, 1999); and, of course, the invertebrates. In the past 5 years, studies of innate immunity have revealed several non-cytokine molecules that are common to the innate defense systems of both invertebrates and vertebrates. Indeed, these data (see reviews by Hoebe et al., 2004; Hoffman et al., 1999; Medzhitov and Janeway, 1998) provide strong evidence of evolutionary parallels and conservation of ancient host defense pathways involving Toll receptors and NF-κB signaling in organisms (e.g., Drosoph-

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ila) that have been separated from mammals by hundreds of millions years. Given the availability of the molecular tools that allowed this new information to be generated, it would seem worthwhile to determine whether such receptors and signaling pathways might be subject to regulation by those mammalian-like neuroendocrine mediators that also appear to have been phylogenetically conserved in invertebrates. Although it is clear that the basic pathways by which the nervous system can regulate the immune system in mammals are operative in teleosts, amphibians, reptiles, and birds, the extent to which products of the immune system can affect the brain and behavior of ectothermic vertebrates is virtually unexplored. Clearly, one of the more exciting developments in our understanding of neural-immune networks in mammals has been the reproducible demonstration that pro-inflammatory cytokines (e.g., IL-1, IL-6, TNFα) induced, for example, by microbial products act as bi-directional communication signal molecules between the immune system and the CNS (Felten et al., 1991; Fleshner et al., 1995; Krueger and Majde, 1994; Parsadaniantz et al., 1994, 1997). There are reports that deliberate infection of lizards (Kluger et al., 1975) or amphibians (Lefcort and Eiger, 1993) with pathogens can evoke behavioral responses consistent with IL-1 mediation (e.g., thermoregulatory behavior resulting in the selection of a higher temperature in the classic study of Kluger et al., 1975). Research on the direct involvement of cytokine homologues in such CNS responses in all classes of ectothermic vertebrates other than teleosts fish (Engelsma et al., 2002; Holland et al., 2002) has yet to be initiated. That recombinant cytokines, appropriate molecular probes, and monoclonal anti-cytokine antibodies will be invaluable for addressing fundamental questions about the phylogeny of neural immune system interactions is sharply revealed by a report that in the frog Xenopus, IL-β and its type 1 receptor, as detected by immunohistochemistry using polyclonal anti-human antibodies, are expressed in neural tissue prior to the development of cells of the immune system (Jelaso et al., 1998). Although these investigators performed Western blotting to “confirm” the IL-1 and IL-1 receptor specificity of the anti-human polyclonal antibodies they used in their Xenopus system, the availability of monoclonal antibodies directed against recombinant Xenopus IL-1 would have eliminated “putative” as a descriptor of the frog “IL-1” they were examining. Until such reagents are available, the issue of whether IL-1 and other pro-inflammatory cytokines were first used (phylogenetically speaking) as regulatory molecules by the nervous system and were subsequently co-

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opted by the adaptive immune system, or vice versa, remains purely speculative. This issue might best be addressed in invertebrates and agnathans in which no true adaptive immune system exists. As previously pointed out, some authors have written extensively about hemocyte-derived homologues of IL-1α and IL1β, IL-2, IL-6, and TNF-α and β (Ottaviani et al., 1995c). Their reports are primarily, if not exclusively, based on cross-reactions with anti-mammalian cytokine antibodies. While these mediators do appear to affect cell migration, phagocytosis, and the induction of biogenic amines and nitric oxide synthase in the species of origin (e.g., freshwater snails), we must reiterate that the appropriate molecular studies confirming that these invertebrate mediators are really cytokine homologues are lacking. Similarly, since we last reviewed this research area (Cohen and Kinney, 2001), we have not become aware of substantive reports that these hemocyte-derived mediators, whatever they are, can affect the nervous system and behavior in the homologous (or even heterologous) invertebrate species. Clearly, appropriate studies in invertebrates and ectothermic vertebrates could prove extremely interesting with respect to the evolution of these mediators themselves as well as to the phylogeny of neural-immune interactions. Information in this area may come from unexpected areas of research. For example, axons of the central nervous system in adult mammals do not regenerate spontaneously after injury, partly because of the presence of oligodendrocytes that inhibit axonal growth (Eitan et al., 1992). This is not the case in fish, where spontaneous regeneration of the optic nerve has been correlated with the presence of factors cytotoxic to oligodendrocytes. This cytotoxic factor from the fish optic nerve has been described by Eitan et al. (1992) as an IL-2–like cytokine. One important outcome of fully understanding neural-immune interactions is the development of behavioral and/or psychopharmacological strategies to maintain optimal health status in humans. There is, however, another related but largely overlooked application of understanding the impact of psychosocial and physical stressors on immune function, namely that eliminating or “controlling” or simply understanding stressors that may be encountered by feral animals may prove important for the health of both captive and wild populations. The following few examples (some of which are discussed elsewhere [Cohen, 2006]) should bring this point home: • Briggs et al. (1996) reviewed the impact of potential immunomodulatory effects of handling stress on the morbidity and mortality of seabirds

•

•

•

•

that are cleaned after having suffered the misfortune of being contaminated by an oil spill. An important method for tracking avian species during their migrations involves capturing the birds (e.g., with mist nets) and then banding them before release. Trapping and handling wild birds clearly influence glucocorticoid levels (Romero and Wingfield, 1999; Romero et al., 1997); the short- and long-term consequences of these well-intentioned manipulations on immune functions of these birds (many of whom are weakened by the flight and lack of feeding) certainly need to be considered by those directing this information-gathering conservation effort. In their paper describing the innervation of the spleen of the beluga whale, Romano et al. (1994) pointed out that in general, the incidence of cetacean strandings, on the rise in recent years, is thought to be associated with an intense but as yet unidentified stressor. As such, the capacity of the immune system to respond to cues from the CNS may prove critical not only for understanding the reasons for such strandings, but also in the rehabilitation of stranded animals. Consequent to certain behavioral circumstances, individual subdominant male wolves may be ostracized from the pack to spend the rest of their lives as solitary animals. Whether this naturally occurring, but forced, isolation of a social animal is accompanied by immunomodulation, and whether the resulting solitary life is then shortened by opportunistic pathogens, is an intriguing scenario that begs to be written. In the section of this chapter dealing with amphibians, we discussed possible connections among environmental contaminants, neuroendocrine function, immunosuppression, and susceptibility to disease-causing pathogens. In a related vein, Herbst and Klein (1995) have speculated that the relationship between environmental contaminants and the increasing prevalence of green turtle fibropapillomatosis (an epizootic, apparently virally induced, disease) may involve immunosuppression associated with environmental contaminants (e.g., endocrine disruptors) or “stressors” such as altered temperature or salinity. Aluru et al. (2004) found that in the Artic char, PCB disrupts the HPI axis. The immunological consequences of the associated downregulation of brain glucocorticoids warrants exploration in normal and stressed fish. Similar considerations apply to other taxa, including mammals (Lawrence and Kim, 2000). These

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speculations certainly provide an ecologically valid rationale for better understanding the mechanisms underlying neural-immune system cross-talk in endothermic as well as ectothermic species. As a rhetorical aside, if fish, amphibians, and reptiles are indeed the pre-verbal canaries in the coal mine, are their warnings being taken seriously enough, in a non-anthropocentric sense, by Homo sapiens occupying leadership positions? • It is well known that environmental temperature modulates immunity in ectothermic vertebrates (Clem et al., 1984, 1991; Józkowicz and Plytycz, 1998). Indeed, a recent study (Alcorn et al., 2002) points out that sockeye salmon reared throughout their entire lifecycle at either 8° or 12°C are quite different creatures in terms of both their innate and adaptive immune response capacity. Scientists are in fundamental agreement about the phenomenon of global warming; ecological consequences are already being noted. What is not known, however, is whether the immune system (e.g., via cytokine mediators) plays a role in the neuroendocrine processes that eventuate in a change in a fish’s or frog’s core temperature and thereby affect its defense systems. If it does, it puts an interesting psychoneuroimmunological twist with an evolutionary spin on one of the world’s most serious and thorny problems. As a final note, captivity can impose potentially stressful environmental conditions (e.g., crowding, isolation, handling, and possible disturbances in circadian activity) (Dulak et al., 1989; Worley and Jurd, 1979) that are immunomodulatory in laboratory animals (Moynihan et al., 1994). Given that zoological specimens are similarly affected by these and other stressors, an understanding of the physiological impact of these conditions would be of benefit to both the animals bred and/or maintained in captivity and to the institutions involved in maintaining them. Minimizing events detrimental to the animals’ selfmaintenance of health status would also minimize the necessity for intervention by animal handlers. As more species from more taxa are being represented primarily by captive specimens, ensuring the individuals’ continued health becomes more critical; one way to ensure this may be to better understand how to enable the organism to maintain its own defense against pathogens.

Acknowledgments This manuscript was written with the support of grants from the NSF (IRCEB-00138) and the NIH (R24-AI-059830).

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Sonetti, D., Ottaviani, E., and Stefano, G. B. (1997), Opiate signaling regulates microglia activities in the invertebrate nervous system. Gen. Pharmacol., 29, 39–47. Spieler, R. E. (1979). Daily rhythms of circulating prolactin, cortisol, thyroxine, and triiodothyronine levels in fishes: a review. Rev. Can. Biol., 38, 301–315. Spooner, C. E., and Winters, W. D. (1966). Distribution of monoamines and regional uptake of DL-norepinephrine-7–H3 in the avian brain. Pharmacologist, 8, 189. Stansberg, C., Subramaniam, S., Collet, B., Secombes, C. J., and Cunningham, C. (2005). Cloning of the Atlantic salmon (Salmo salar) IL-1 receptor associated protein. Fish Shell. Immunol., 19, 53–65. Stave, J. B., and Roberson, B. S. (1985). Hydrocortisone suppresses the chemiluminescent response of striped bass phagocytes. Dev. Comp. Immunol., 9, 77–84. Stefano, G. B., Cadet, P., and Scharrer, B. (1989a). Stimulatory effects of opioid neuropeptides on locomotory activity and conformational changes in invertebrate and human immunocytes: evidence for a subtype of ∂ receptor. Proc. Natl. Acad. Sci. USA, 86, 6307–6311. Stefano, G. B., Digenis, A., Spector, S., Leung, M. K., Bilfinger, T. V., Makman, M. H., Scharrer, B., and Abumrad, N. N. (1993). Opiatelike substances in an invertebrate, an opiate receptor on invertebrate and human immunocytes, and a role in immunosuppression. Proc. Natl. Acad. Sci. USA, 90, 11099–11103. Stefano, G. B., Leung, M., Zhao, X., and Scharrer, B. (1989b). Evidence for the involvement of opioid neuropeptides in the adherence and migration of immunocompetent invertebrate hemocytes. Proc. Natl. Acad. Sci. USA, 86, 626–630. Stefano, G. B., Shipp, M., and Scharrer, B. (1991a). A possible immunoregulatory function for [Met]-enkephalin-Arg6–Phe7 involving human and invertebrate granulocytes. J. Neuroimmunol., 31, 97–103. Stefano, G. B., Smith, E., and Hughes, T. (1991b). Opioid induction of immunoreactive interleukin-1 in Mytilus edulis and human immunocytes: an interleukin-1–like substance in invertebrate neural tissue. J. Neuroimmunol., 32, 29–34. Sumpter, J. P., Pottinger, T. G., Rand-Weaver, M., and Campbell, P. M. (1994). The wide-ranging effects of stress on fish. In K. G. Davey, R. E. Peter, and S. S. Tobe (Eds.), Perspectives in comparative endocrinology (pp 535–538). Ottawa: National Research Council of Canada. Tripp, R. A., Maule, A. G., Schreck, C. B., and Kaattari, S. L. (1987). Cortisol mediated suppression of Salmon lymphocyte responses in vitro. Dev. Comp. Immunol., 11, 565–576. Trout, J. M., and Mashaly, M. M. (1995). Effects of in vitro corticosterone on chicken T- and B-lymphocyte proliferation. Brit. Poultry Sci., 36, 813–820. Turpen, J. B., and Smith, P. B. (1989). Precursor immigration and thymocyte succession during larval development and metamorphosis in Xenopus. J. Immunol., 142, 41–44. Vacchio, M. S., Papadopoulos, V., and Ashwell, J. D. (1994). Steroid production in the thymus: implications for thymocyte selection. J. Exp. Med., 179, 1835–1846. van den Burg, E. H., Metz, J. R., Spanings, F. A., Wendelaar Bonga, S. E., and Flik, G. (2005). Plasma alpha-MSH and acetylated betaendorphin levels following stress vary according to CRH sensitivity of the pituitary melanotropes in common carp, Cyprinus carpio. Gen. Comp. Endocrinol., 140, 210–221. Varas A., Torroba M., and Zapata A. G. (1992). Changes in the thymus and spleen of the turtle Mauremys caspica after testosterone injection: a morphometric study. Dev. Comp. Immunol., 16, 165–174.

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Verburg-van Kemenade, B. M. L., Nowak, B., Engelsma. M. Y., and Weyts, F. A. A. (1999). Differential effects of cortisol on apoptosis and proliferation of carp B lymphocytes from head kidney, spleen and blood. Fish Shell. Immunol., 9, 405–415. Verburg-van Kemenade, B. M. L., Weyts, F. A. A., Debets, R., and Flik, G. (1995). Carp macrophages and neutrophilic granulocytes secrete an interleukin-1–like factor. Dev. Comp. Immunol., 19, 59–70. Vleck, C. M., Vertalino, N., Vleck, D., and Bucher, T .L. (2000). Stress, corticosterone, and heterophil to lymphocyte ratios in free-living Adelie penguins. Condor, 102, 392–400. Wang, R., and Belosevic, M. (1995). The in vitro effects of estradiol and cortisol on the function of a long-term goldfish macrophage cell line. Dev. Comp. Immunol., 19, 327–336. Wang, T., Holland, J. W., Bols, N., and Secombes, C. J. (2005). Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss. FEBS J., 272, 1136–1147. Wechsler S. J., McAllister, P. E., Hetrick, F. M., and Anderson, D. P. (1986). Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus. Vet. Immunol. Immunopath., 12, 305–311. Wendelaar Bonga, S. E. (1997). The stress response in fish. Physiol. Rev., 77, 591–625. Weyts, F., Flik, G., Cohen, N, and Verburg-van Kemenade, B. M. L. (1999). Interactions between the immune system and hypothalamo-pituitary-interrenal axis in fish. Fish Shell. Immunol., 9, 1–20. Weyts, F. A. A., Flik, G., Rombout, J. H. W. M., and Verburg-van Kemenade, B. M. L. (1998a). Cortisol induces apoptosis in activated B cells, but not in thrombocytes or T cells of common carp, Cyprinus carpio L. Dev. Comp. Immunol., 22, 551–562. Weyts, F. A. A., Flik, G., and Verburg-van Kemenade, B. M. L. (1998b). Cortisol inhibits apoptosis in carp neutrophilic granulocytes. Dev. Comp. Immunol., 22, 563–572. Weyts, F. A. A., Verburg-van Kemenade, B. M. L., and Flik, G. (1998c). Characterization of corticoid receptors in carp, Cyprinus carpio L., peripheral blood leukocytes. Gen. Comp. Endocrin., 111, 1–8. Weyts, F. A. A., Verburg-van Kemenade, B. M. L., Flik, G., Lambert, J. G. D., and Wendelaar Bonga, S. E. (1997). Conservation of apoptosis as an immune regulatory mechanism: effects of cortisol and cortisone on carp lymphocytes. Brain Behav. Immun., 11, 95–105. White, B., and Nicoll, C. (1981). Hormonal control of amphibian metamorphosis. In L. Gilbert and E. Frieden (Eds.), Metamorphosis: a problem in developmental biology (pp. 363–395). New York: Plenum Press. Wick, G., Brezinschek, H. P., Hala, K., Dietrich, H., Wolf, H., and Krömer, G. (1990). The obese strain of chickens: an animal model with spontaneous autoimmune thyroiditis. Adv. Immunol., 47, 433–500. Wick, G., Hu, Y., Schwarz, S., and Krömer, G. (1993). Immunoendocrine communication via the hypothalamo-pituitary-adrenal axis in autoimmune diseases. Endocr. Rev., 14, 539–563. Wojtaszek, J. S. (1992). Seasonal changes of circulating blood parameters in the grass snake, Natrix natrix natrix L. Comp. Biochem. Physiol., 103A, 461–471.

Wojtowicz, A., and Plytycz, B. (1997). Seasonal changes of the gutassociated lymphoid tissues in the common toad, Bufo bufo. J. Nutrit. Immunol., 52, 57–65. Woodhams, D. C., Rollins-Smith, L. A., Carey, C., Reinert, L., Tyler, M. J., and Alford, R. A. (2006). Population trends associated with skin peptide defenses against chytridiomycosis in Australian frogs. Oecologia., 146, 531–540. Worley, R. T. S., and Jurd, R. D. (1979). The effect of a laboratory environment on graft rejection in Lacerta viridis, the European green lizard. Dev. Comp. Immunol., 3, 653–665. Yada, T., and Nakanishi, T. (2002). Interaction between endocrine and immune systems in fish. Int. Rev. Cytol., 220, 35–92. Yamaguchi, A., Teshima, C., Kurashige, S., Saito, T., and Mitsuhasi, S. (1981). Seasonal modulation and antibody formation in rainbow trout (Salmo gaidneri). In J. B. Solomon (Ed.), Aspects of developmental and comparative immunology (p. 483). Oxford: Pergamon Press. Yin, Z., Lam, T. J., and Sin, Y. M. (1995). The effects of crowding stress on the non-specific immune response in fancy carp (Cyprinus carpio L.). Fish Shell. Immunol., 5, 519–529. Zapata, A., and Leceta, J. (1986). Seasonal variations in the immune response of the tortoise Mauremys caspica. Immunology, 57, 483–487. Zapata, A., Varas, A., and Torroba, M. (1992). Seasonal variations in the immune system of lower vertebrates. Immunol. Today, 13, 142–147. Zapata, A., Villena, A., and Cooper, E. L. (1982). Direct contacts between nerve endings and lymphoid cells in the jugular body of Rana pipiens. Experientia, 38, 623–624. Zentel, H. J., Nohr, D., Albrecht, R., Jeurissen, S. H. M., Vainio, O., and Weihe, E. (1991). Peptidergic innervation of the bursa fabric: interrelation with T-lymphocyte subsets. Internat. J. Neurosci., 59, 177–188. Zentel, H. J., and Weihe, E. (1991). The neuro-B cell link of peptidergic innervation of the bursa fabric. Brain Behav. Immun., 5, 132–147. Zou, J., Bird, S., Truckle, J., Bols, N., Horne, M., and Secombes, C. (2004). Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss). Eur. J. Biochem., 271, 1913–1923. Zou, J., Carrington, A., Collet, B., Dijkstra, J. M., Yoshiura, Y., Bols, N., and Secombes, C. (2005). Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1type cytokine characterized functionally in fish. J. Immunol., 175, 2484–2494. Zou, J., Peddie, S., Scapigliati, G., Zhang, Y., Bols, N. C., Ellis, A. E., and Secombes, C. J. (2003). Functional characterisation of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss. Dev. Comp. Immunol., 27, 813–822. Zou, J., Wang, T., Hirono, I., Aoki, T., Inagawa, H., Honda, T., Soma, G. I., Ototake, M., Nakanishi, T., Ellis, A. E., and Secombes, C. J. (2002). Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss. Dev. Comp. Immunol., 26, 161–172. Zuk, M., Simmens, L. W., Rotenberry, J. T., and Stoehr, A. M. (2004). Sex differences in immunity in two species of field cricket. Can. J. Zool., 82, 627–634.

P A R T

I NEURAL AND ENDOCRINE EFFECTS ON IMMUNITY COBI HEIJNEN

INTRODUCTION This section contains a challenging overview of the developments in the research on mechanisms involved in the neuroendocrine-immune circuitry. Since the intriguing paper of J. Edwin Blalock in 1985, proposing a theory about shared mediators and shared receptors in immune and neuroendocrine systems, the field of psychoneuroimmunology (PNI) has made enormous progress (Blalock et al., 1985). At the time, the major challenge was to prove first of all that leukocytes express bonafide receptors for neurotransmitters, neuropeptides, and hormones, and second, that leukocytes can produce the same mediators that were known as classical neuroendocrine mediators. On the other hand, there was a scientific debate going on around the hypothesis that neurons express receptors for immune mediators like cytokines and that cytokines can be produced by cells of the neuroendocrine system. Nowadays, the dust has settled, and we are no longer questioning the validity of the concept of shared mediators and receptors in neuroendocrine and immune systems. Today’s challenge is to define the functional importance of this neuroendocrine-immune circuitry for health and disease. The chapters in this section show that neuroendocrine-immune communication encompasses complete regulatory circuits on many functional levels: • on the level of the whole organism • on the level of interorgan communication • on the level of intercellular communication within the immune system

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I. Neural and Endocrine Effects on Immunity

On the level of communication between immune cells, Chapter 5, by Ganea and Delgado, describes the production and function of the neuropeptide vasoactive intestinal peptide (VIP).VIP is described here as a powerful anti-inflammatory neuropeptide that is produced not only by neurons but also by T-cells. Production of VIP can lead to the differentiation of T-cells into so-called T regulatory cells (Treg), which are of paramount importance for the induction and maintenance of tolerance during proinflammatory autoimmune diseases like rheumatoid arthritis. From these data one could hypothesize that the (auto)immune activation of the T-cells may lead to the production of VIP, which will contribute to the induction or activation of Tregs. This will, in turn, suppress the level of activation of the T-cell, which subsequently will lead to less VIP production by the T-cell. Interesting examples of regulatory neuroendocrine-immune circuitries on the level of organ systems are described in Chapter 2, by Sanders and Kavelaars; Chapter 4, by Van der Kleij and Bienenstock; and Chapter 3, by Czura, Rosas, and Tracey. Sanders and Kavelaars start from the original observation of Hugo Besedovsky et al. that immune activation of the spleen leads to catecholamine secretion by sympathetic nerves in the spleen, which subsequently leads to a down regulation of immune activation (Besedovsky et al., 1983). Recent research revealed that the picture is more complex. Sympathetic activation and stimulation of beta2-adrenergic receptors on immune cells is not always immunosuppressive. Both in the innate as well as in the adaptive immune response, it has been shown that, depending on timing and context, beta-adrenergic activation of immune cells can be either stimulatory or suppressive. Elegant work from the Sanders group describes the molecular mechanisms underlying the immunoenhancing effects of catecholamines on both B- and T-cells. Van der Kleij and Bienenstock describe the role of neuropeptides like VIP, Substance P and CGRP for nervous system-immune communication within the mucosal immune system in, e.g., the gastrointestinal tract, the urinary tract, and the airways. These authors elucidate the central role of mast cells not only as a target for neuropeptides, but also as a source for conveying information on inflammatory activity within these organ systems to the nervous system. Neuropeptides activate mast cells, and mast cells secrete mediators that activate neurons in an intricate network of interactions with important consequences for the activity of the end organs like the gut or the airways. Disturbances in these circuitries are related to diseases like asthma or inflammatory bowel disease. The importance of cholinergic signaling as a distinct counter-regulatory mechanism to restrain excessive inflammatory cytokine production for the prevention of endotoxic shock, tissue injury, and perhaps autoimmunity is described in Chapter 3, by Czura et al. The inflammatory reflex is described in this chapter as the capacity of the vagus nerve to respond to low concentrations of cytokines in peripheral tissue with activation of a cholinergic efferent arm that has potent anti-inflammatory capacities in models of, e.g., endotoxemia and ischemia. Acetylcholine stimulation of the alpha-7 subunit of nicotinic acetylcholine receptors on macrophages is identified as the immunosuppressive route of the inflammatory reflex. Clinically, the use of cholinergic agonists to inhibit pro-inflammatory cytokine secretion has numerous possible applications.


A more classical hormonal inflammatory feedback mechanism is represented by the loop of cytokine production, activation of the hypothalamus-pituitary-adrenal-axis, and glucocorticoid mediated inhibition of inflammation. In Chapter 8, Gorby and Sternberg describe beautifully the concept that disturbances in this feedback mechanism could be involved in the inflammatory activity in autoimmune diseases like rheumatoid arthritis and the disease-associated behavioral symptoms like sensitivity to stress and depression. Although the role of glucocorticoids in autoimmunity has received most of the attention, we should not forget the importance of sex steroids and their metabolism in autoimmune diseases as described in Chapter 9, by Cutolo and Calzia. The well-known changes in the clinical symptoms of autoimmune diseases during various phases of pregnancy form the most clear-cut example of the regulatory capacity of sex steroids. From the initial discovery that leukocytes produced neuropeptides, including opioids, in vitro after proper stimulation, Machelska and Stein continued to work on this phenomenon, despite a lot of skepticism at the time. Now they show convincingly in Chapter 6 that leukocytes produce these opioids like endorphins and enkephalins in vivo during inflammatory conditions. More importantly, inhibition of production of the leukocyte-derived opioids leads to more pronounced hyperalgesia of the animal and increased inflammation. These in vivo studies clearly show the functional importance of opioid production by the immune system in intra- and inter-organ communication. I think we can consider these findings as another example of a feedback loop using shared mediators and receptors, in this case to dampen inflammatory pain. Until now I have mainly commented on the influence of the production of shared mediators by neurons and cells of the immune system. However, the other important players in the circuitry are the shared receptors. In various chapters of this section, the authors paid attention to the importance of regulation of receptor function for determining the outcome of neuroendocrine-immune communication. In Chapter 1, Schoneveld and Cidlowski elegantly describe the latest state of knowledge on the function and regulation of glucocorticoid receptors (GR) in cells of the immune system. Activation of GR, which are in fact transcription factors, can lead to transactivation and transrepression of genes in the immune cell, depending on the vicinity, availability, and balance of various transcriptional elements in the cell. However, an additional means of regulation occurs at the receptor site itself, e.g., by post-translational modification of the GR via modifications like phosphorylation at different sites or ubiquitination of the GR leading to an alteration of receptor functioning. In Chapter 2, by Sanders and Kavelaars, the regulation of β2-adrenergic receptor expression and sensitivity is described. Here, the authors describe that inflammation can alter the sensitivity of receptors for catecholamines by modulation of the intracellular kinases, like G protein coupled receptor kinases that regulate phosphorylation and internalization of adrenergic receptors. In Chapter 7, McCusker and Kelley make a crucial step forward when considering the consequences of simultaneous activation of more than one receptor on a target cell. The ultimate effect of regulatory hormones, neurotransmitters, and cytokines will be determined by the concerted action of these mediators on multiple receptors expressed by the same cell. The molecular approach used in these studies reveals an important

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level of interaction that, because of the complexity of the phenomenon, has been studied only sparsely. By now we know a lot about the activities of separate hormones and receptors, but in real life, activation of, e.g., sensory neurons will lead to a simultaneous secretion of many neuropeptides like Substance P, calcitonin-gene-related peptide, norepinephrine, acetylcholine, etc. Conversely, the cell or the organ will be bombarded by many immune mediators such as a myriad of pro-inflammatory cytokines. The cell will respond to most of the neuroendocrine and immune mediators since it expresses receptors for these molecules. This will activate several intracellular signaling pathways that will be counteracted or enhanced by each other, leading finally to the net physiological outcome of cellular activity. We are only now beginning to understand this overwhelming complexity, and the challenge in the coming decade will be to use signalomic, metabolomic, and proteomic approaches to further unravel the hierarchy in the neuroendocrine-immune circuitry. The progress of the field of psychoneuroimmunology becomes very clear in this section of Psychoneuroimmunology, Fourth Edition, since nearly every chapter deals with the effect of neuroendocrine-immune circuitry from in vitro to in vivo to translation to pathological conditions. In this respect, it is intriguing to read Chapter 10, by Straub and colleagues, who argue that neuroendocrine-immune circuitry has developed because of its evolutionary importance for the survival of the species. This means that the driving force for development of the circuitry should be understood from the point of view of defense against acute infections in individuals during and before the reproductive age. The authors propose that, in diseases that mostly occur among the elderly, the neuroendocrine-immune circuitry may lose its protective role and transform into a counterproductive system. This hypothesis is important because it may alter our idea of the way the neuroendocrine-immune circuitry should be therapeutically targeted in diseases of the elderly. Chapter 11, by Sood et al., and Chapter 12, by Avraham and Ben Eliyahu, highlight neuroendocrine-immune influences on cancer progression. Sood and co-workers describe elegantly the pivotal importance of angiogenesis for cancer progression and metastasis and the effect of the neuroendocrine mediators, e.g., catecholamines, on this process. Moreover, it appears that tumor cells themselves are sensitive to neuroimmune regulation. Tumor cells express adrenergic receptors and respond to catecholamines with increased metalloprotease production that enhances the invasive capacity of these cells. In Chapter 12, Avraham and Ben Eliyahu continue to describe the negative effects of stress and stress hormones on cancer metastasis. These authors concentrate on the fact that the stress associated with surgery to remove the primary tumor via catecholaminergic suppression of NK cell activity may, in fact, facilitate the dissemination of single tumor cells and the formation of micrometastases. Taken into account that there are now at least two levels described for potentiation of metastasis formation by catecholamines, the need for a randomized trial with beta-adrenergic blockers during surgery for the inhibition of micrometastasis formation is obvious. At the end of my introduction, I would like to stress that as PNI scientists, who have come so far as to

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make the translation from fundamental studies on neuroendocrine-immune circuitry in vitro to the description of this intricate interplay of shared mediators and receptors in pathological conditions, we should now together step forward and enter the field of neuro-immune therapeutic interventions. Cobi J. Heijnen

References Besedovsky, H., Del Rey, A., Sorkin, E., Da Prada, M., Burri, R., and Honegger, C. (1983). The immune response evokes changes in brain noradrenergic neurons. Science, 221, 564–566. Blalock J. E., Harbour-McMenamin, D., and Smith, E. M. (1985). Peptide hormones shared by the neuroendocrine and immunologic systems. J. Immunol., 135, 858s–861s.


C H A P T E R

1 Glucocorticoids and Immunity: Mechanisms of Regulation ONARD J. L. M. SCHONEVELD AND JOHN A. CIDLOWSKI

I. II. III. IV. V. VI.

INTRODUCTION 45 IMMUNITY 46 GLUCOCORTICOIDS 48 GLUCOCORTICOID RECEPTOR 48 REGULATION OF GENE EXPRESSION BY GR 50 INHIBITORY EFFECTS OF GLUCOCORTICOIDS ON THE IMMUNE SYSTEM 54 VII. PERSPECTIVES 56

nisms, glucocorticoids act at multiple levels in the immune system, including the regulation of cytokine signaling, cytokine receptor synthesis, and other critical mediators of the immune response. Because of their clinical importance and diverse roles as antiinflammatory therapeutics, it is of paramount importance to understand how glucocorticoids affect the immune system. This chapter will provide a concise overview of the immune response as well as glucocorticoid signaling, and how these two systems interact.

ABSTRACT I. INTRODUCTION

Glucocorticoids are among the most effective pharmaceuticals used to treat inflammatory and allergic diseases. Their therapeutic effects are, however, accompanied by a number of severe side effects, including fat redistribution, weight gain, hyperglycemia, and osteoporosis. Glucocorticoids exert their effects mainly through the glucocorticoid receptor, a liganddependent transcriptional regulator that activates or represses target gene expression. While the adverse effects of glucocorticoid treatment are thought to be primarily caused by activation of catabolic genes, the anti-inflammatory effects are largely thought to be mediated by the transrepressive properties of glucocorticoids. Transrepression of gene expression can be achieved by association of GR with either negative regulatory sequences in target genes, or proinflammatory transcription factors such as NFκB, AP1, and NF-AT, thereby preventing the upregulation of pro-inflammatory genes. Via these repressive mechaPSYCHONEUROIMMUNOLOGY, 4E VOLUME I

Glucocorticoids are among the most prescribed immunosuppressive drugs and have been used in the clinic for over 50 years (Dickey, 1976; Galon et al., 2002). When they were initially used in this setting, very little was known about the mechanisms underlying the anti-inflammatory effects of glucocorticoids, although the side effects had already been recognized (Clark, 1968). Years of research in addition to the development of molecular biological tools have led to significant progress in the field. Despite our better understanding and the development of non-steroidal anti-inflammatory and immunosuppressive drugs, glucocorticoids are still among the most prescribed immunosuppressive medications. This chapter will give a concise overview of the immune response and mechanisms of glucocorticoid signaling. In addition we will outline several examples where

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glucocorticoids elicit a myriad of anti-inflammatory responses at multiple levels in the immune cascade.

II. IMMUNITY To protect against invasion by pathogenic organisms, our body has developed an array of complex protective processes. In principle, two general immune mechanisms exist: innate immunity, in which the host’s existing genes determine responses to pathogens by recognition of molecular patterns that are distinct from the host, and the adaptive immune system, where the response depends on gene rearrangements to generate new receptors that are specific for one particular antigen. The immune response can be divided in three stages: the recognition phase, the activation phase, and the effector phase. Although the innate and adaptive immune systems are very different in terms of rapidity and specificity of the response, there is crosstalk between both mechanisms (Chaplin, 2003).

A. Innate Immunity The innate response is a broadly expressed system and can therefore respond very rapidly to microbial infections. It responds prior to the development of the adaptive immune response and acts as a first response against invading pathogens. By definition, the innate response consists of all the defense mechanisms that are encoded by the host’s germ-line genes. These include (1) the body’s physical barriers like the skin and the gastrointestinal tract, (2) molecules that recognize foreign antigens such as blood proteins of the complement system and cell-surface receptors that recognize molecular patterns, (3) molecules and cells that direct effector cells to the site of inflammation like vascular endothelial cells and cytokines, and (4) effector cells that eliminate foreign pathogens like phagocytes. The innate immune system responds only to molecular patterns found in microbes and products thereof such as dsRNA found in replicating viruses (Alexopoulou et al., 2001), and complex carbohydrates such as lipopolysaccharides (LPS). An important system for recognizing foreign antigens is formed by the complement system (Morgan and Harris, 2003). Its most predominant role involves recognizing pathogens, pathogenic products, or altered host cells, and targeting them for degradation. The complement system consists of over 25 plasma and cell-surface proteins that recognize pathogen-associated molecular patterns and relay this information via a signaling cascade onto the central component C3. Activation of

this system occurs through three pathways (Liszewski et al., 1996): In the classical pathway, C3 becomes activated in response to antigen-antibody complexes that are formed during the humoral response (Reid and Porter, 1981). The mannose-binding lectin pathway is activated upon recognition of pathogen-related molecular patterns by lectin proteins (Reid and Turner, 1994). In contrast, the alternative pathway is not activated by a specific signal, but is turned on by spontaneous activation of C3, or its non-specific interaction with other proteins (Fearon and Austen, 1975). Once activated, target-bound C3 can elicit several responses including (1) induction of cell lysis by the formation of a membrane-bound attack complex, (2) opsonization of the antigen, and (3) targeting of the antigen to the adaptive immune system (Sim and Tsiftsoglou, 2004). Besides the complement system, recognition of microbial antigens also takes place via Toll-like receptors (TLR) located on the membrane of certain subclasses of leukocytes (Pasare and Medzhitov, 2004). To date there are 10 known TLRs, each recognizing a different class of antigens. TLR4, for instance, recognizes LPS, potent inducers of inflammation and cytokine production that are derived from the cell wall of gramnegative bacteria (Janeway and Medzhitov, 2002). Upon activation of TLRs, the TLR-mediated MyD88dependent signaling is initiated, resulting in activation of the nuclear factor κB (NFκB) and activating protein 1 (AP-1) pathways that in turn trigger the host’s innate defense system (Wang et al., 2001). This results in the induction of pro-inflammatory genes and the production of pro-inflammatory cytokines and chemokines. Chemokines stimulate endothelial cells to produce selectins that mediate the loose attachment of leukocytes to the vascular cells at the site of inflammation (Gonzalez-Amaro and Sanchez-Madrid, 1999). Leukocytes are then activated by chemokines produced by the endothelial cells and start producing high-affinity integrins. These molecules recognize intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1), which are expressed on endothelial cells in response to pro-inflammatory cytokines (Gahmberg, 1997). Subsequently, the leukocytes can migrate through inter-endothelial cell junctions from the blood into the tissue (Bianchi et al., 1997). At the site of infection, the effector cells of the innate immune system (e.g., neutrophils and macrophages) can “ingest” microbes and kill them in a process known as phagocytocis.

B. Adaptive Immunity In contrast to the innate response, the adaptive immune response develops in reaction to an infection

1. Glucocorticoids and Immunity: Mechanisms of Regulation

by the selection of B- and T-lymphocytes that recognize particular antigens. Because of the large number of potential antigens, the adaptive immune system generates very few cells with specificity for each antigen (Chaplin, 2003). Once activated, the responding cells need to proliferate in order to provide an adequate response. Therefore, this response is much more specific but also much slower than the innate immune system. However, due to its ability to memorize the response to a specific antigen, it is able to react faster and more vigorously after repeated exposures to the antigen (Rocha and Tanchot, 2004; Stockinger et al., 2004). The adaptive immune response is initiated in the peripheral lymphoid tissues (i.e., lymph nodes, spleen, and the cutaneous and mucosal immune systems) where the antigens are concentrated. The first step in the adaptive immune response is the presentation of antigens at the cell surface of so-called antigenpresenting cells, including dendritic cells, monocytes, macrophages, and also B-cells (Del-Val and Lopez, 2002). Among those, dendritic cells are the most predominant class of antigen presenting cells and have the special function to stimulate the proliferation of naïve T-cells into Th1 or Th2 effector cells (Degli-Esposti and Smyth, 2005). Antigens produced intracellularly, such as virus-infected cells, are presented in a complex with MHC-1 molecules to Tlymphocytes. The first step in the presentation of intracellular antigens is the generation of peptide fragments by the proteasome (Bogyo et al., 1997). In the endoplasmic reticulum, these oligopeptides are then tethered to the MHC class I receptors and transported to the cell surface. Antigens of exogenous origin, such as bacteria, need to be ingested by phagocytosis or endocytosis, after which they are cleaved by lysosomal enzymes to generate peptide fragments that can be tethered to MHC class II receptors. The effector cells of the adaptive response are the T- and B-lymphocytes, and the natural killer (NK) cells that can be discriminated by the receptor type they express on the cell surface. T-cells contain receptors that interact with antigens displayed on the MHC receptors of antigen-presenting cells. Besides this antigen-specific T-cell receptor, T-cells express a complementarity domain (CD) on their cell surface that interacts with subunits of the MHC complex. CD8+ T-cells recognize MHC class I molecules and induce a response against intracellular pathogens, whereas T-helper cells (CD4+) recognize antigens that are complexed with MHC class II molecules and evoke the cellular and humoral immune responses (Williams et al., 1991). The identity of the cytokines present at the site of activation determine whether the acti-

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vated CD4+ T-cells differentiate into Th1 or Th2 cells (Mosmann and Coffman, 1989). Th1 cells produce a different set of cytokines than Th2 cells, resulting in a distinct immune response. The Th1 cells induce a cellmediated immune response, whereas the Th2 cells are involved in the humoral and allergic responses. Besides activation of the T-cell receptor complex, a co-stimulatory signal is required for T-cell activation. The first signal, derived from the antigen, determines specificity, while the second signal, which is a product of the innate immune response, ensures that a response is executed when needed. The latter can be a costimulator, cytokine, or breakdown products of the complement system. (Bretscher, 1992). By synergistic activation of the T-cell receptor complex and the co-stimulatory molecules, the transmembrane CD3 complex with which the T-cell receptor associates can initiate a mitogen-activated protein kinase (MAPK) signaling cascade resulting in the activation of genes involved in lymphocyte proliferation and differentiation (Pitcher and van Oers, 2003). B-cells are lymphocytes derived from the bone marrow and are characterized by the production of immunoglobin (Ig) molecules. The variable region of these molecules constitutes the antigen-binding domain, whereas the constant carboxy-terminal end forms the Fc domain that conducts the effector functions. Surface Ig molecules capable of recognizing a specific antigen are assembled in the B-cell receptor complex. Like T-cells, B-cell activation requires the synergistic activation of the B-cell receptor complex by the antigen and by the co-receptor complex through binding of the complement factor C3d (Fearon and Carroll, 2000). This results in activation of a downstream MAPK cascade that activates a set of genes involved in cell proliferation and maturation. In contrast to the phagocytes of the innate immune system, natural killer (NK) cells recognize intracellular antigens, like virus-infected cells. All nucleated cells normally express the class I MHC molecules that inhibit cell lysis by NK cells. Target cells in which the level of MHC molecules is reduced as a result of infection or transformation into a cancer cell can trigger the activation of natural killer cells. Also, the CD16 receptor of natural killer cells can recognize target cells that have been coated with certain classes of IgG antibodies. Once activated, the NK cell can lyse the bound target cell by releasing the content of its granules, which contain perforin and granzymes that make pores and induce apoptosis in the target cells, respectively (Smyth et al., 2005). In addition, the NK cells produce IFN-γ to activate macrophages.

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In contrast to the innate immune system, the adaptive immune response has the ability to generate longterm protection against repeated exposure to a certain antigen. After antigen-stimulation, a subset of the effector B-cells differentiates into memory B-cells and plasma cells, whereas a subset of the T-cells develops into CD4 and CD8 memory T-cells (Rocha and Tanchot, 2004; Stockinger et al., 2004).

III. GLUCOCORTICOIDS Endogenous glucocorticoids are produced by the adrenal gland under the control of the hypothalamus and pituitary, constituting the hypothalamicpituitary-adrenal gland axis (HPA). When activated by stress factors such as exposure to endotoxins, corticotropin releasing factor (CRF) and arginine vasopressin (AVP) are released from the hypothalamus, stimulating the pituitary to release adrenocorticotrophic hormone (ACTH) into the blood (Schreiber et al., 1993). In turn, ACTH stimulates the release of cortisol from the adrenal cortex into the blood, initiating the activation of downstream pathways. Named for their role in maintaining glucose homeostasis, glucocorticoids can regulate many catabolic genes, including phosphoenolpyruvate carboxykinase involved in gluconeogenesis (Scott et al., 1998), and also carbamoylphosphate synthetase that is a key enzyme in the urea cycle (Christoffels et al., 2000). In contrast to these transactivating functions, glucocorticoids have transrepressive activities on inflammatory pathways, and are widely applied to treat inflammatory responses such as asthma, arthritis, and eczema. One of the major drawbacks on the long-term use of glucocorticoids as anti-inflammatory agents is the concomitant catabolic response in many tissues of the body. The enzymes that play a role in protein catabolism often contain glucocorticoid-response elements (GREs) in their regulatory sequences and are upregulated upon treatment with glucocorticoids. In the liver, for example, induction of protein catabolism is accompanied by increases in amino acid-catabolizing, gluconeogenic, and ammonia-detoxifying enzymes. A considerable amount of research is now focusing on dissociating the repressive from the transactivating effects of glucocorticoids. Besides the endogenous glucocorticoids that are produced in the adrenal cortex, many synthetic glucocorticoids have been developed demonstrating a different benefit-risk ratio; however, significant side effects due to upregulation of genes remain (Belvisi et al., 2001; Coghlan et al., 2003).

IV. GLUCOCORTICOID RECEPTOR A. Glucocorticoid Receptor Expression The effects of glucocorticoids are mediated by the glucocorticoid receptor (GR) that is expressed in almost all tissues (Rousseau and Baxter, 1979). Although multiple isoforms exist, GR is encoded by a single gene. This gene is composed of nine exons and is located on chromosome 5 (Encio and Detera-Wadleigh, 1991). Three different promoters drive the expression of this gene resulting in three mRNAs differing in the 5’ untranslated region. Based on their GC-richness and the fact that they are expressed in most cell types, the 1B and 1C promoters have been suggested to act as housekeeping-gene promoters (Breslin et al., 2001). In contrast, the 1A promoter contains a non-consensus GRE directly upstream of a transcription-factor binding site for both c-Myb and c-Ets and is regulated by glucocorticoids. In many cell types, GR expression is downregulated by glucocorticoids (Rosewicz et al., 1988), except in immature thymocytes and Tlymphoblasts where GR is upregulated. Upregulation of GR seems to be important for the pro-apoptotic effects of glucocorticoids in these cells. It has been suggested that depending on the type of transcription factor (c-Myb or c-Ets) that is bound to the response element adjacent to the GRE, the gene is either repressed or activated by glucocorticoids (Geng and Vedeckis, 2004). Splice variants of exon nine give rise to the α and β isoform of GR that, although identical up to amino acid 727, differ at their carboxy termini. GRα, the most predominant isoform, contains a 50 amino acid carboxy terminal end that composes part of the ligand-binding domain. In contrast, in GRβ, the carboxy terminal end is replaced by 15 amino acids that have not been reported to bind any ligand (Yudt et al., 2003). The physiological relevance of GRβ is a topic of controversy. Besides these splice variants, different GR isoforms result from alternative-translation initiation due to the presence of a weak Kozak sequence in front of the first AUG and the presence of multiple AUGs in the GR coding region (Lu and Cidlowski, 2004; Lu and Cidlowski, 2005; Yudt and Cidlowski, 2001). The ratio of the different isoforms varies between cell types, and each of these isoforms regulates a specific set of genes. It has been proposed that this provides an additional mechanism to regulate the glucocorticoid response (Lu and Cidlowski, 2005).


B. Protein Structure The GR belongs to the steroid receptor family that also includes the estrogen receptors, the androgen receptors, thyroid receptors, and mineralocorticoid receptors. Similar to other steroid receptors, GR is a modular protein, with each domain having separate functions (Figure 1). The N-terminal domain contains activation function 1 (AF-1) that can activate target genes in a hormone-independent fashion (Hollenberg and Evans, 1988; Miesfeld et al., 1987). The central domain harbors the DNA-binding domain and a nuclear-localization signal (NL1). Two zinc ions in the DNA-binding domain are coordinated by two sets of four cysteins to constitute two zinc fingers (Luisi et al., 1991). Three amino acids within the zinc-finger loop constitute the P-box, which contacts the DNA and governs specificity (Eriksson and Nilsson, 1998; Umesono and Evans, 1989). The core nuclear localization motif of NL1 is located adjacent to the DNAbinding domain. In addition, two short clusters of amino acids increase the strength of the nuclearlocalization signal (Savory et al., 1999). The carboxyteminus of GR contains a second but weaker activation function (AF-2), a second nuclear localization signal (NL2), and GR’s ligand-binding domain. Since NL2 is located within the ligand-binding domain, it is not very well defined. However, there are reports suggesting that NL2 is an inefficient nuclear-localization signal (Cadepond et al., 1992; Jewell et al., 1995). Crystalstructure analysis of hGRα has shown that the ligandbinding domain contains α-helices and β sheets that form a hydrophobic pocket in which the ligand can

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bind. In the absence of hormone, GR is localized in the cytoplasm in a complex with HSP90 and other proteins to keep it in a conformation poised to bind its ligand (Pratt and Toft, 1997). Glucocorticoid binding produces a conformational change in GR and causes it to dissociate from this complex and rapidly translocate to the nucleus, where it can regulate expression of target genes (Freedman and Yamamoto, 2004; Howard and Distelhorst, 1988).

C. Post-translational Modifications Post-translational modifications like phosphorylation and ubiquitination can alter protein function and, therefore, provide an additional means for regulation. Bodwell and colleagues have identified seven phosphorylation sites in mouse GR (Bodwell et al., 1991). All but one of these sites were found to be located within the N-terminal transactivation domain of GR. In addition, hormonal induction increased the phosphorylation status at all but two sites, Ser150 and Thr159, suggesting a role of phosphorylation in ligand-dependent transactivation (Bodwell et al., 1995). However, the effects of GR phosphorylation on its capacity to transregulate gene expression were ambiguous. Data from Mason and Housley that were later confirmed by Jewell and colleagues have shown that mutation of all seven phosphorylation sites in mouse GR did not have any major effects on its transactivation capacity from an MMTV reporter construct in COS-1 cells (Mason and Housley, 1993; Jewell et al., 1995; Webster et al., 1997). In agreement Almlof et al. have shown that mutation of all five serine residues in

FIGURE 1 Schematic representation of the hGRα. GR is a modular protein, functionally consisting of three major domains. The N-terminal domain contains the strong activation function, AF1, that is important for relaying the transcription-activation signal onto the basal transcription machinery. The DNA-binding domain contains two zinc fingers that are responsible for receptor dimerization and for contacting the DNA in a sequence-specific manner. Although the hinge region does not act as a functionally important domain, it does contain a nuclear localization signal that is important for receptor translocation to the nuclear compartment. The ligandbinding domain is important for ligand binding, but contains in addition, a second transactivation function, AF2, and a second nuclear localization signal.

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human GR, which are homologous to amino acids that can be phosphorylated in mouse GR, did not result in a significantly different transactivation capacity of AF1 from a 2xGRE-TK promoter in COS-7 cells (Almlof et al., 1995). However, when these experiments were repeated using a 2xGRE-TATA reporter, it was found that mouse GR mutated at single or multiple phosphorylation sites exhibited a decreased transactivation capacity relative to wild type GR in COS-1 cells (Bodwell et al., 1998; Webster et al., 1997). Thus, these results suggested that the phosphorylation status of GR can alter its transactivation potential in a promoterspecific manner. Transcription factors, being part of signal transduction pathways, are mobile proteins within the cell. Cytoplasmic-nuclear shuttling of the GR is mediated by the import and nuclear export machinery that recognizes nuclear-localization signals and nuclearexport signals respectively (Black et al., 2001). Posttranslational modification can mask or reveal such localization sequences, thereby regulating the subcellular distribution of GR. Harreman and colleagues have shown that phosphorylation of residues adjacent to the SV40 nuclear-localization signal can indeed determine the subcellular localization of a recombinant protein (Harreman et al., 2004). However, Jewell and colleagues showed that the nuclear translocation of mouse GR mutated at single, multiple, or all phosphorylation sites in response to dexamethasone was indistinguishable from the wild type receptor, indicating that these phosphorylation sites do not play a major role in hormone-inducible translocation (Jewell et al., 1995; Webster et al., 1997). In contrast, a report by Wang et al. suggested that there might be an effect of GR’s phosphorylation status on its subcellular distribution (Wang et al., 2002). Using phosphorylationsite specific antibodies, they have shown that in stably transfected U2OS cells without hormone treatment, GR proteins phosphorylated at Ser203 or at Ser211 are both confined to the cytosolic fraction. However, following 1hr dex induction, the Ser211 form, which has been associated with the transcriptionally active subpopulation of the receptor, was found both in the cytoplasm and in the nucleoplasm, whereas the Ser203 phosphorylated form of GR was confined only to the cytosolic fraction. The discrepancy between the above findings may stem from differences in the cell types used or from stable versus transiently transfected cells. Besides influencing the transactivation properties and subcellular localization of GR, phosphorylation may affect the stability of the protein. Indeed, Webster et al. have shown that a decreased degree of phosphorylation affects receptor half-life (Webster et al.,

1997). How exactly this takes place is unclear, but it could involve ubiquitination (Wallace and Cidlowski, 2001). In cells transfected with GR constructs in which the cDNA is placed under control of a heterologous promoter, addition of glucocorticoid ligand downregulates GR expression. The absence of regulatory sequences in these constructs suggests down regulation at the post-transcriptional level. Indeed, it has been reported that mouse GR is degraded by the ubiquitinproteasome pathway (Wallace and Cidlowski, 2001).

V. REGULATION OF GENE EXPRESSION BY GR A. Transactivation of Genes Activation of gene expression by GR through DNAbinding involves glucocorticoid-response elements (GREs). These sequences, located in the regulatory regions of target genes, consist of two GRE-half sites separated by a three-base pair linker. The consensus sequence of a GRE has been defined as the imperfect palindrome GGTACAnnnTGTTCT. GR uses amino acids in the P-box of the DNA-binding domain to recognize the GRE’s nucleic acids from the major groove. A GR monomer first binds the most conserved 3’ half of the GRE after which a second monomer binds the 5’ half to form a DNA-bound dimer (Tsai et al., 1988). Once bound, GR can utilize several mechanisms to activate transcription (Figure 2). One mechanism involves the interaction of GR with TFIID, a key component of the basal transcription machinery, providing a means to directly regulate gene expression (Ford et al., 1997). However, in this case the GRE needs to be in direct proximity to the TATA-box. Another mechanism is provided by coactivators that can act as transcriptional bridges to interact with the basal transcription machinery (Glass and Rosenfeld, 2000; Rosenfeld and Glass, 2001). Members of the p160 family of transcription factors, for instance, contain NR-boxes with which they can interact with GR (Ding et al., 1998) and subsequently recruit secondary coactivators that can interact with the basal transcription machinery (Wang et al., 1998). Alternatively, these coactivators can possess histone acetyltransferase activity to loosen the interaction between histones and the DNA and thereby regulate transcription-factor access to the promoter (Vo and Goodman, 2001). As for an example, this is the case in the mouse mammary tumor virus gene (MMTV) promoter that contains positioned nucleosomes that hinder transcriptionfactor binding to the DNA. GR, however, is still able to bind its GRE in this repressive environment and


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FIGURE 2 Mechanisms of transactivation and transrepression by GR. In absence of glucocorticoid hormone, GR is complexed in the cytoplasm with heat-shock proteins and other proteins. Once bound by ligand, the glucocorticoid receptor migrates through the nuclear-pore complex to the nucleus. In the nucleus, GR can employ several mechanisms to regulate transcription. GR can bind directly to GREs and interact with general transcription factors to activate transcription. In addition, GR can recruit coactivators that can activate the basal transcription machinery or it can recruit coactivators with histone acetyltransferase activity to modify the local chromatin structure from a repressive into a permissive state. Transrepression can be achieved by interfering with transcription-factor binding to overlapping response elements. In addition, GR can interact with the DNA-bound transcription factor to render a transcriptionally inactive protein complex, or it can interact with a transcription factor to prevent it from binding the DNA.

recruits a set of coactivators with histone-acetyl transferase activity to the nucleosome-bound DNA. These coactivators displace the nucleosome, enabling transcription-factor binding adjacent binding sites to activate gene transcription (Archer et al., 1992; Blomquist et al., 1996; Deroo and Archer, 2001).

B. Transrepression of Genes When a GRE borders or overlaps with another transcription-factor binding site, the two transcription factors have to compete for DNA binding. Hence, activation of one would result in decreased transregula-

tion of the other. This is nicely exemplified in the rat α-fetoprotein gene where a GRE overlaps with an HNF4 binding site. Activation of GR strongly reduced the activity of this gene (Nakabayashi et al., 2001). Another example is the osteocalcin gene where the GRE overlaps the TATA-box and has to compete with TATA box-binding protein for DNA binding (Meyer et al., 1997). To date, we are only aware of one publication in which the nGRE sequence directs gene repression (Ou et al., 2001). In the neuronal serotonin receptor (5-1HT1A) gene, two directly repeated GRE-half sites are separated by a hexamer. When cells were treated with glucocorticoids, GR could repress expression of

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this gene by 40%. However, when this sequence was modified into a classical GRE, it increased gene expression by three- to four-fold. Besides these direct effects, GR can also exert its repressive effects by interacting with other transcription factors, including NFκB, AP-1, and nuclear factor of activated T-cells (NF-AT). NFκB plays an important role in regulating the expression of several proinflammatory genes (Barnes and Karin, 1997). The NFκB signaling pathway is activated by cytokines such as interleukin 1 (IL-1) and tumor-necrosis factor α (TNF-α) (Janeway and Medzhitov, 2002), but also by lipopolysaccharides (Janeway and Medzhitov, 2002), and UV (Adachi et al., 2003). The NFκB transcription

factor is composed of homo- and heterodimers of subunits from the NFκB/Rel family of transcription factors (p50, p52, RelA, c-Rel, and RelB) (Menetski, 2000). Interaction with the inhibitory subunit IκB retains NFκB in the cytoplasm via its ankyrin motif (Beg and Baldwin, 1993). Phosphorylation of IκB triggers ubiquitination and subsequent degradation by the proteasome (Karin and Ben-Neriah, 2000). Due to its constitutive phosphorylation, IκB has a half-life of only 1 to 2 hours (Rice and Ernst, 1993). Activation of the NFκB pathway activates IκB kinase (IKK) (Figure 3). The resulting hyperphosphorylation of IκB results in an increased rate of IκB degradation and releases NFκB for signaling to the nucleus. In the nucleus,

FIGURE 3 GR-mediated repression of NFκB signaling. In its inactive state, NFκB is retained in the cytoplasm in complex with IκB. Activation of the NFκB pathway by lipopolysaccharides or cytokines activates IκB kinase and leads to phosphorylation and subsequent degradation of IκB. The released NFκB can subsequently translocate to the nucleus to bind its response element in the regulatory regions of pro-inflammatory target genes to activate transcription. Glucocorticoids have been reported to repress NFκB signaling through multiple mechanisms. Activation of GR by glucocorticoid binding results in translocation to the nucleus where it can activate the expression of IκB and shifts the balance of NFκB localization towards the cytoplasmic compartment. GR can also interfere with NFκB signaling by interacting with NFκB and either prevent it from binding its response element or form a transcriptionally inactive protein complex on the DNA. An alternative but less-favored mechanism involves the competition between NFκB and GR for binding CBP/p300 that is present in the cell in limiting amounts.


NFκB can bind to regulatory regions of target genes and activate transcription of genes involved in the inflammatory process, including cytokines such as TNF-α (Collart et al., 1990) and IL-1β (Cogswell et al., 1994), but also inflammatory mediators such as cyclooxygenase-2 (COX-2) (Inoue and Tanabe, 1998; Nakao et al., 2002), inducible nitric oxide synthase (iNOS), and the cytosolic phospholipase A2 gene (cPLA2) (Morri et al., 1994). Several hypotheses have been postulated as to how glucocorticoids inhibit the pro-inflammatory actions of NFκB. First, due to the presence of a GRE in its regulatory region, glucocorticoids can upregulate the expression of IκB (Auphan et al., 1995; Scheinman et al., 1995a), thereby shifting the balance towards the cytoplasmic-localized NFκB. Second, it has been proposed that GR and NFκB compete for binding to CREB-binding protein (CBP)/p300. Since CBP/p300 is present in the cell in limiting amounts, activation of GR can repress the NFκB pathway. However, since NFκB and signal transducers of activated T-cells (STATs) are also targets for CPB/p300 but do not transrepress AP-1 activity, it is unlikely that such competition is the cause for glucocorticoid-mediated transrepression of these transcription factors (Karin and Chang, 2001). Indeed, McKay and Cidlowski have shown that although GR bound by antagonist RU486 is not able to interact with CBP/p300, it retains its ability to repress NFκB mediated transactivation (McKay and Cidlowski, 2000). These data, therefore, suggest that rather than acting as a coactivator for which NFκB and GR have to compete, CBP/p300 acts as a scaffolding protein that integrates NFκB and GR signaling. In the third model, activated GR can interact with NFκB via protein–protein interactions rendering the NFκB incapable of binding its response elements (McKay and Cidlowski, 1998). This model has become more and more accepted as the predominant mode of GR-mediated repression (Figure 2). Indeed, it has been reported that glucocorticoids can repress NFκB action by disturbing the interaction of p65 with the basal transcription machinery, rather than being dependent on the levels of coactivators in the cells (De Bosscher et al., 2000). Furthermore, it has been shown that GR and NFκB physically interact (Ray and Prefontaine, 1994; Scheinman et al., 1995b). Deletion analysis has shown that multiple GR domains are required for its transrepressive effect on NFκB (McKay and Cidlowski, 1998). An additional mechanism was suggested when it was shown that GR activation does not prevent NFκB from binding its response elements on the IL-8 promoter, but rather acts by recruiting repressor proteins to the target site on the DNA (Nissen and Yamamoto, 2000). Moreover, GR was able to recruit

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the transcriptional coactivator glucocorticoid receptor interacting protein-1 (GRIP-1) to the collagenase gene promoter and inhibit the transcriptional activator NFκB, turning GRIP-1 into a co-repressor (Rogatsky et al., 2001). Similar repressive mechanisms by glucocorticoids have been reported for other pro-inflammatory transcription factors. The AP-1 transcription factor, for instance, is involved in the regulation of several pro-inflammatory genes, including many cytokines (Johnson and Lapadat, 2002). AP-1 proteins are composed of homo- and heterodimeric proteins of the Jun, Fos, ATF, or Maf family of transcription factors (Angel and Karin, 1991). AP-1 dimers containing Jun recognize the AP-1 sequence (5’ TGAGCTCA) in the regulatory regions of target genes, while ATF-containing complexes recognize cAMP-responsive elements (5’ TGACGTCA). Differential expression of the AP-1 subunits allows regulation of the AP-1 activity (Bakiri et al., 2000; Chiu et al., 1989; Passegue and Wagner, 2000; Szabowski et al., 2000). Additional regulation can be achieved through AP-1 phosphorylation (Karin, 1995; Karin, 1998). Activation of AP-1 can be initiated by several stimuli, including growth factors (Quantin and Breathnach, 1988; Ryder and Nathans, 1988), TNF-α (Brenner et al., 1989), IL-1 (Goldgaber et al., 1989; Muegge et al., 1989), phorbol esters, and also by environmental stressors such as UV irradiation (Karin, 1998). These stimuli activate MAPK cascades that phosphorylate AP-1 and increase its activity. Once activated, AP-1 can bind to its response elements and regulate gene transcription of several inflammatory genes, including the collagenase gene (Schule et al., 1990). When the composition of the AP-1 complex is altered, different responses to different stimuli can be elicited. It has been shown, for instance, that a Jun-Fos heterodimer has a higher affinity for DNA than a Jun-Jun homodimer (Halazonetis et al., 1988). Glucocorticoids can negatively regulate the AP-1 mediated inflammatory response. It has been reported that GR can employ similar mechanisms to repress AP-1 as the ones repressing NFκB (Schule et al., 1990). Another transcription factor serving as a target of glucocorticoid action is the nuclear factor of activated T-cells (NF-AT). NF-AT was originally characterized as a transcription factor driving the transcription of the human IL-2 gene (Shaw et al., 1988). Four members of the NF-AT family have been identified so far (Hoey et al., 1995), regulating many genes that are activated upon T-cell activation (Kel et al., 1999). Activation of these genes involves NF-AT binding to its respective sites in regulatory regions, often acting synergistically with other transcription factors, including AP-1 in the IL-2 enhancer (Vacca et al., 1992) and Oct-1 in the IL-3

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enhancer (Duncliffe et al., 1997). In the IL-4 enhancer, NF-AT does not require cooperative interaction to drive transcription. Instead, cytoplasmic NF-AT becomes dephosphorylated in response to calcium and translocates to the nucleus to bind its response element in the IL-4 gene in order to activate transcription. Glucocorticoid treatment induces the formation of a complex between GR and NF-AT in the nucleus, thereby preventing NF-AT from binding the DNA (Chen et al., 2000).

VI. INHIBITORY EFFECTS OF GLUCOCORTICOIDS ON THE IMMUNE SYSTEM A. The Hypothalamic-Pituitary-Adrenal Gland Axis Transcription factors can regulate many genes. Therefore, the inhibition of pro-inflammatory transcription factors by glucocorticoids modulates a myriad of responses. Studies in animal models have suggested that defects in the HPA axis may increase the susceptibility to inflammatory disorders. Lewis rats, for instance, have reduced corticotropin-releasing hormone synthesis, rendering them more susceptible to streptococcal cell wall-induced arthritis (Sternberg et al., 1989a; Sternberg et al., 1989b). In agreement, supplementing glucocorticoids to these rats, thus restoring their physiological level, did reverse this phenotype. Studies in humans have shown that patients suffering from hypocortisolemia exhibit an increase in IL-2 synthesis by T-cells (Sauer et al., 1994). These examples show that defects in HPA-axis function may lead to elevated activation of inflammatory pathways. The central role of glucocorticoids in controlling the immune response suggests that there is a tight mutual regulation between the pro- and antiinflammatory pathways. Indeed, the inflammatory response is equipped with a classical feedback mechanism, triggering the release of the anti-inflammatory glucocorticoids. During the inflammatory response TNF-α, IL-1, and IL-6 mediate most of the feedback regulation on the HPA-axis (Akira et al., 1990; Hesse et al., 1988; van Deventer et al., 1990). These cytokines can independently as well as synergistically stimulate the release of adrenocorticotrophic hormone (ACTH) from the hypothalamus and pituitary, thus resulting in an increased production of glucocorticoids, thereby counteracting the inflammatory response (Adcock, 2001; Chrousos, 1995). Thus, defects in the functioning of the HPA-axis make individuals more susceptible to inflammatory diseases (Morand and Leech, 2001),

stressing the importance for the development of treatments for inflammatory diseases.

B. Upregulation of Anti-inflammatory Genes In the human body, many genes involved in catabolic processes are positively regulated by glucocorticoids, underscoring the role of GR in maintaining glucose homeostasis. However, besides these activating functions, glucocorticoids have an inhibitory effect on the immune system. One mechanism involves the activation of genes that negatively regulate the inflammatory response. For example, the lipocortin-1 (Hayashi et al., 2004), the secretory leukocyte protease inhibitor (SLPI) (Hayashi et al., 2004), and the glucocorticoid-induced leucine zipper (GILZ) (Cannarile et al., 2001; Mittelstadt and Ashwell, 2001; Riccardi et al., 1999) genes are all upregulated by glucocorticoids. However, most of the genes regulated by glucocorticoids and involved in the inflammatory response lack GREs or nGREs in their regulatory sequences. This paradox was reconciled when the major antiinflammatory actions of glucocorticoids were found to be mediated by a direct inhibitory interaction of the GR with pro-inflammatory transcription factors such as NFκB (Ray and Prefontaine, 1994), AP-1 (Adcock, 2001), and NF-AT (Chen et al., 2000). These transcription factors are activated by many stimuli and are considered of paramount importance in the inflammatory response (Baldwin, 2001). Over the years, it has become increasingly clear that, in contrast to NSAIDs, glucocorticoids act on multiple levels in the immune response in order to exert their repressive effects (Figure 4). Although the precise mechanisms in these responses are not always clear, transcription factors are thought to play a central role.

C. Glucocorticoid-mediated Post-transcriptional Regulation Glucocorticoids can increase mRNA turnover of target genes by increasing the expression of certain ribonucleases. These ribonucleases have been reported to act upon AU-rich regions in the 3’ UTR of certain genes, including GM-CSF (Bickel et al., 1990) and COX-2, thereby reducing mRNA half-life and subsequent protein synthesis (Newton et al., 1998; Nishimori et al., 2004; Ristimaki et al., 1996). A similar mechanism has been described for the interferon-β gene, in which the 3’ UTR is a target for glucocorticoids to increase interferon-β mRNA turnover (Peppel et al., 1991).


FIGURE 4 Glucocorticoids act on multiple levels in the immune response. By activating the expression of antiinflammatory proteins, GR can inhibit the immune response. GR can inhibit the transcriptional activity of pro-inflammatory transcription factors. This leads to altered cytokine production, reduced expression of cytokine receptor. Combined, these modifications affect many processes in the immune response including the development of T cells, but also the migration of leukocyte to the site of inflammation by inhibiting the expression of adhesion molecules.

Interleukin 5 (IL-5) is predominantly produced by Th2 cells to regulate differentiation and proliferation of eosinophils (Clutterbuck et al., 1989; Hamid et al., 1991; Ying et al., 1995). Since asthma is characterized by eosinophil infiltration of the airways, IL-5 is regarded as a major target for anti-asthma therapies (Djukanovic et al., 1990). Glucocorticoids have been shown to inhibit IL-5 synthesis, but the exact mechanism is unknown (Corrigan et al., 1993; Rolfe et al., 1992). Since dexamethasone does not inhibit IL-5 transcription, regulation most likely takes place via a posttranslational mechanism. It has been suggested that glucocorticoids upregulate the synthesis of mRNA destabilizing factor, thereby shortening IL-5 mRNA half-life (Staples et al., 2003).

D. Glucocorticoids Modulate the Innate Immune Response The vascular endothelium plays a crucial role in the immune response by directing leukocytes to the site of inflammation. Endothelial cells express a set of adhesion molecules on their cell surface, including ICAM-1, endothelial-leukocyte adhesion molecule 1 (ELAM-1) (Cronstein et al., 1992), and E-selectin (Brostjan et al., 1997), all of which are involved in directing leukocytes to the site of inflammation (Caldenhoven et al., 1995; Cronstein et al., 1992). Glucocorticoids possess the ability to negatively regulate the expression of these adhesion molecules, thus providing a mechanism of

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inhibiting the inflammatory response. The ICAM-1 and E-selectin genes, for instance, contain NF-κB binding sites in their regulatory regions. Association of GR to NF-κB does not prevent NF-κB from binding the ICAM-1 gene. Instead, a transcriptionally inactive transcription-factor complex is formed on the DNA (Brostjan et al., 1997; Liden et al., 2000). Besides these inhibitory effects of glucocorticoids, certain proinflammatory genes related to the innate immune response are upregulated by glucocorticoids (Galon et al., 2002). TLR-2, for instance, contains binding sites for NFκB, STAT, and GR, which have been shown to synergistically activate gene transcription (Hermoso et al., 2004). Furthermore, microarray analysis using peripheral blood mononuclear cells suggested that while genes involved in the adaptive immune response are downregulated by glucocorticoids, a number of genes of the innate immune response are upregulated by glucocorticoids (Galon et al., 2002). Such upregulation of pro-inflammatory genes by glucocorticoid hormones suggests an interesting mechanistic discrimination between the innate and the adaptive immune response. It has been suggested that glucocorticoids stimulate the clearance of antigens by promoting the innate immune response, while inhibiting the adaptive immune response (Galon et al., 2002).

E. Glucocorticoids Affect Cytokine Production Cytokines, being signaling molecules between different cells, are produced at multiple levels during the immune response. There are many reports indicating a glucocorticoid-mediated inhibition of cytokine expression including, but not limited to, IL-1 through IL-6, IL-8, IL-11, IL-12, IL-16, IFN-γ, and TNF-α (Almawi et al., 1991; Amano et al., 1993; Arima et al., 1999; Blotta et al., 1997; Chen et al., 2000; Culpepper and Lee, 1985; Elenkov et al., 1996; Haynesworth et al., 1996; Kunicka et al., 1993; Paliogianni and Boumpas, 1995; Tobler et al., 1992). T-cell activation, for instance, triggers intracellular signaling cascades, including the NFκB, AP-1 and NF-AT pathways. These transcription factors can regulate the expression of cytokine genes. The IL-2 gene, for instance, has binding sites for all these three transcription factors, allowing synergistic action of the transcription factors (Durand et al., 1988; Paliogianni et al., 1993; Vacca et al., 1992). Administration of glucocorticoids inhibits expression of the IL-2 gene, most likely through protein–protein interaction with AP-1, thereby preventing it from forming a protein complex on the DNA that can activate transcription (Paliogianni et al., 1993).

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F. Glucocorticoids Can Regulate the Expression of Cytokine Receptors Glucocorticoids have been shown to modulate the expression of several cytokine receptors including those for TNF-α, IL-1, IL-2, IL-6, IL-12, IFN-γ, and GMCSF (Almawi et al., 1996; Wiegers and Reul, 1998). The IL-4 receptor, for instance, is downregulated by dexamethasone. Nuclear run-on assays indicated that this regulation did not take place at the transcriptional level. Instead, mRNA stability assays showed that dexamethasone decreased mRNA stability of the receptor (Mozo et al., 1998). Another example is the IL-12 receptor that is primarily expressed on T-cells and NK cells (Desai et al., 1992). IL-12 binding to the receptor induces intracellular signaling cascades regulating the generation and stability of Th1 and Th2 responses. It has been established that dexamethasone can decrease the expression of the IL-12 receptor at the cell surface, although the exact mechanism remains largely unclear (Wu et al., 1998).

G. Glucocorticoids Modulate the Adaptive Immune Response Naïve T helper cells serve as precursors of Th1 and Th2 cells in the adaptive immune response. The differentiation of a Th precursor cell into a Th1 or a Th2 cell depends on the cytokines that are locally present. The production of IL-12 by antigen-presenting cells has been shown to direct the response into a Th1 response (Trinchieri, 1995), whereas IL-4 has been reported to develop the Th2 response (Paul and Seder, 1994). Th1 helper cells produce IL-2, IFN-γ, and TNF-β and are involved in regulation of the cellular immunity by activating cytotoxic T-cells, natural killer cells, and macrophages. In contrast, Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 cytokines that regulate humoral immunity by promoting the proliferation and activation of mast cells and eosinophils, and by promoting B-cell differentiation into antibody-secreting cells (Mosmann and Sad, 1996). Besides stimulating the response into the Th1 or Th2 direction, these cytokines simultaneously inhibit development of the reciprocal phenotype (Mosmann and Sad, 1996). Glucocorticoids can shift the Th1/Th2 balance into the direction of the Th2-mediated humoral immunity (Elenkov et al., 1996). The main underlying mechanism of this shift results from the selective repression of Th1 cytokine production as well as the upregulation of Th2-cytokine synthesis mainly through inhibition of IL-12 production by antigen-presenting cells (Blotta et al., 1997). In addition, glucocorticoids inhibit the expression of the

IL-12 receptor in T-cells and NK cells (Elenkov, 2004; Wu et al., 1998). Increased glucocorticoid production from endogenous origin due to various stress factors may have a great impact on the outcome of the immune response. Cellular responses to viral infections, such as common cold and Herpes simplex virus, may be seriously impaired in such cases. Psychological stress can, for instance, increase the susceptibility to respiratory infections in humans (Cohen et al., 1991). Furthermore, it has been suggested that impairment of the host’s cellular response by stress-associated immunomodulation can affect the defense against herpes simplex virus (Sainz et al., 2001).

VII. PERSPECTIVES The glucocorticoid receptor acts as a liganddependent transcriptional regulator and plays an important role in the regulation of catabolic genes to maintain glucose homeostasis. In addition, it is also an important negative regulator for counteracting the effects of the immune response. Today, glucocorticoids are among the most powerful antiphlogistic drugs, but their severe adverse effects rival their beneficial therapeutic effects. Research over several decades trying to uncouple the transactivating from the transrepressive effect has led to the development of synthetic glucocorticoids that have lower adverse effects but are usually less effective. It has become increasingly clear that the glucocorticoid receptor employs diverse mechanisms to inhibit pro-inflammatory signaling pathways. First, the receptor itself is the subject of regulation at the transcriptional, post-transcriptional, and post-translational levels. Second, it can positively regulate anti-inflammatory genes and downregulate pro-inflammatory genes by direct binding to its response element on the regulatory regions of target genes. Also, it can inhibit the activity of proinflammatory transcription factors by formation of a protein complex that is either transcriptionally inactive or cannot bind DNA. Furthermore, the effects of the antiphlogistic mechanisms of glucocorticoids become apparent at multiple levels throughout the immune response. A better in-depth understanding of the regulatory mechanisms of the pro- and anti-inflammatory pathways as well as the complex crosstalk between these two pathways may help to develop better therapeutic regimens for the drugs currently available and assist in defining new therapeutic targets for the ongoing development of safe and effective antiinflammatory drugs.


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cooperativity between nuclear factor of activated T cells and AP-1 enhancer elements. J. Exp. Med., 175, 637–646. van Deventer, S. J., Buller, H. R., ten Cate, J. W., Aarden, L. A., Hack, C. E., and Sturk, A. (1990). Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood, 76, 2520–2526. Vo, N., and Goodman, R. H. (2001). CREB-binding protein and p300 in transcriptional regulation. J. Biol. Chem., 276, 13505– 13508. Wallace, A. D., and Cidlowski, J. A. (2001). Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. J. Biol. Chem., 276, 42714–42721. Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J., and Chen, Z. J. (2001). TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature, 412, 346–351. Wang, J. C., Stafford, J. M., and Granner, D. K. (1998). SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4. J. Biol. Chem., 273, 30847–30850. Wang, Z., Frederick, J., and Garabedian, M. J. (2002). Deciphering the phosphorylation “code” of the glucocorticoid receptor in vivo. J. Biol. Chem., 277, 26573–26580. Webster, J. C., Jewell, C. M., Bodwell, J. E., Munck, A., Sar, M., and Cidlowski, J. A. (1997). Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the receptor protein. J. Biol. Chem., 272, 9287–9293. Wiegers, G. J., and Reul, J. M. (1998). Induction of cytokine receptors by glucocorticoids: functional and pathological significance. Trends Pharmacol. Sci., 19, 317–321. Williams, M. E., Chang, T. L., Burke, S. K., Lichtman, A. H., and Abbas, A. K. (1991). Activation of functionally distinct subsets of CD4+ T lymphocytes. Res. Immunol., 142, 23–28. Wu, C. Y., Wang, K., McDyer, J. F., and Seder, R. A. (1998). Prostaglandin E2 and dexamethasone inhibit IL-12 receptor expression and IL-12 responsiveness. J. Immunol., 161, 2723– 2730. Ying, S., Durham, S. R., Corrigan, C. J., Hamid, Q., and Kay, A. B. (1995). Phenotype of cells expressing mRNA for TH2-type (interleukin 4 and interleukin 5) and TH1-type (interleukin 2 and interferon gamma) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am. J. Respir. Cell Mol. Biol., 12, 477–487. Yudt, M. R., and Cidlowski, J. A. (2001). Molecular identification and characterization of a and b forms of the glucocorticoid receptor. Mol. Endocrinol., 15, 1093–1103. Yudt, M. R., Jewell, C. M., Bienstock, R. J., and Cidlowski, J. A. (2003). Molecular origins for the dominant negative function of human glucocorticoid receptor beta. Mol. Cell. Biol., 23, 4319–4330.


C H A P T E R

2 Adrenergic Regulation of Immunity VIRGINIA M. SANDERS AND ANNEMIEKE KAVELAARS

I. EARLY EVIDENCE THAT THE NERVOUS AND IMMUNE SYSTEMS COMMUNICATE WITH EACH OTHER 63 II. SYMPATHETIC INNERVATION OF LYMPHOID TISSUE AND NOREPINEPHRINE RELEASE 64 III. EVIDENCE FOR ADRENERGIC RECEPTOR EXPRESSION ON IMMUNE CELLS 65 IV. EVIDENCE THAT NOREPINEPHRINE REGULATES IMMUNE CELL ACTIVITY IN VIVO 66 V. EVIDENCE FOR ADRENERGIC RECEPTOR REGULATION OF IMMUNE CELL ACTIVITY IN VITRO 71 VI. RELEVANCE TO HEALTH AND DISEASE 75

tion, epinephrine (EPI) is stored in the adrenal medulla, released systemically in response to stress, and also binds to the β2AR. Thus, both NE and EPI are important regulators of immune cell activity, with NE being released in response to both antigen and stress, and EPI being released primarily in response to stress alone. Thus, mechanisms are in place within lymphoid tissue that allow for a message to be delivered from the brain to cells of the immune system to maintain homeostasis when the host is threatened by antigen entry, and this message can be modified during times of stress or behavioral conditioning. In this way, a neurotransmitter-mediated adjustment in the level of immune cell activity has the potential to limit susceptibility or resistance to disease.

ABSTRACT Changes in immune system structure and function are associated with both stress and behavioral conditioning, suggesting that cells of the immune system are influenced by mediators released from the nervous and/or endocrine systems. The evidence to confirm that a communication exists between the sympathetic nervous system and immune system is well established. First, primary and secondary lymphoid organs are innervated with sympathetic nerve fibers in the parenchyma. Second, the sympathetic neurotransmitter norepinephrine (NE) is released from the nerve terminals following exposure to an antigen. Third, all immune cell types, with the possible exception of Th2 cells, express the beta-2-adrenergic receptor (β2AR) that binds NE to activate signaling intermediates within the cell. And finally, stimulation of the β2AR by NE regulates the level of leukocyte activity. In addiPSYCHONEUROIMMUNOLOGY, 4E VOLUME I

I. EARLY EVIDENCE THAT THE NERVOUS AND IMMUNE SYSTEMS COMMUNICATE WITH EACH OTHER A number of recent review articles have documented and discussed the evidence in support of the existence of a neuroimmune connection (Glaser and KiecoltGlaser, 2005; Harbuz, 2003; Kohm et al., 2001; Lombardi et al., 2002; Schorr and Arnason, 1999; Steinman, 2004). There are three landmark findings that are considered the cornerstone of psychoneuroimmunology. The study by Ader and Cohen, in which they showed the effect of taste aversion conditioning on a humoral immune response (Ader and Cohen, 1975), indicated that behavior influenced immunity and that immunity influenced behavior. These results

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suggested that a bi-directional relationship between behavior and immunity existed, and that such a relationship would have biological relevance for the treatment of disease. The stage was now set to explore the mechanisms responsible for mediating this communication, i.e., the mechanisms by which the immune system alerts the brain that it is responding to an antigen, as well as those mechanisms by which the brain regulates the level of immune cell activity that develops. The study by Besedovsky and Del Rey was the first to show that the activated immune system was able to release a soluble product that precipitated a change in the firing rate of neurons in a specific location within the brain, the hypothalamus (Besedovsky et al., 1977; Besedovsky et al., 1983). The importance of this finding was that the hypothalamus is the brain region that controls activation of organ system pathways that allow for the brain to communicate with the periphery. These organ system pathways include the sympathetic nervous system (SNS), which releases the neurotransmitter NE from nerve terminals and EPI from the adrenal medulla, and/or the hypothalamic-pituitaryadrenal axis, which releases a variety of hormones such as corticosteroids. Besedovsky and Del Rey were also the first to show that the SNS, via the stimulation of a receptor that could bind NE, was able to regulate the magnitude of an antibody response (Besedovsky et al., 1979; Del Rey et al., 1981). Thus, a brain-toimmune circuit was now in place to explain how behavior might affect immunity via the sympathetic nervous system. There is also an immune-to-brain circuit that explains how behavior might be affected by the activated immune system, but this topic will not be discussed herein. Instead, the reader is referred to the following excellent reviews on this topic (Besedovsky and Del Rey, 1996; Tracey, 2002; Watkins and Maier, 2005) and to the appropriate chapter in this book.

II. SYMPATHETIC INNERVATION OF LYMPHOID TISSUE AND NOREPINEPHRINE RELEASE The SNS maintains homeostasis by regulating the activity of organ systems that are not under voluntary, conscious control, e.g., the cardiovascular and respiratory systems. The SNS is associated with the “flight or fight” response, which involves immediate regulation of cardiovascular, respiratory, and metabolic functions during times of critical need, such as when fleeing or fighting an attacker. It seems as no surprise then that the immune system might also be under the regulatory

control of the SNS, especially since the cells of the immune system are involved in guarding against reactions to self and eliminating anything that is non-self. But before this assumption would be proven, it was imperative to first show that lymphoid organs were innervated with sympathetic nerve fibers so that the labile neurotransmitter would be delivered within close proximity to immune cells. Also, because epinephrine is released systemically, primarily during a response to physical or psychological stress, and is less labile than NE, it was necessary to show that a high enough concentration of epinephrine in the plasma would reach the microenvironment of immune cells to exert an effect on immune cell activity. Both human and animal primary and secondary lymphoid organs are innervated with sympathetic nerve fibers (Felten and Felten, 1991; Meltzer et al., 1997; Panuncio et al., 1998). A dense perivascular network of sympathetic nerve fibers is detectable within the splenic white pulp, thymus, and lymph nodes of mice and humans. Nerve endings that terminate in the parenchyma of the white pulp are especially numerous in the periarteriolar lymphoid sheath (PALS) and are found adjacent to both CD4+ and CD8+ T-cells, as well as in the vicinity of macrophages and B-cells (Felten et al., 1987; Felten et al., 1998). The level of NE released at a nerve terminal may reach a local concentration of approximately 1 × 10−5 to 5 × 10−4 M (Shimizu et al., 1994), which becomes lower as NE is either taken back into the nerve terminal, degraded by enzymes, or moves away from the nerve terminal to create a concentration gradient. Also, epinephrine that is released during a stress response can reach a concentration of 1 × 10−7 M in the plasma, which is considerably lower than that of NE due to the systemic nature of its release. Thus, a mechanism was now in place by which NE could be released into, and epinephrine could attain a high enough concentration within, the microenvironment in which immune cells responded to antigen. Consequently, cells in these two compartments, blood vs. lymphoid organs, may be exposed to different concentrations of NE and subsequently may respond differently. It was now important to determine if the introduction of antigen into the body was associated with SNS activation and release of NE within lymphoid tissue, independently of a stress response that induces a release of both NE and epinephrine. Systemic infection, LPS-, or infectious microorganisms increased the rate of NE release in the spleen during the first 12 hours of exposure (Kohm et al., 2001). Non-infectious types of immune stimuli, e.g., particulate or soluble protein antigens, also influenced the rate of NE release in lymphoid organs. Sheep erythrocytes increased the

2. Adrenergic Regulation of Immunity

level of sympathetic nerve activity and the rate of NE release in the spleen (Besedovsky et al., 1979), without disrupting the homeostatic mechanisms responsible for maintaining a constant level of NE tissue content (Fuchs et al., 1988). Likewise, a soluble protein antigen, TNP-KLH, increased the rate of NE release within the spleen and bone marrow in an antigen-specific manner between 9–18 hours following immunization (Kohm et al., 2000), suggesting that initial antigen distribution, uptake, processing, presentation, and cytokine synthesis/release occurred before the SNS was activated. In the latter study, the increase in sympathetic nerve activity was partially blocked by the ganglionic-blocker chlorisondamine, suggesting that both pre- and postganglionic mechanisms are likely involved in regulating NE release following an immune insult. Thus, activation of the SNS is one mechanism by which the brain appears to communicate with cells of the immune system to potentially regulate the magnitude of an immune response. As a result of an immune response to antigen, SNS activation appears to be initiated from either within the central nervous system (CNS) or outside the CNS at sympathetic nerve terminals and post-ganglionic sites (Straub, 2004). It is interesting to speculate that severe infections, which pose an immediate threat to survival, may require participation of the CNS to orchestrate SNS-mediated immune regulation peripherally, while less severe infections or autoimmune reactions may require the participation of local regulatory mechanisms alone.

III. EVIDENCE FOR ADRENERGIC RECEPTOR EXPRESSION ON IMMUNE CELLS Adrenergic receptors bind catecholamines, such as NE and epinephrine. Adrenergic binding sites have been identified on immune cell subsets that are saturable, reversible, high affinity, and almost exclusively of the β2AR subtype, as determined using radioligand binding analysis (Sanders et al., 2001). In addition, single nucleotide polymorphisms associated with the β2AR gene are becoming evident in cells from rheumatoid arthritis patients (Xu et al., 2005). In contrast, evidence for the expression of the αAR subtype by immune cells appears to be limited to specific cell subsets and is primarily of the α1AR subtype (Kavelaars, 2002).

A. Cells of the Innate Immune System Data show that cells of the innate immune system— i.e., polymorphonuclear cells (PMN), mast cells, monocyte/macrophages, and natural killer (NK)

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cells—express one and/or another AR subtype. Initially, radioligand binding analyses that were performed on isolated subsets of innate immune cells showed that all cells expressed the β2AR (Elenkov et al., 2000). These findings were confirmed recently by analysis of mRNA expression (de Coupade et al., 2004). However, it remains unclear as to whether other βAR subtypes are expressed by PMN. A recent study using human PMN detected no β1AR mRNA (de Coupade et al., 2004), while functional studies using human peripheral blood monocytes and monocyte-derived macrophages revealed the expression of not only the β2AR, but also the β1AR (Speidl et al., 2004). Dendritic cells derived from bone marrow appear to express the α1AR (Maestroni, 2000), α2AR, β1AR, and β2AR (Maestroni et al., 2003). In contrast, NK cells express the β2AR exclusively (Jetschmann et al., 1997). Expression of the AR subtypes on cells of the innate immune system is not static, and is subject to regulation by cell activation and/or various hormones. For example, during sepsis, the β2AR number expressed on a phagocytic liver Kupffer cell is markedly increased (Hahn et al., 1998). Likewise, the β2AR number on a human PMN is higher on cells obtained from females in comparison to cells from males, suggesting that innate immune cell interactions with sexual hormones may regulate expression of the β2AR on these cells (de Coupade et al., 2004). Few data are available with respect to the presence of the αAR subtype on cells of the innate immune system. Cell lines of monocytic origin express a detectable level of the α1AR, as determined on the mRNA level, but this subtype is undetectable on peripheral blood cells from healthy individuals (Heijnen et al., 2002; Rouppe van der Voort et al., 2000). Upon activation, however, the level of the α1AR expressed on monocytic cell lines changes (Heijnen et al., 2002; Rouppe van der Voort et al., 1999), and expression is induced on freshly isolated human peripheral blood monocytes (Kavelaars, 2002). Moreover, there is evidence that in certain clinical conditions, e.g., in children with juvenile idiopathic arthritis, a functional α1AR is expressed on cells in the peripheral blood, as indicated by increased production of IL-6 and TNF-α after exposure of cells to an α1AR agonist. Because the cytokines are likely secreted by monocytes in the peripheral blood, this finding suggests that monocytes represent the immune cell subset that expresses the α1AR (Heijnen et al., 2002; Rouppe van der Voort et al., 2000). Other studies report that murine peritoneal macrophages express a α2AR, as determined functionally, and that human PMN express about 500 α2AR per cell, as determined using radioligand binding analysis (Miles et al., 1996; Panosian and Marinetti, 1983;

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Spengler et al., 1990). In contrast, the α1AR was undetectable on PMN (Panosian and Marinetti, 1983).

B. Cells of the Adaptive Immune System 1. T-Cells CD8+ T-cells have not been studied as much as CD4+ T-cells, but they do express βAR binding sites (Sanders et al., 2001). In contrast, the animal and human CD4+ T-cell has been well characterized for expression of the β2AR (Sanders et al., 2001). However, the CD4+ T-cells analyzed in most of these studies represent a population of cells that may contain all three subsets of CD4+ T-cells, namely the naïve, Th1, and Th2 cell. Because radioligand binding analysis is difficult to perform on a purified population of naïve CD4+ T-cells due to low cell number, RT-PCR analysis was used to show that murine naïve CD4+ T-cells express mRNA for the β2AR (Swanson et al., 2001). Clones of murine Th1 cells express the β2AR and accumulate cAMP upon exposure to a β2AR agonist, but clones of Th2 cells do not (Sanders et al., 1997). The latter conclusion was initially suggested earlier when it was reported that different CD4+ T-cell lines (not necessarily Th1 or Th2) differentially accumulated cAMP in response to a β2AR agonist, even though all of the cell lines accumulated cAMP in response to other ligands such as histamine or PGE2 (Khan et al., 1985a; Khan et al., 1985b). Indirectly, these findings suggested that murine CD4+ Tcell subsets might differentially express the β2AR, even though all subsets express other receptors capable of activating cAMP. Expression of the β2AR on murine primary Th1 and Th2 cells generated from a naïve cell precursor activated under Th1- or Th2-polarizing conditions remains unknown, although preliminary data confirm the findings using clones of differential expression (Sanders et al., unpublished data). These findings may be difficult to confirm using human cells since human CD4+ T-cells do not polarize as clearly as mouse cells do under Th1and Th2-polarizing conditions (Abbas et al., 1996; Romagnani, 1994; Romagnani, 2000). Nonetheless, conflicting data exist when using human cells, with some suggesting the absence of a β2AR (Borger et al., 1998) and others suggesting the presence (Goleva et al., 2004; Heijink et al., 2003). However, as techniques are improved to purify out human CD4+ T-cells that secrete only Th1- or Th2-like cytokines, it may be possible to solve this question. Upon CD4+ T-cell activation, via either mitogen activation, TCR stimulation, or contact sensitization, the level of β2AR expression either increases (Madden et al., 1989; Radojcic et al., 1991; Ramer-Quinn et al.,

1997; Sanders and Munson, 1985b; Westly and Kelley, 1987;) or decreases (Cazaux et al., 1995; Radojcic et al., 1991), but remains undetectable on Th2 cell clones (Ramer-Quinn et al., 1997). Stimulation of the β2AR on a CD4+ T-cell that expresses the receptor increases both the intracellular concentration of cAMP and adenylate cyclase activity (Sanders et al., 2001). Thus, the β2AR appears to be maintained as the naïve T-cell differentiates into a Th1 cell, but is repressed as the naïve cell differentiates into a Th2 cell. The mechanism responsible for mediating the differential expression of the β2AR by these two effector cell subsets remains unknown. 2. B-Cells Radioligand binding analysis shows that a B-cell expresses almost twice as many receptors as a CD4+ T-cell, and that the receptor is of the β2AR subtype (Sanders et al., 2001). Expression of the β2AR by a purified population of naïve antigen-specific B-cells has also been reported, using both radioligand binding, immunofluorescence, molecular, and biochemical analyses (Kohm and Sanders, 1999). Thus far, no data have been reported to show expression of either the β1AR or β3AR on B-cells. A few radioligand binding analyses show expression of the αAR on B-cells (Goin et al., 1991; McPherson and Summers, 1982; Titinchi and Clark, 1984), but the results may be misleading since platelets, which express the αAR at a high level, were not removed from the lymphocyte samples before analysis. The β2AR on a B-cell is functional since exposure to a β2AR agonist enhances both the intracellular accumulation of cAMP and adenylate cyclase activation (Sanders et al., 2001). Taken together, the innate and adaptive immune cell data suggest that all immune cell types, except for Th2 cells, express an AR subtype, and that expression occurs at different densities (see Figure 1).

IV. EVIDENCE THAT NOREPINEPHRINE REGULATES IMMUNE CELL ACTIVITY IN VIVO After infection, immune cells of the innate and adaptive immune system protect the host by destroying the infectious pathogen and by providing longterm pathogen-specific protection. The innate immune system acts almost immediately in an antigen nonspecific manner and does not generate memory cells that would recognize a specific antigen upon reexposure. The adaptive immune system protects the host within a few days of antigen entry in an antigen-

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β2 PMN

β1

Innate Immune Cells β2 β ? β1 β2 α α1 β1 2 α1 α 1 2

Human Mono Mac

Murine Mac

Dendritic Cell

β2 NK Cell

Adaptive Immune Cells β2

β2

?

β2

β2

Naive CD4+ T-cell

CD4+ Th1 Cell

CD4+ Th2 Cell

CD8+ T-cell

B-cell

FIGURE 1 Adrenergic receptor phenotype on cells of the innate and adaptive immune systems. Each cell type shows only the subtype designation for the adrenergic receptor. A question mark indicates that the findings are inclusive in both humans and animals; e.g., murine and human Th2 cells are reported to not express the β2AR, while some human Th2 cells appear to express the receptor (see III. B. 1). The same question holds true for expression of the α2AR by murine macrophages. Mono = monocyte; Mac = macrophage.

specific manner and generates long-term protection in the form of memory. Both arms of the immune system are characterized by either cell-mediated or humoraltype responses. Cell-mediated responses are those that can be passively transferred to an infected host, i.e., in the form of cells from infected donors to promote a protective response in a host either directly via cell– cell contact to kill infected targets or indirectly via the release of cytokines to enhance the activity of other immune cells. On the other hand, humoral responses are those that can be passively transferred in the form of soluble proteins contained in serum, usually in the form of antibodies that are made and released by B-cells. Pharmacological evidence for involvement of the sympathetic nervous system in regulating the level of both cell-mediated and humoral immune responses was initially obtained from studies in which the chemical neurotoxin 6-hydroxydopamine (6-OHDA) was used to reversibly deplete NE from peripheral sympathetic nerve terminals in adult mice. Such NE-depleted mice had either enhanced, suppressed, or unaltered immune responses in response to antigen when compared to mice in which NE remained intact (Kohm et al., 2001), suggesting that NE needed to be released from nerve terminals within the microenvironment of immune cells responding to antigen so that the level of an immune response could be regulated. However, different findings may result from either the initial burst of NE displaced by 6-OHDA as it is taken into a nerve terminal, which may have exerted an effect on

cells before NE depletion was established, the dose and/or time of 6-OHDA administration, and/or the type of immune response being measured. Such caveats have made it difficult to conclude whether the disparate findings reflected different aspects of a related phenomenon or reflected differences in the responsiveness of different cell subsets to NE during different stages of an immune response. Nonetheless, the following data provide evidence that NE plays a role in regulating the magnitude of innate and humoral immunity.

A. Innate Immunity The majority of studies on the effects of acute stress in humans have focused on changes that occur in the number of specific cell subsets that are primarily within the human peripheral blood compartment. The reason for the attention to number changes within peripheral blood as opposed to lymphoid tissue that is commonly used from animals is clear, namely that human peripheral blood is more easily accessible and cell-counting techniques are not very complicated. It is well known that acute stress results in a reliable and rapid increase in the number of circulating NK cells and a slower increase in the number of PMN [reviewed in (Benschop et al., 1996b; Elenkov et al., 2000)]. In contrast, less is known about how stress affects the number and/or distribution of specific innate immune cell subsets during the course of a clinical condition. However, more recent studies have explored the effect of stress and AR stimulation on innate immunity in sepsis and models of NK cell-dependent defense against tumor metastasis. In this chapter, we will focus on the general effects of NE, EPI, and AR stimulation on innate immunity. 1. NK Cells The role of AR signaling in NK cell-mediated killing of tumor cells will be discussed in detail in Chapter 12, authored by Ben Eliyahu and Avraham. The effects of stress on NK cell distribution and function will be discussed here briefly. Acute stress in humans, e.g., the stress experienced during either the delivery of a public speech or a first parachute jump, or the mimicking of acute stress via the infusion of an adrenergic agonist such as EPI, results in a rapid and transient increase in the number of NK cells in peripheral blood (Benschop et al., 1994; Benschop et al., 1996a; Kappel et al., 1998; Schedlowski et al., 1993; Tonnesen et al., 1987). This effect of both acute stress or EPI infusion is evident within 5–30 minutes and is prevented completely by administration of a β2AR antagonist

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(Benschop et al., 1994; Benschop et al., 1996a; Schedlowski et al., 1993). Thus, an increase in the level of circulating NE/EPI results in an increased number of circulating NK cells via stimulation of the β2AR. Although no formal proof exists to show that the effect on NK cell distribution is mediated directly via β2AR stimulation on an NK cell, the consensus is that NE/ EPI binding directly to the β2AR expressed on an NK cell results in the effect. In contrast, the stress-induced increase in PMN number in the peripheral blood was not inhibited completely by administration of a βAR antagonist (Benschop et al., 1996b), suggesting that other endogenous stress signals, such as glucocorticoids, are involved in the PMN distributional response to stress. Despite a vast body of literature describing the effects of acute stress and infusion of βAR agonists on innate immune cell distribution, little is known with respect to the mechanisms underlying this phenomenon. The first question that needed to be addressed concerned the source of the NK cells that enter the circulation, and the second concerned the mechanisms by which β2AR stimulation increased the number of NK cells in the peripheral blood. Based on studies performed in both humans and animals, it is now well established that the major source for the increased number of NK cells is the marginal pool of transiently immobilized leukocytes located along the blood vessel wall, as opposed to cells from the spleen or bone marrow. This transient immobilization results from changes in the interaction of adhesion molecules expressed on both endothelial cells and leukocytes, resulting in cell adherence to the blood vessel wall and creation of a shearing force to the blood flow. Also, because the blood flow is lower in venules than in arterioles, the marginal pool of leukocytes is present predominantly in venules. Although there is no direct evidence that NE/EPI reduce the adherence of cells to a blood vessel wall in vivo, there is evidence from an in vitro study to show NK cell adherence to cultured vascular endothelium is inhibited in the presence of a β2AR agonist (Benschop et al., 1993). 2. PMN/Monocytes/Macrophages/Dendritic Cells Acute administration of NE or EPI changes the number of PMN in the blood, an effect that is not only dependent on the βAR, but also involves the αAR (Dhabhar and McEwen, 1999). However, it remains unclear as to whether the AR subtypes involved in PMN distribution are on the PMN itself or on other cells. On a functional level, NE-depleted non-stressed animals show an enhanced early resistance to the intracellular pathogen Listeria monocytogenes (LM),

presumably due to increased PMN infiltration into infected organs during the early innate immune response (Rice et al., 2001). Similarly, an increase in NE that is induced by acute stress is associated with an increase in LM burden in infected organs at 3 days after infection, which results from reduced PMN migration (Cao et al., 2002). Again, NE depletion prevented this stress-induced increase in LM burden in infected organs. Thus, the possible underlying mechanism responsible for the increase in LM burden appears to involve an inhibitory action of NE on PMN migration and/or activity. As mentioned earlier, behavioral conditioning modulates the level of immune cell activity. In a recent study, the role of the SNS in the conditioning of the PMN response to activation by poly-IC was investigated (Chao et al., 2005). Poly-IC administration to animals, in combination with a specific smell, increases PMN activity. This effect was subsequently evoked upon re-exposure to the smell alone, and was prevented when NE was depleted. Similarly, it has been shown that ablation of the peripheral stores of NE has a marked effect on the defense against bacterial infection. For example, NE depletion prior to infection with Pseudomonas aeruginosa results in an improved phagocytic response of peritoneal cells and an increased influx of monocytes into the peritoneal cavity, leading to reduced dissemination of this microorganism (Straub et al., 2005c). The level of an innate immune response in vivo is modulated not only by endogenous adrenergic agonists such as NE and EPI, but also by pharmacological agents such as β2AR agonists. Interestingly, LPSinduced cytokine production in animals following administration of a β2AR agonist is dependent on the type of cytokine produced and the type of tissue analyzed (Eijkelkamp et al., 2004). In vivo, administration of a β2AR agonist decreases the LPS-induced production of IFN-γ by peritoneal cells in the small intestine and lung. IL-10 production, however, is modulated in a tissue-specific manner so that LPS-induced IL-10 in the peritoneal cavity is increased, but decreased in the ileum and the lung. These tissue site- and cytokinedependent differences in the effect induced of βAR activation is adding information to help better the understanding of how NE depletion either enhances or suppresses susceptibility to infection. Dendritic cells are called Langerhans cells when they reside in the skin. It appears that an NE/EPI may participate in a normal contact hypersensitivity response since the administration of a β2AR antagonist enhanced the normal response, suggesting an inhibitory influence of NE/EPI under normal circumstances (Seiffert et al., 2002). This finding is supported by


another report that NE inhibited Langerhans cell mobility (Maestroni, 2000).

B. Adaptive Immunity 1. T-Cells Findings indicated that a suppressed Th1 cellmediated contact hypersensitivity response occurred when NE was depleted either before or after sensitization in comparison to NE-intact control mice (Madden et al., 1989), suggesting that NE is needed for the generation of an optimal response at both the naïve and effector T-cell stages. This result was confirmed when mice were genetically deficient for the enzyme dopamine beta-hydroxylase, which is required for the synthesis of NE from dopamine, and which provides another means to deplete NE stores (Alaniz et al., 1999). These NE-deficient mice showed less protection against the Th1-promoting pathogens Listeria monocytogenes or Mycobacterium tuberculosis, and a significant decrease in the level of the Th1 cytokine IFN-γ produced, again suggesting that NE plays a role to upregulate the magnitude of a Th1 cell-mediated immune response. However, a 10-fold higher level of dopamine in these mice (Alaniz et al., 1999) may have caused the decrease in CD4+ T-cell function instead of the lack of NE (Kouassi et al., 1987). In contrast, NE may exert a suppressive effect on Th1 cell activity during a normal contact hypersensitivity response, possibly via an effect on the level of IL-12 produced by dendritic cells or on the ability of the cells to migrate to the lymph node (Maestroni et al., 2003; Seiffert et al., 2002). Taken together, most studies indicate that NE exerts an enhancing effect on either early naïve CD4+ T-cell development into a Th1 cell and/or the amount of IFN-γ secreted by the resulting Th1 cell. Analysis of T-cells that infiltrate the inflamed synovium of RA patients shows that there is a bias to a Th1-like cell phenotype and the production of IFN-γ (Feldmann et al., 1996). Likewise, animal models of arthritis are clearly Th1-cell dependent (Firestein, 2003). Blockade of the β2AR in rodents prior to, and during, arthritis delays the onset and reduces the severity of joint injury (Levine et al., 1988), suggesting that NE stimulation of the β2AR on a Th1 cell may exacerbate the disease process. The latter effect also appears to depend on the time when NE is released during the disease process, the level of sympathetic innervation within a specific tissue compartment, and the induction of anti- versus pro-inflammatory cytokines (Harle et al., 2005). β2AR stimulation on human peripheral blood mononuclear cells from RA patients induces a higher level of cAMP accumulation

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than does the stimulation on cells from healthy individuals (Lombardi et al., 1999). This increased sensitivity of the β2AR appears to be independent of a change in the level of β2AR expression, but may be associated with a 50% decrease in the expression and activity of the G-protein–related kinase 2 (GRK2) (Lombardi et al., 1999), which phosphorylates the β2AR upon agonist binding to desensitize the receptor (Benovic et al., 1986; Ferguson, 2001). Cells from rodents with adjuvant-induced arthritis also express less GRK2, and this effect is specifically found to exist in CD4+, but not CD8+, T-cells (Lombardi et al., 2001). Collectively, these findings in vitro suggest that the increased response to a β2AR agonist by human peripheral blood mononuclear cells from RA patients results from reduced GRK2 expression in immune cells, possibly Th1 cells, indicating that NE may use a similar mechanism in vivo. The role that NE plays in a Th2 cell-mediated response is less clear. When two different strains of mice, C57Bl/6J (Th1 cell-slanted strain) and Balb/c (Th2 cell-slanted strain), were depleted of NE and immunized with KLH, splenic cells from both strains of mice produced significantly higher levels of IL-2 and the Th2 cytokine IL-4 following reactivation in vitro when compared to cells isolated from immunized NE-intact controls (Kruszewska et al., 1995), suggesting that NE may help to produce a normal Th2 response. However, the mechanism by which this effect on Th2 cells occurs may be indirectly mediated by a direct effect on macrophages. In mice receiving a thermal burn injury, elevated plasma NE levels were associated with an increase in the production of the chemokine CCL2 production by macrophages, chemokine CCL3 production by T-cells, and the subsequent development of a predominant Th2-like response, effects that were prevented by NE depletion (Takahashi et al., 2004; Takahashi et al., 2005). This finding suggests that NE may influence susceptibility to infections after a thermal burn by regulating macrophage and T-cell chemokine secretion and a subsequent increase in Th2 cell accumulation at the site of injury, as has been described previously (Kobayashi et al., 1998). To date, few studies have shown a direct correlation between the levels and timing of NE exposure and/or β2AR stimulation with the number of CD8+ T-cells. In one study, administration of a monoamine oxidase inhibitor to tumor-bearing rats increased both NE levels and the percentage of CD8+ T-cells in the spleen (ThyagaRajan et al., 1999). With respect to the effect of NE/EPI exposure and/or β2AR stimulation on CD8+ T-cell activity, a few animal studies have provided some insight into what might be expected in humans.

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When mice were depleted of NE before sensitization with trinitrochlorobenzene (TNCB), the generation of hapten-specific CD8+ T-cell cytotoxicity decreased upon TNCB challenge in comparison to controls (Madden et al., 1989), suggesting that NE is required for the generation of cytotoxicity after initial antigen exposure. In contrast, exposure of mice to restraint stress decreased the generation of a CD8+ T-cell response to either HSV or influenza virus infection, and this effect was partially mediated by a βARinduced mechanism (Dobbs et al., 1993; Sheridan et al., 1998). Thus, a definitive answer about the effect of NE on murine CD8+ T-cell cytotoxicity remains unknown. In humans, certain types of physical and psychological stress have been shown to induce an increase in CD8+ T-cell number that is blocked by the administration of a βAR antagonist, suggesting that both CA release and βAR stimulation are involved in these stress-induced immune changes (Benschop et al., 1996a). In women exposed to a public-speaking stress, CD8+ T-cell number increased along with increased levels of NE, EPI, and cortisol (Mathews et al., 1983), suggesting that a concomitant increase in cortisol with NE does not alter the NE-induced increase on these particular immune parameters. A number of these studies also showed that a β2AR-subtype specificity exists for these effects since they were either induced by a β2AR selective agonist or blocked by a βARselective antagonist, but not by a β1AR-selective antagonist. In contrast, when the β2AR agonist terbutaline was administered to healthy subjects for 7 days, CD8+ T-cell numbers decreased, while cell numbers increased upon acute exposure (Maisel et al., 1990). Chronic exposure to a β2AR agonist in asthmatic individuals did not change the number of bronchial CD8+ T-cells (Roberts et al., 1999), while an increase in number was found in HIV-infected individuals (Sondergaard et al., 2000), suggesting that a disease process may influence the response to β2AR stimulation. Also, CD62L was increased on CD8+ T-cells from individuals exposed to dynamic exercise (Mills et al., 1999), suggesting that more naïve-type T-cells are localized into the circulation during exercise. Thus, in humans, acute exposure to NE/EPI appears to increase CD8+ T-cell number in the circulation, while chronic exposure appears to decrease. Taken together, NE may either enhance or suppress CD4+ and CD8+ T-cell function, depending on the specific type of immune response in which T-cell function was evaluated, the type of antigen-presenting cell involved in the response, the responsiveness of the APC itself to NE, and/or the time at which the AR is stimulated in relation to antigen activation.

C. B-Cell The role played by NE in regulating the magnitude of an antibody response has been implicated in studies conducted in NE-depleted mice. Data show that either a decrease (Ackerman et al., 1991; Cross et al., 1986; Fuchs et al., 1988; Hall et al., 1982; Kasahara et al., 1977a; Kasahara et al., 1977b; Livnat et al., 1985; Madden et al., 1989) or increase (Besedovsky et al., 1979; Chelmicka-Schorr et al., 1988; Miles et al., 1981) in the Th cell-dependent antibody response occurs after NE depletion. In a chronic depression mouse model, IgG levels increased acutely when the NE levels were elevated, but decreased chronically when the NE levels had returned to normal (Silberman et al., 2003). Also, anti-HSV IgM levels increased in young, but not old, mice that were exposed to moderate exercise training, and the increase was inhibited by blockade of the βAR, suggesting that NE was involved in elevating the antibody level (Kohut et al., 2004). Taken together, the majority of results in mice suggest that NE exerts a regulatory effect on the level of antibody produced by enhancing the endogenous activity of the immune cells that generate the response. Thus, the presence and concentration of NE at the time of antigen exposure may be a determining factor in the development of a normal primary and secondary antibody response to a T-cell-dependent antigen. The possibility exists, however, that the initial burst of NE that was released by 6-OHDA may have exerted an effect on immune cells before an NE-depleted state was obtained. To address this possibility, antigen-specific Th2 cells and B-cells were adoptively transferred into severe combined immunodeficient (scid) mice that were depleted of NE by 6-OHDA prior to cell transfer and immunization (Kohm and Sanders, 1999). A lower serum level of antigen-specific IgM and the neutralizing antibody isotype IgG1 was measured in these mice as compared to reconstituted NE-intact mice, an effect that was prevented by the administration of a β2AR-selective agonist at the time of immunization. Secondary immunization of these mice induced serum levels of antigen-specific IgG1 that were delayed in reaching a control level of response. It is important to note that NE depletion did not alter T- and B-cell trafficking to the spleen, but after immunization, NE depletion did decrease follicular expansion and germinal center formation when compared to NE-intact controls. Taken together, these data suggest that NE is needed to obtain a normal level of IgM and Th2 celldependent IgG1 during a primary and secondary Th2 cell-dependent antibody response in vivo. Because the β2AR is expressed on both the Th1 and B-cell, it has been more difficult to determine the effect

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of NE on the IgG2a response that develops due to the effect that NE has on the level of IFN-γ released by the Th1 cell. However, when mice deficient in dopamine beta-hydroxylase were studied, they exhibited normal lymphocyte development, but an impaired ability to produce Th1 cells and IFN-γ–dependent IgG2a (Alaniz et al., 1999), suggesting that NE is necessary for the development of Th1 cell immunity and subsequent IgG2a production, a finding that has been confirmed in vitro (Swanson et al., 2001). A recent study reported that stress diminishes the IgG2a response, but that this diminution appears to result from a stress-induced depletion of splenic NE that can be reversed pharmacologically (Kennedy et al., 2005), again suggesting that NE may exert an enhancing effect on IgG2a production. In contrast, mice exposed to either herpes simplex or influenza virus at the time of restraint stress had decreased numbers of virus-specific IgM-, IgG1-, and IgG2a-secreting cells in the lung, mediastinal LN, and spleen, but increased numbers were found in cervical LN cells, in a partially β2AR-mediated manner (Sheridan et al., 1998). The latter finding suggested that tissue-specific regulation of the antibody response may exist in response to changes in NE levels within lymphoid organs. Few in vivo human studies have shown directly that exposure to either NE or a β2AR agonist alters a specific isotype of antibody. In one study, the administration to asthmatics of either a β2AR agonist alone increased levels of serum IgG, but did not affect the level of any other isotype, including IgE (Manfield and Nelson, 1982). However, if the β2AR agonist was administered with cortisol, an increase in the level of serum IgG was measured. This finding suggests that a synergy may exist in humans between the β2AR agonist- and cortisol-induced signals to affect an immune cell function, such as IgG production. In contrast, peripheral blood cells obtained from women asked to perform a public-speaking task, which resulted in elevated levels of NE but not cortisol, produced less IgM and IgG in response to mitogen stimulation in vitro than when cells were taken before the stressful event (Matthews et al., 1995). At first glance, these findings may seem to contradict each other, in that β2AR stimulation alone appears to increase IgG in one study, but decrease IgG in the other. But again, the study designs need to be taken into account before the data are compared. In the first situation, the study was performed entirely in vivo and in individuals with asthma who had been chronically exposed to a specific allergen. In the other situation, the NE-exposed cells were cultured ex vivo and were activated with a polyclonal stimulant instead of a specific antigen. In addition, disparate results may also stem from variables

such as the tissue source of human cells for study, i.e., peripheral blood B-cells vs. tonsil B-cells. Thus, the effect of NE exposure and β2AR stimulation in vivo on human antibody production remains unclear, but the mechanism by which the effect occurs may depend on the presence or absence of a clinical disease, the presence or absence of another hormone, the type of activation stimulus used to induce a functional response, the length of time for β2AR stimulation, and/or the time of β2AR stimulation in relation to antigen receptor stimulation. The findings summarized thus far suggest strongly that NE is released upon antigen exposure from nerve terminals located within lymphoid tissue to serve as a messenger from the brain to cells of the immune system. This message is heard by the β2AR that is expressed on immune cells and is translated into an intracellular signal that regulates the magnitude of a specific immune cell response.

V. EVIDENCE FOR ADRENERGIC RECEPTOR REGULATION OF IMMUNE CELL ACTIVITY IN VITRO A. Innate Immunity 1. NK Cells Although data in the literature with respect to the in vivo effects of β2AR activation on NK cell activity are somewhat contradictory, in vitro studies clearly suggest that exposure of NK cells to a β2AR agonist reduces NK cell activity (Gan et al., 2002; Hellstrand et al., 1985; Hellstrand et al., 1989; Takamoto et al., 1991). Little is known, however, about the cellular mechanisms responsible for this effect. It has been suggested that treatment of NK cells with NE reduces the ability of NK cells to bind to a target cell, possibly via a mechanism involving reduced CD16 expression on NK cells. In addition, when IL-2–maintained NK cells were exposed to NE, the production of IFN-γ and TNF-α was inhibited, affecting NK cell maturation into fully functional cytotoxic cells (Gan et al., 2002). 2. PMN A key role in host defense against invading microorganisms is played by the PMN, which is a phagocytic cell that releases reactive oxygen species and anti-microbial proteins/peptides to kill bacteria. Activation of PMN by a variety of stimuli results in a “respiratory burst” that results in superoxide anion production, which is suppressed following exposure to a βAR agonist (O’Dowd et al., 2004). Moreover, there

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is evidence that a β2AR agonist inhibits the adhesion of PMN to vascular endothelium cultured in vitro (Bloemen et al., 1997), an effect which may contribute to the reduced tissue infiltration of PMN after treatment with a βAR agonist (Johnson and Rennard, 2001). In contrast, other data suggest that β2AR stimulation on PMN stimulates cell motility, which may result in increased accumulation of PMN at sites of inflammation (de Coupade et al., 2004). In this case, NE was not acting as a chemotactic factor in the classic sense; i.e., NE does not cause directed migration of PMN towards itself. However, NE does enhance non-directed migration, also known as chemokinesis, but inhibits directed migration of PMN, or chemotaxis, toward the chemokine IL-8 (de Coupade et al., 2004). Thus, NE may specifically enhance PMN non-directed, as opposed to directed, migration. 3. Macrophages The best-described effect of an adrenergic agonist on macrophage/monocyte function involves the level of cytokine produced. In most studies, LPS is used to induce the production of both pro- and anti-inflammatory cytokines, such as TNF-α, IL-12, IL-6, and IL-10. Agents that induce an increase in cAMP, including a β2AR agonist, inhibit the LPS-induced secretion of IL12 by monocytes and dendritic cells activated in vitro (Elenkov et al., 1996; Panina-Bordignon et al., 1997). In addition, the production of the pro-inflammatory cytokine TNF-α is sensitive to inhibition by a βAR agonist (Hetier et al., 1991; van der Poll et al., 1994). In contrast, IL-10 IL-8 production is enhanced by the addition of an βAR agonist to LPS-activated human monocytes or mouse peritoneal macrophages (Kavelaars et al., 1997; van der Poll et al., 1997a; van der Poll et al., 1997b). These effects of a βAR agonist on LPS-induced cytokine production by macrophages/monocytes is mimicked by other agents that raise the intracellular level of cAMP (Kavelaars et al., 1997), suggesting that the cAMP/protein kinase A (PKA) signaling pathway is responsible for the observed effects of βAR stimulation on the production of these cytokines and chemokines. There is also evidence that macrophages not only respond to a β2AR agonist, but also to αAR agonists. It should be noted, however, that although the response to a βAR agonist takes place under most circumstances in vivo, special endogenous situations likely occur that cause these cells to respond to an αAR agonist, also. For example, elicited peritoneal macrophages from specific pathogen-free mice respond to an α2AR agonist with increased secretion of TNF-α (Spengler et al.,

1990). In addition, when THP-1 cells, which represent a human monocytic cell line, are exposed to an α1AR agonist, the ERK and NK-κB pathways within the cell are activated to produce IL-6 (Bierhaus et al., 2003). However, when similar experiments were performed using peripheral blood monocytes from healthy individuals, an α1AR agonist did not have any effect on these cells (Rouppe van der Voort et al., 2000). There are, however, conditions in which we can detect an α1AR-induced increase in cytokine production by peripheral blood monocytes. For example, in vitro stimulation with LPS can induce expression of the α1AR, and subsequent triggering of this receptor with a specific agonist results in increased phosphorylation of ERK. Similarly, peripheral blood cells from children with juvenile idiopathic arthritis, a chronic inflammatory disease, express α1AR mRNA and respond to α1AR stimulation in vitro with an increase in production of the cytokines IL-6 and TNF-α (Heijnen et al., 1996). More recently, NE/EPI have been shown to modulate not only cytokine production by monocytes, but also the expression of matrix metalloproteases [(MMPs) (Speidl et al., 2004)]. MMPs degrade collagen, proteoglycans, and elastin, thus playing a role in various disease states, such as joint destruction in arthritic patients, rupture of atherosclerotic plaques, and macrophage-assisted tumor cell invasion into surrounding normal tissue (Speidl et al., 2004). Both primary human monocytes and monocyte-derived macrophages respond to LPS by increasing the secretion of MMPs, which is further enhanced by NE/EPI and inhibited by addition of a β1antagonist, but not a β2AR antagonist. These data suggest that monocyte/macrophages not only express a functional β2AR, but also a functional β1AR. 4. Dendritic Cells In contrast to the large number of studies describing modulation of monocyte/macrophage function by NE/EPI, little is known with respect to dendritic cells. One study shows that NE enhances skin dendritic cell migration from the site of antigen deposition to the draining lymph nodes in an α1/β2AR-dependent manner (Maestroni et al., 2003). In this response, NE may act as a chemotactic factor, although it may be that NE stimulates DC motility, rather than directed migration. In the same study, it was suggested that immature, but not mature, DCs express the αAR. Acute restraint stress in mice increases DC migration to the skin, an effect that is prevented by NE depletion (Maestroni et al., 2003). In addition, exposure of bone marrow-derived DCs to NEs reduces either LPS-


or protein antigen (KLH)–induced IL-12 release, but facilitates IL-10 release (Maestroni, 2002). Thus, in contrast to the effect described for NE on cytokine production by macrophages, the effect of NE on DC cytokine production is not exclusively mediated via the β2AR, since a β2AR antagonist reverses only part of the effect.

B. Adaptive Immunity 1. T-Cells An intracellular elevation in cAMP, the second messenger activated by β2AR stimulation, inhibits mature T-cell proliferation by decreasing IL-2 expression and secretion (Chen and Rothenberg, 1994; Mary et al., 1989; Tamir and Isakov, 1994; Wacholtz et al., 1991) and IL-2 receptor expression (Feldman et al., 1987; Krause et al., 1991; Tamir and Isakov, 1994). These findings suggest that there may be an effect of cAMP alone on naïve T-cell proliferation and IL-2 production, even though naïve CD4+ T-cells were not specifically isolated in these studies. When naïve CD4+ T-cells were isolated and activated, IL-2 secretion was decreased by exposure to NE, an effect that was prevented when a β2AR antagonist, but not β1AR or αAR antagonist, was added to the culture (Ramer-Quinn et al., 2000). These results suggested that NE and β2AR stimulation affect the ability of an activated naïve T-cell to produce IL-2 and, possibly, to expand in number. However, when the NE- or β2AR agonist-exposed naïve T-cells were cultured with either antigen/irradiated APC or antiCD3/anti-CD28, they produced an equal number of viable cells after 5 days in culture even though they produced less IL-2 (Swanson et al., 2001), suggesting that an early decrease in IL-2 after β2AR stimulation may affect initial naïve T-cell expansion, but that this effect on IL-2 may either dissipate over time or simply limit the number of T-cells that will go on to differentiate into an effector cell. In human T-cells activated via CD3, the β2AR inhibits the activation of NF-κB via a mechanism that involves the stabilization of its inhibitor protein, IκBα (Loop et al., 2004). In addition, high levels of NE appear to induce apoptosis and cell death (Bergquist et al., 1998; Del Rey et al., 2003), suggesting that other mechanisms for a diminished immune cell functional response may exist. These findings prompted study designs that would determine if NE and β2AR stimulation affected the ability of a naïve T-cell to develop into a Th1 or Th2 cell. Although the effect on Th2 cell development remains unclear, data show that NE stimulation of the β2AR on a naïve CD4+ T-cell does not affect the number of Th1 cells that develop under defined Th1-driving conditions, but does allow for the Th1 cells that develop

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to produce more IFN-γ per cell upon reactivation (Swanson et al., 2001). IL-12 appears to be essential for β2AR stimulation to exert this enhancing effect on resulting Th1 cell function, suggesting that the IL-12R and β2AR signaling pathways may interact with each other to mediate the enhancing effect. In this particular study, NE and β2AR stimulation had no effect on the level of IL-12 secreted by APC that activated the naïve T-cell, although the effect of NE on IL-12 production and Th1 development has been reported to be suppressed (Panina-Bordignon et al., 1997). Thus, NE may also have an effect on IL-12 production, which must be considered when evaluating any effects of NE on the development of a Th1 response in vivo. The effect of NE, β2AR stimulation, and cAMP on effector cell function itself, as opposed to naïve T-cell function, has been an area of active investigation. In Th1 cells, an increase in intracellular cAMP inhibits the production of IL-2 (Gajewski et al., 1990; Munoz et al., 1990; Novak and Rothenberg, 1990) and IFN-γ (Gajewski et al., 1990). For the mouse Th1 cell that expresses the β2AR, it appears that the timing of β2AR stimulation in relation to cell activation may play a role in determining the effect on Th1 cell activity. For example, exposure of Th1 cells to NE or a β2AR-selective agonist before their activation decreases both IL-2 and IFN-γ production (Sanders et al., 1997). However, stimulation either at the time of, or after, cell activation appears to be either without effect or induces a small increase in IFN-γ (Ramer-Quinn et al., 1997). In contrast, the effect of a cAMP increase in Th2 cells is either no change (Betz and Fox, 1991; Gajewski et al., 1990; Katamura et al., 1995; Novak and Rothenberg, 1990; Paliogianni and Boumpas, 1996; Pochet and Delespesse, 1983; Yoshimura et al., 1998), an inhibition (Borger et al., 1996a; Parker et al., 1995), or enhancement (Betz and Fox, 1991; Borger et al., 1996b; Crocker et al., 1996; Lacour et al., 1994; Naito et al., 1996; Wirth et al., 1996) of IL-4 and IL-5 production. However, it appears that NE has no effect on Th2 cell activity, likely because the Th2 cell does not express the β2AR (Sanders et al., 1997). Thus, an elevation in intracellular cAMP within each effector CD4+ T-cell subset is able to affect T-cell activity, but NE and β2AR stimulation affect only naïve T-cell and Th1 cell activity, with the effect on Th1 cells depending on the time of β2AR stimulation in relation to cell activation. In vitro exposure of human PBMC to dexamethasone, EPI, NE, or terbutaline induced a decrease in IFN-γ production, but an increase in IL-4 and IL-10, due primarily to a decrease induced in IL-12 production by APC (Agarwal et al., 1998a; Agarwal et al., 1998b; Agarwal et al., 2000; Elenkov et al., 1996; PaninaBordignon et al., 1997). These findings indicate that a

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decrease in IL-12 induced by either NE, a β2AR agonist, or cortisol prevents Th1 cell differentiation, resulting in a possible shift to Th2 cell development. However, when human HIV-infected PBMC were exposed in vitro to NE, stimulation of the β2AR decreased IFN-γ, IL-10, and IL-4 production and enhanced HIV replication in T-cells, an effect that was prevented when these cytokines were added exogenously to the cultures (Cole et al., 1998). Thus, the latter finding suggested that NE stimulation of the β2AR on T-cells may enhance HIV pathogenesis by inhibiting the production of all cytokines that would prevent HIV replication, namely IFN-γ, IL-10, and IL-4. Exposure of murine spleen cells to NE increased the generation of CD8+ T-cell lytic activity (Hatfield et al., 1986), perhaps by inhibiting the production of TNF-α, an effect that was reversed by the addition of exogenous cytokine (Kalinichenko et al., 1999). In contrast, EPI and a βAR agonist decreased the generation of CD8+ T-cell lytic activity (Cook-Mills et al., 1995; Strom et al., 1973). However, the timing of exposure to an NE/EPI or agonist in relation to the stage of CD8+ Tcell differentiation may be relevant to the outcome in cell activity. For example, the above-cited studies were conducted using cells exposed to AR ligands during the generation of the CTL response. If ligands were added after the generation of CTL, i.e., during the effector stage of the response to antigen, a decrease in CTL activity occurred (Cook-Mills et al., 1995; Strom et al., 1973) that may have been due to a cAMP-induced decrease in the TCR-dependent release of cytotoxic granules (Takayama et al., 1988). Thus, the role of NE and/or β2AR stimulation in modulating CD8+ T-cell activity remains uncertain in both humans and animals, but may be influenced by the time of β2AR stimulation in relation to the stage of CD8+ T-cell differentiation. 2. B-Cells β2AR stimulation and elevation of cAMP have been shown to affect B-cell proliferation (Blomhoff et al., 1987; Cohen and Rothstein, 1989; Diamantstein and Ulmer, 1975; Holte et al., 1988; Johnson et al., 1981; Muraguchi et al., 1984; Muthusamy et al., 1991; Vischer, 1976; Watson, 1975; Whisler et al., 1992) by either inhibiting early biochemical events and proliferation (Blomhoff et al., 1987; Holte et al., 1988; Muthusamy et al., 1991) or enhancing B-cell proliferation (Cohen and Rothstein., 1989; Li et al., 1989; Whisler et al., 1992). For a comprehensive review of the very early history of findings in this area, please refer to the following review article (Sanders and Munson, 1985a). A pharmacologic characterization of the enhancing effect of NE on the IgM response shows that either NE alone or

NE in the presence of the αAR antagonist phentolamine produced an enhanced IgM response, while in the presence of the βAR antagonist propranolol produced a control level response, suggesting that βAR activation was responsible for the enhancement (Sanders and Munson, 1984a). Selective β2AR stimulation with the agonist terbutaline enhanced the antibody response with a similar magnitude and kinetics to that produced by NE, and this enhancement was blocked with propranolol (Sanders and Munson, 1984b), suggesting that β2AR activation specifically was responsible for mediating the enhancing effect of NE. IgG1 production in vitro by B-cells cultured in the presence of an activating stimulus and a source for IL-4 is enhanced following β2AR stimulation and elevation of cAMP when compared to control cells (Kasprowicz et al., 2000; Podojil and Sanders, 2003; Roper et al., 1990; Roper et al., 2002; Stein and Phipps, 1991a; Stein and Phipps, 1991b; Stein and Phipps, 1992). However, the mechanism by which this enhancement was induced remained unknown until recently. Before one attempts to understand the mechanism by which β2AR stimulation regulates the level of IgG1 produced, a working knowledge of how the production of IgG is regulated at the molecular level is essential. First, Bcell activation in conjunction with specific cytokines induce class switch recombination (CSR) at the DNA level so that a B-cell will differentiate into a cell that produces an antibody isotype other than IgM. The CSR triggers the production of germline γ1 mRNA before the DNA completely rearranges for production of the mature form of IgG1 mRNA and protein that will be secreted from the cell (Stavnezer-Nordgren and Sirlin, 1986). The level of mature IgG1 produced by a B-cell is regulated by activity at the 3′-IgH enhancer region that is contained within the IgH locus in the mouse (Khamlichi et al., 2000). DNA sequences contained within the 3′-IgH enhancer region bind various transcription factors, including the B-cell–specific transcription factor Oct-2, which is known to regulate activity of the 3′-IgH enhancer in association with the coactivator OCA-B (Tang and Sharp, 1999). Both Oct-2 and OCA-B are induced when the activation receptor CD40 is stimulated on the B-cell (Pinaud et al., 2001; Stevens et al., 2000). Deletion of Oct-2 decreases the level of serum IgG1 in mice (Corcoran and Karvelas, 1994; Humbert and Corcoran, 1997), while deletion of OCA-B decreases serum IgG1 and germinal center formation (Kim et al., 1996; Nielsen et al., 1996; Schubart et al., 1996). Taken together, these data indicate that 3′-IgH enhancer activity is regulated by the transcription factor Oct-2 and its co-activator protein OCA-B, and that these proteins determine the rate at which IgG1 is produced after


switching. Therefore, regulation of an IgG1 response can occur at the level of either CSR and/or 3′-IgH enhancer activity. The Th2 cell that helps a B-cell to make IgG1 is not affected by β2AR stimulation since it does not express the receptor. Thus, the effect of β2AR stimulation on the B-cell alone could be ascertained because any contribution of an indirect effect that may be exerted on Th2 cell activity was eliminated. Using a model system of purified splenic naïve B-cells cultured in the presence of CD40L and IL-4, data show that β2AR stimulation increases the amount of IgG1 produced per B-cell, without affecting the number of cells that switch to produce IgG1, as compared to B-cells not exposed to β2AR stimulation (Kasprowicz et al., 2000; Podojil and Sanders, 2003; Podojil et al., 2004). β2AR stimulation mediates this enhancing effect in two ways: first, via a direct pathway from the β2AR that regulates the level of 3′-IgH enhancer activity (Podojil and Sanders, 2003); and second, via an indirect pathway that first upregulates expression of the co-stimulatory molecule CD86 (also known as B7-2), which upon stimulation with CD28, activates another signaling pathway in the Bcell that also regulates the level of 3′-IgH enhancer activity. More specifically, the OCA-B is upregulated by β2AR activation of PKA and subsequent phosphorylation of CREB (Podojil et al., 2004). Using chromatin immunoprecipitation, the increase in OCA-B translated into a higher level of OCA-B binding to the 3′IgH-enhancer region of the IgH locus (Podojil et al., 2004), suggesting that the coactivator was binding to a transcription factor that could activate 3′-IgH enhancer activity directly. Additionally, the level of Oct-2, the transcription factor that binds to the 3′-IgH enhancer, is increased indirectly via β2AR stimulation as a consequence of the β2AR-induced increase in CD86 expression (Podojil et al., 2004). Specifically, CD86 stimulation increases the level of Oct-2 expression and binding to the 3′-IgH enhancer via a newly characterized signaling pathway that involves NF-κB activation (Podojil et al., 2004). Thus, the β2AR-induced indirect increase in the level of the transcription factor Oct-2, along with the β2AR-induced increase in the level of its coactivator protein OCA-B, bind cooperatively to the 3′IgH enhancer region to increase the rate at which IgG1 mRNA is produced (Podojil et al., 2004). If this effect from β2AR stimulation in vitro is what happens normally during a B-cell response to antigen in vivo when NE is released within the microenvironment of B-cells responding to antigen during a Th2–B-cell interaction, then immune reactions studied in vitro in the absence of NE reflect responses that involve immune cells alone, and may not reflect what happens in vivo when

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NE is released to stimulate the β2AR. This probability is supported by in vivo data showing that antigen induces an increase in CD86 expression on a B-cell, but that NE depletion prevents this increase in CD86 from occurring (Kohm et al., 2002), suggesting that NE may regulate the level of a key co-stimulatory molecule that is involved in the co-stimulation of not only T-cells to stimulate cytokine production, but is also involved in direct signaling to the B-cell itself to regulate the level of IgG1 produced. For a more detailed description of how the study of the role played by the β2AR in enhancing the IgG1 response led to the discovery of the signaling pathway activated by CD86, please refer to the following review (Podojil et al., 2005). Taken together, these findings suggest that NE exerts a regulatory effect on the level of IgG1 produced by enhancing the endogenous activity of the B-cells that are activated to generate the response. These findings also suggest that signaling pathways activated in a B-cell through stimulation of an immunoreceptor (CD86) and a neuroreceptor (β2AR) converge to regulate the magnitude of an IgG1 response.

VI. RELEVANCE TO HEALTH AND DISEASE An understanding of the cellular, biochemical, and molecular mechanisms by which NE regulates the level of immune cell activity will potentially lead to the development of therapeutic approaches that will alter the etiology and/or progression of immune system-related diseases. Such therapeutic approaches will be important as one’s immune system encounters a multitude of exogenous and endogenous antigens. It will also be important to understand how one’s level of immunocompetence might affect different components of the nervous system that regulate the level of immune cell activity, e.g., the level of locally secreted NE within lymphoid tissue and/or the level of expression for AR subtypes on immune cells. To date, a few clinical examples support a role for a neuroimmune interrelationship in the etiology or progression of a disease state, and they will be discussed below. As summarized above, expression of the βAR on cells of the innate immune system contributes to suppression of innate immunity. Thus, during acute psychological or physical stress, it is likely that partial suppression of NK cell cytolytic activity and macrophage/PMN anti-microbial activity will occur. Is there evidence that the β2AR-mediated suppression of innate immunity takes place in clinically relevant situations? We think that the answer is yes. For example, the welldescribed effects of acute stress on, or the administra-

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tion of a β2AR agonist to, laboratory animals not only suppresses NK cell function, but also enhances tumor cell metastasis [reviewed in (Ben Eliyahu, 2003)]. In addition, the suppression of NK cell activity and the increase in metastasis formation precipitated by surgical stress is prevented by administration of a βAR antagonist, again indicating the clinical relevance of these findings to cancer therapy (Melamed et al., 2005). Additional details concerning the clinical relevance of βAR stimulation on innate immune cell activity during times of stress are discussed in Chapter 12 of this book, authored by Ben Eliyahu and Avraham. It is known that infections are a common complication of stroke, and that a large part of the mortality in stroke patients is due to complications that develop because of the infection. In an animal model of stroke, immune cell activity is suppressed, particularly the ability of NK and T-cells to produce IFN-γ (Prass et al., 2003). Moreover, the stroke-induced suppression of IFN-γ production was prevented by the administration of a βAR antagonist, as was the level of infection, suggesting that normal regulation of NK and T-cell activity by NE is dysregulated. Also, patients with chronic obstructive pulmonary disease (COPD), who are chronically exposed to a βAR agonist to relax constricted airway smooth muscles, may suffer the effect of β2AR-induced reduction in the number of PMN and an inhibition of PMN activity and mediator release. Moreover, there is evidence that a β2AR agonist promotes PMN apoptosis and reduces integrin expression on the cell surface [reviewed in (Johnson and Rennard, 2001)]. Thus, the effect of sympathetic neurotransmitters on immune cells that are essential for the clearance of infections needs to be considered, especially when the level of neurotransmitter may be more or less than normal. Evidence for the involvement of a neuroimmune interaction in the pathogenesis of an inflammatory disease, such as rheumatoid arthritis (RA), is suggested by a collection of clinical observations. First, the distribution of joint inflammation is mostly symmetric (Mitchell and Fries, 1982), as is the distribution of sympathetic nerves. Second, the joints in a patient with unilateral paralysis are spared from the inflammatory process on the affected side alone, suggesting that arthritis is limited to joints in which the SNS remains intact (Thompson and Bywaters, 1962). Third, NE and immune cells that express the β2AR are involved in regulating the intensity of joint inflammation (Straub et al., 2005a; Straub and Harle, 2005b). And finally, stress often precedes disease exacerbation (Baker, 1982; Herrmann et al., 2000), suggesting that the SNS and HPA are activated to release NE/EPI and/or corticosteroids, respectively. Therefore, in RA patients where

the systemic level of IFN-γ is elevated, as is the local level at the inflammatory sites (Park et al., 2001; Yin et al., 1999), it is possible that disease onset and/or progression is promoted by a mechanism that involves an SNS-induced, β2AR-mediated effect on a naïve CD4+ T-cell to increase the level of IFN-γ produced by the Th1 cells that develop, as has been reported for murine naïve CD4+ T-cells (Swanson et al., 2001). In this manner, Th1 cells secreting a higher level of IFN-γ might accumulate within the synovial fluid to promote the inflammatory process. Such a finding would indicate that a neuroimmune process was involved in the disease process and, therefore, should be controllable via pharmacological therapeutic approaches. Such findings may also have relevance for the design of vaccination protocols in which the goal might be to increase IFN-γ production by a limited number of Th1 cells that may develop under conditions where IL-12 is limited. The administration of a β2AR drug may have beneficial effects for patients with other inflammatory autoimmune diseases. For example, the administration of a β2AR agonist to patients with multiple sclerosis suppresses pro-inflammatory cytokine production, which may help to reduce the inflammatory response in these patients. Interestingly, there is evidence from patients with multiple sclerosis (Giorelli et al., 2004; Vroon et al., 2005), as well as from patients with rheumatoid arthritis, that immune cells are more sensitive for regulation of the immune response by a βAR agonist, an effect which may be beneficial (Lombardi et al., 1999). Based on this information, it was shown that the administration of a βAR agonist to rats with adjuvant arthritis increases production of the anti-inflammatory cytokines IL-10, TGF-β, and IL-1RA, but reduced production of the pro-inflammatory cytokines TNF-α and IFN-γ at mucosal sites (Cobelens et al., 2002). More importantly, however, results show that administration of a βAR agonist to rats with adjuvant arthritis, together with oral administration of a tolerogenic antigen, suppresses disease symptoms, while administration of either the antigen or the β2AR agonist alone did not have an effect. These data suggest that administration of a βAR agonist during an inflammatory disease process may modify the cytokine milieu so that tolerogenic processes can develop. An understanding of the regulatory mechanism by which CD86 and/or β2AR stimulation increases the level of IgG1 produced by a murine B-cell may help to explain how acute stress has been linked to an enhancement of IgG immunity in humans. A recent study reported that the drug ephedrine exacerbates lupus symptoms in lupus-prone mice via a mechanism that appears to involve stimulation of the bAR on a B-cell

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to increase the level of IgG (Hudson et al., 2005), suggesting that NE may regulate the severity of this disease process. In acute stress, an increase in the level of NE/EPI released from lymphoid organ nerve terminals, above that released by antigen alone, might stimulate a higher number of the β2AR on a B-cell to induce the B-cell to produce a higher level of IgG, as reported in mice (Kohm and Sanders, 1999; Podojil and Sanders, 2003; Podojil et al., 2004). While in chronic stress, the β2AR may be downregulated, thus preventing optimal β2AR stimulation on a B-cell and, consequently, preventing optimal IgG production to occur. However, a recent study reported that chronic stress in mice is associated with no change in NE levels, but is associated with an increase in β2AR expression on immune cells and an increased sympathetic effect on T- and B-cell responses (Edgar et al., 2003). The latter findings may have relevance for vaccination protocols in which the goal might be to raise the level of humoral immunity in new recruits to the armed forces, especially when vaccinations occur just prior to deployment when stress levels have been elevated for days. An increase in sympathetic tone that is induced by the anxiety of surgery may suppress immune cell function to slow down wound healing, and thus increase the chance for wound infection (Glaser et al., 1999; Glaser et al., 2005; Kiecolt-Glaser et al., 1998; Rozlog et al., 1999). Likewise, an increase in sympathetic tone during exercise may enhance the intensity and duration of a humoral immune response to antigen, and consequently affect the likelihood of the development of infectious conditions such as colds and flu (Gleeson et al., 2004). Interventions that include exercise may decrease anxiety, depression, and other psychological states, which appear to have a negative effect on immunity, but increase humoral immunity. Also, the activation or suppression of sympathetic tone during psychological and/or behavioral interventions for clinical conditions such as either depression or HIV infection may affect immune status and the ultimate survival of the affected individual (Robinson et al., 2000). Also, if NE plays a role in regulating immune cell function, then an age-related decline in lymphoid tissue innervation (Bellinger et al., 1992; Madden, 2000) may contribute to the age-associated increase in the incidence of autoimmunity, cancer, and susceptibility to infection (Biondi and Zannino, 1997; Caruso et al., 2004; Kiecolt-Glaser and Glaser, 1999). An understanding of this neuroimmune interaction would be useful in the design of targeted therapeutic approaches for aged individuals who might manifest changes in the level of adrenergic receptors and/or their ligands at the time of flu or pneumococcal vaccination.

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3 Cholinergic Regulation of Inflammation CHRISTOPHER J. CZURA, MAURICIO ROSAS–BALLINA, AND KEVIN J. TRACEY

I. INTRODUCTION 85 II. BRAIN-IMMUNE COMMUNICATION 86 III. THE CHOLINERGIC ANTI-INFLAMMATORY PATHWAY IN MEDICINE 89 IV. PERSPECTIVES 93

in order to prevent shock, tissue injury arthritis, and the other complications of excessive cytokine activity. These redundant anti-inflammatory mechanisms are integral to the host response because they effectively inhibit the release of cytokines by macrophages and other cells. Macrophage deactivating factors accumulate at the local site of infection (e.g., prostaglandin E2 and spermine) and are released systemically (e.g., IL10, TGF-β, glucocorticoids). Local accumulation of prostaglandin E2 (PGE2) inhibits synthesis of proinflammatory cytokines and restrains acute cytokine responses (1). Spermine is a ubiquitous biogenic molecule that accumulates at sites of infection or injury and inhibits the activity of several cytokines at a posttranscriptional level, including TNF, IL-1, and the macrophage inflammatory proteins 1α and 1β (MIP1α, MIP-1β) in macrophages and monocytes (2–5). Anti-inflammatory cytokines such as IL-10 and transforming growth factor β (TGF-β) also serve to downregulate inflammatory responses. IL-10 deactivates macrophages in culture; in trauma patients, TNF levels are higher when IL-10 levels are depressed, an indication of pending septic complications (6–8). Elevated levels of TGF-β, a potent inhibitor of monocyte activation, have been observed in monocytes derived from immunosuppressed trauma patients (9;10). There also are mechanisms to inhibit the activity of cytokines that have already been released. For example, TNF receptor fragments cleaved from the cell surface are released as soluble factors that bind and neutralize the cytokine activity of TNF. Surprisingly, recent evidence now implicates a critical controlling role for the nervous system in both detecting and suppressing inflammation via the

I. INTRODUCTION The innate immune system is the front line of host defense against tissue injury and infection. Infectious agents and tissues damaged by injury release chemotactic factors that recruit leukocytes to the site of injury, which recognize and neutralize endogenous and exogenous cellular debris and invading pathogens. These activated cells release pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, and HMGB1, which amplify the inflammatory response, modulate wound healing and tissue remodeling, and activate specific immune responses. In the majority of cases, these and other inflammatory responses culminate in the resolution of infection and healing of the wound. Occasionally, however, the innate immune response can spiral out of control, leading to widespread cytokine release that spills out of the local environment to cause shock, tissue injury, diffuse coagulation, organ ischemia, and even death. Excessive pro-inflammatory cytokine activity has a causative role in human disease, as evidenced by the success of anti-TNF strategies for treatment of rheumatoid arthritis and Crohn’s disease. Evolution has conferred upon mammals several distinct counter-regulatory mechanisms that “normally” restrain the pro-inflammatory cytokine response PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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“inflammatory reflex.” Inflammation is sensed by afferent fibers in the vagus nerve, which innervates the reticuloendothelial system and other major organs. Low levels of cytokines produced in tissues activate an afferent vagus nerve signal that elicits the central nervous system responses to inflammation (e.g., fever, sickness behaviors). Indeed, an intact vagus nerve is required to mount appropriate physiological responses to inflammation, because vagotomy ablates cytokineinduced fever in response to exposure to low doses of IL-1 in the abdomen. Since it innervates the liver, spleen, lungs, and other visceral organs that act as filters or routes of entry for pathogens or their products, the vagus nerve is uniquely positioned to anatomically modulate and integrate a range of potential infectious or injurious threats. The sensory fibers terminate in the dorsal vagus complex in the medulla oblongata; the efferent vagus fibers originate in the dorsal vagus complex (and particularly the nucleus tractus solitarius). Together these vagus nerve nuclei can interact with forebrain regions, the hypothalamus, and amygdala, to sense peripheral physiological activity, including immune status, integrate the information, and coordinate appropriate responses to maintain homeostasis. We discovered that the efferent arm of the inflammatory reflex is a vagus nerve-dependent cholinergic anti-inflammatory pathway which inhibits pro-inflammatory cytokine synthesis in animal models of endotoxemia, ischemia, and other types of inflammation. Electrical stimulation of the vagus nerve inhibits cytokine release and protects animals against local inflammation, as well as systemic inflammation associated with endotoxemia, hypovolemic shock, and septic peritonitis. Pharmacological stimulation of this pathway using either peripheral cholinergic agonists that target cytokine-secreting cells, or using centrally acting compounds that cross the blood-brain barrier and activate efferent vagus nerve activity, exert significant protection against cytokine-mediated diseases (11–13). Acetylcholine, the principal neurotransmitter of the vagus, interacts with the α7 subunit of nicotinic acetylcholine receptors specifically expressed on macrophages to inhibit the release of proinflammatory cytokines including TNF, IL-1, IL-6, IL18, and HMGB1, without affecting the release of the anti-inflammatory cytokines such as IL-10 (13;14). Disruption of this cholinergic anti-inflammatory pathway, either by surgical vagotomy or genetic knockout of the α7 subunit, renders animals more sensitive to inflammatory stimuli than their wild-type counterparts, indicating that the cholinergic anti-inflammatory pathway exerts tonic regulatory effects on innate immune responses (14;15). Cholinergic agonists that inhibit

macrophage release of pro-inflammatory cytokines significantly improve survival in animal models of local and systemic inflammation, together indicating that the α7 subunit of the nicotinic acetylcholine receptor is an experimental therapeutic target for diseases characterized by excessive pro-inflammatory cytokine activity (13;14;16).

II. BRAIN-IMMUNE COMMUNICATION The nervous system is composed of the somatic motor system, which controls voluntary movements, and the autonomic motor system, which controls involuntarily visceral functions. Two divisions of the autonomic nervous system—the parasympathetic and the sympathetic—continuously regulate basic physiological responses, including heart rate and blood pressure, respiratory rate, gastrointestinal motility, and body temperature. The autonomic nervous system detects the physiologic status of the major body systems and relays this information to the primitive brain, including the limbic system, which interprets this information and coordinates sympathetic and parasympathetic neural responses to maintain homeostasis. This physiological regulatory activity occurs subconsciously, with the hypothalamus acting as a gatekeeper, letting less than 1% of ascending information to penetrate to higher brain centers. New insights have revealed a basic neural pathway that reflexively monitors and adjusts the inflammatory response, not unlike the regulation of instantaneous heart rate. Inflammatory stimuli activate afferent pathways within the vagus nerve that are relayed to the hypothalamus. Like the baroreflex, in which the stretching of the carotid or aortic sinuses elicits an efferent response to prolong the time to the next heartbeat, inflammatory input can activate an anti-inflammatory response that rapidly inhibits the subsequent production of pro-inflammatory cytokines. Thus, the nervous system can gather sensory information about inflammatory or invasive events from multiple local sites, and integrate this data with information about other aspects of health and function. Therefore, the central nervous system is a central coordinator of the immunological homeostasis.

A. Anatomy of the Vagus Nerve The vagus nerve, one of 12 pairs of cranial nerves, is the predominant neural effector of the parasympathetic nervous system that comprises 75% of all parasympathetic nerve fibers. The vagus nerve is particularly well positioned to interface the immune and central

3. Cholinergic Regulation of Inflammation

nervous systems because it innervates the organs that are portals of entry or filters for pathogens and their products. The vagus nerve has motor functions in the larynx, diaphragm, stomach, and heart; and sensory functions in the ears, tongue, and visceral organs, including the liver. Inflammatory signals in peripheral tissues activate afferent signals in the vagus nerve, which are relayed to the hypothalamus and stimulate the release of ACTH and the development of fever after administration of endotoxin (17;18). The vagus nerve contains three fiber types: highly myelinated A fibers, which have low activation thresholds; lightly myelinated B fibers; and unmyelinated C fibers, which have high activation thresholds. The B and C fibers have been implicated in regulation of heart rate (19–21); no specific function has been attributed to the vagal A fibers, but our recent results suggest that the A fibers may contribute to the regulation of cytokine release. In agreement with this possibility, vagus nerve inhibition of cytokine release is specific and can be dissociated from the vagus nerve regulation of the heart rate (Huston, J., unpublished data). Moreover, the cholinergic anti-inflammatory pathway has a low activation threshold, as would be expected from an A-fiber dependent pathway. Percutaneous stimulation of the vagus via carotid massage is sufficient to inhibit systemic inflammatory responses to endotoxin challenge (Huston, J., unpublished data). Ongoing studies may delineate an immunologicalregulatory role for the vagus nerve A fibers, which are heavily myelinated and therefore most sensitive to activation.

B. Sensing Peripheral Inflammation The central nervous system receives sensory input from the immune system through both humoral and neural routes. Blalock suggested in 1984 that the immune system functions as a “sixth sense” that detects microbial invasion and produces molecules to relay this information to the brain (22;23). IL-1, TNF, and other immunological mediators can gain access to brain centers that are devoid of a blood-brain barrier, known as circumventricular regions, or can penetrate other regions of the brain by active transport systems. Very low levels of IL-1 and endotoxin in the tissues can also activate afferent vagus nerve signals at thresholds well below the serum levels that are required to achieve signaling to the brain via the humoral route. Activation of the vagus nerve by these extremely low concentrations can mediate sickness behaviors, including food aversion and fever. Both the humoral and neural routes for immune system to nervous system communication have been implicated in the develop-

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ment of fever, anorexia, activation of hypothalamicpituitary responses to infection and injury, and other behavioral manifestations of illness. 1. Humoral Mechanisms of Immune to Brain Communication TNF and IL-1 are actively transported across the blood-brain barrier, and IL-1α directly enters the brain through circumventricular organs such as the pineal gland, area postrema, median eminence, and the neural lobe of the pituitary, where the blood-brain barrier is non-existent or discontinuous (24–26). IL-1α also penetrates these areas of the brain through a putative transport mechanism in the vasculature (25;27). Microglia and endothelial cells of the cerebral vasculature express IL-1 receptors (28;29), as well as the coreceptors for endotoxin, CD14 (30) and Toll-like receptor 4 (TLR4) (31). Once cytokines gain access to circumventricular organs, they can induce widereaching effects through second messengers such as cyclooxygenase (COX2), which processes arachadonic acid to release prostaglandin E2 (PGE2); prostaglandin receptors localize to areas of the brain that play important roles in peripheral inflammatory responses such as activation of the HPA axis and fever (32–34). High molecular weight, blood-borne inflammatory mediators therefore can directly inform the central nervous system of inflammatory conditions in the periphery. The humoral route seems to be a predominant means for immune-to-brain communication when circulating cytokine levels are high. 2. Neural Mechanisms of Immune to Brain Communication The vagus nerve is a mixed nerve composed of approximately 80% sensory fibers relaying information between the brain from the head, neck, thorax, and abdomen. The central nervous system can become informed about the status of inflammation via afferent neural signals, even when blood cytokine levels are low, because locally increased levels of cytokines in tissues can activate an afferent signal in the vagus nerve. Sensory innervation of immune organs by ascending fibers traveling in the vagus nerve, as well as other pain and ascending sensory pathways, provides important input about the status of invasive and injurious challenges in distributed body compartments (19). Importantly, these neural inflammation-sensing pathways function at lower detection thresholds and activate responses even when the inflammatory agents are present in tissues at levels that are not high enough to reach the brain through the bloodstream. Linda

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Watkins and colleagues provided critical insight into the sensory role of afferent vagus nerve fibers by observing that vagotomy blunts the development of fever in animals exposed to low, intra-abdominal doses of IL-1 or endotoxin (18;35–37). It is not entirely clear how the vagus nerve “detects” the presence of inflammatory mediators, but cells isolated from the vagus nerve can express IL-1 receptor mRNA, and discrete IL-1 binding sites have been identified on “glomus” cells (38;39). Electrophysiological studies indicate that vagus nerve signals can also be activated by mechanoreceptors, chemoreceptors, temperature sensors, and osmolarity sensors, each of which can be activated at an inflammatory locus (40). The sensory afferent cell bodies of the vagus nerve reside in the nodose ganglion and transmit information to the area postrema and nucleus tractus solitaries (NTS), two regions that are active during peripheral inflammation. Ascending vagus nerve signals excite second-order neurons within the NTS (41). The NTS forms the apex of a feedback loop that modulates visceral activity through two mechanisms: NTS neurons inhibit a subset of neurons within the dorsal motor nucleus of the vagus (DMV) that activate the viscera and the digestive tract. The NTS also suppresses visceral activity by activating inhibitory DMV efferent neurons. The NTS and DMV both contain blood vessels that lack a functional blood-brain barrier, thereby allowing these brain regions to respond to either diffusible circulating factors such as LPS, TNF, and IL-1 as well as to signals arriving via the ascending vagus nerve (42). Information projecting from the NTS can in turn be relayed to the higher brain via autonomic feedback loops, direct projections to the medullary reticular formations, or through the parabrachial nucleus and the locus ceruleus. Connections from the latter emanate to the hypothalamus, the amygdala (mood regulation), and the entire forebrain [for a review see (43)].

C. The Cholinergic Anti-Inflammatory Pathway The cholinergic anti-inflammatory pathway is a neural mechanism that inhibits pro-inflammatory cytokine release via signals that require the vagus nerve and α7 receptors (12;14;15;44;45). Experimental activation of the cholinergic anti-inflammatory pathway by direct electrical stimulation of efferent vagus nerve inhibits the synthesis of TNF in liver, spleen, and heart, and attenuates serum TNF levels during endotoxemia, ischemia/reperfusion injury, hemorrhagic shock, septic peritonitis, and other dis-

eases associated with excessive cytokine release (12;14;15;44;45). Vagotomy or genetic disruption of the α7 gene significantly exacerbates TNF responses to inflammatory stimuli and sensitizes animals to the lethal effects of endotoxin (15), suggesting that cholinergic anti-inflammatory signals transmitted via the efferent vagus nerve play a role in maintaining immunological homeostasis. The synapse between the cholinergic nervous system and the innate immune system requires the α7 subunit of the acetylcholine receptor, a nicotinic αbungarotoxin-sensitive cholinergic receptor expressed on macrophages and other immunocompetent cells that modulate or participate in the inflammatory response (15). Cholinergic agonists, including nicotine and acetylcholine, significantly inhibit the release of TNF and other cytokines from endotoxin-stimulated human macrophages. Tissue macrophages, not circulating monocytes, produce most of the inflammatory cytokines released systemically during excessive inflammatory responses. The interaction of cholinergic agonists with the acetylcholine receptor inhibits the synthesis of pro-inflammatory cytokines, but not antiinflammatory cytokines (e.g., IL-10) (13–15). Monocytes, which express little or no α7 receptor, are refractory to the cytokine-inhibiting effects of acetylcholine; only supra-physiological concentrations of cholinergic agonists inhibit monocyte cytokine synthesis (14). Macrophage expression of the α7 subunit of nicotinic acetylcholine receptor distinguishes the cholinergic anti-inflammatory pathway from muscarinic receptor activities identified previously on lymphocytes, peripheral blood mononuclear cells, and alveolar macrophages (46;47). Activation of α7 transduction in macrophages inhibits endotoxin-induced NF-κB activation but does not affect the activation of several MAP kinases typically associated with endotoxin signaling (13). Since macrophages are exquisitely sensitive to acetylcholine, it is plausible that other, non-neuronal acetylcholine producing cells (e.g., epithelial cells, T lymphocytes, endothelial cells) might also modulate cytokine synthesis in nearby macrophages (48;49). Most studies to date characterizing the cholinergic– anti-inflammatory pathway have focused on the macrophage/acetylcholine interaction, but other cell types, particularly the endothelium, are also potentially regulated by acetylcholine. Activation of endothelial cells during inflammation, characterized by increased adhesion molecule expression and inflammatory mediator production, plays a critical role in the adhesion and subsequent trafficking of inflammatory leu-


kocytes. Inflammatory mediators such as chemokines present on the surface of the endothelium progressively activate leukocytes rolling across the vasculature; for example, neutrophils become activated upon binding to endothelium-expressed adhesion molecules (50;51). Human microvascular endothelial cells express the α7 subunit of the acetylcholine receptor on the cell surface, and acetycholine significantly blocks TNFinduced adhesion molecule expression and chemokine expression in a concentration-dependent manner (16). Cholinergic signaling via receptor agonists (e.g., nicotine) or direct electrical vagus nerve stimulation inhibits leukocyte migration across the endothelium in vivo; notably, this effect can be blocked with mecamylamine, a negative allosteric modulator of nicotinic acetylcholine receptors, indicating that the effects of cholinergic agonists are not due to the activity of other receptor interactions (16). Acetycholine also modulates intestinal inflammatory responses and decreases histamine release by airway mucosal mast cells (52). Nicotine has anti-inflammatory effects on several other cell types, including monocytes, epithelial cells, T-cells, and neutrophils, but the role of the α7 acetylcholine receptor subunit in these cells has not been specifically defined (53–57). Our recent studies indicate that electrically stimulating the cholinergic anti-inflammatory pathway significantly inhibits TNF synthesis in spleen. Surgical ablation of the common celiac nerve to spleen abolishes the effects of vagus nerve stimulation on serum TNF levels, whereas transection of other abdominal branches of the vagus does not interfere with the cytokine-inhibiting activity of vagus nerve stimulation (Huston, et al. submitted.)

III. THE CHOLINERGIC ANTIINFLAMMATORY PATHWAY IN MEDICINE It now appears that the vagus nerve is an integral component of a reflex loop that can detect and regulate inflammatory responses in real-time. Inflammation activates an ascending signal that can be relayed to the hypothalamus to activate humoral anti-inflammatory mechanisms; efferent vagus nerve signals can rapidly and specifically inhibit macrophages in tissues (58). Physiological anti-inflammatory mechanisms represent efficient systems that have been selected by evolution to control inflammation. Now perhaps, they can be exploited for the treatment of inflammatory disorders. Several approaches have been used successfully

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to treat or prevent inflammatory disease in animals; some of these may well be adapted for therapeutic use in the clinic (13–15).

A. Electrical Vagus Nerve Stimulation The “hard-wired” connection between the nervous and immune systems functions as an antiinflammatory mechanism in several models of systemic and local inflammation. Rats subjected to bilateral cervical vagotomy are more sensitive to subsequent injection of endotoxin than animals that have undergone sham surgery; vagotomized animals develop endotoxemic shock more quickly and exhibit a more robust systemic inflammatory response, as compared to sham animals. Electrical stimulation of the vagus nerve attenuates the release of pro-inflammatory cytokines, including TNF, and IL-1 in murine endotoxemia, and also protects against endotoxemic shock (hypotension) (14;15). Stimulation of the vagus nerve has similar effects in several animal models of systemic inflammatory disease, including peritonitis/sepsis, ischemia/ reperfusion injury, and hypovolemic shock [(12;44); personal observations]. Interestingly, electrical vagus nerve stimulation also attenuates inflammatory responses in animal models of local inflammation, including carrageenan-induced paw edema and the carrageenan air pouch model; these cutaneous inflammation models are characterized by sustained endothelial cell activation (16). Furthermore, electrical stimulation of the vagus nerve increased gastric emptying, activated STAT3 in intestinal macrophages, and reduced intestinal inflammation as measured by neutrophil infiltration into the muscularis layer of the gut (59). The voltage and frequency parameters sufficient to activate the cholinergic anti-inflammatory pathway are beneath the threshold required to activate cardiac vagal fibers, indicating that vagus nerve stimulation may be a practical and efficient means of therapeutically regulating peripheral inflammation in human disease (personal observations). This approach to regulating systemic or local inflammation may have significant therapeutic potential, because vagus nerve stimulators are clinically approved devices used for the treatment of epilepsy and depression in patients refractory to other treatments. This technology to treat surgically refractory epilepsy was developed based on anecdotal observations that carotid massage, which activates vagus nerve firing, reduced seizure. Vagus nerve stimulators have been safely used in thousands of patients, but their effects on the immune response remain unknown.

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B. a7 Agonists: A New Class of Anti-inflammatory Agents α7 receptors are required for the cytokinesuppressing effects of the cholinergic anti-inflammatory pathway in animals. Activation of α7 with acetylcholine or nicotine activates calcium influx in macrophages and suppresses endotoxin-induced release of pro-inflammatory cytokines, including TNF, IL-1, and HMGB1. These observations indicate that cholinergic agonists may modulate inflammatory responses in vivo and may be an effective approach to treat diseases mediated by cytokine excess. Animal models using cholinergic agonists, including the non-specific agonist nicotine, have provided proof-of-concept data that indicate that such an approach may be successful. Although the antiinflammatory properties of nicotine are well described, the precise mechanism by which nicotine inhibits inflammation had not been previously understood. Nicotine is a relatively non-specific α7 agonist that provides significant survival advantages to animals subjected to endotoxemia or peritonitis/sepsis, and suppresses the progression of experimental ulcerative colitis and cutaneous inflammation (13;60–62). Nicotine suppresses leukocyte migration into carrageenan air pouches and decreases inflammatory infiltrate in the ears of mice subjected to the Shwartzmann reaction. The effect of nicotine on inflammatory cell recruitment is dependent upon cholinergic receptors, because the receptor antagonist mecamylamine reverses the protective effects of nicotine (16). The endogenous α7 agonist, acetylcholine, is a physiological anti-inflammatory modulator of HMGB1 release, suggesting that the cholinergic antiinflammatory pathway can be exploited for the treatment of severe sepsis (13). Nicotine recapitulates the inhibitory effects of acetylcholine on HMGB1 release, through a mechanism that requires the α7 subunit of the acetylcholine receptor to inhibit the NF-κB pathway (13). Novel, more specific α7 agonists are also in development and under study. We have found that α7 agonists are effective inhibitors of NF-κB nuclear translocation, cytokine release, and endothelial cell and macrophage activation, in murine models of local (carrageenan air pouch) and systemic (endotoxemia, peritonitis/sepsis) inflammation (13;16). α7 inhibition of the NF-κB pathway may not be the primary mechanism of action of these agents, and additional signal transduction mechanisms leading to macrophage inactivation are under study. Recent in vitro experiments have demonstrated that activation of peritoneal macrophages through the α7 nicotinic receptor is associated with activation of the transcrip-

tion factor STAT3, through a JAK2-dependent mechanism (59). Nicotine also has proven efficacy in several clinical trials; randomized, placebo-controlled trials showed that nicotine treatment was associated with significant remissions in ulcerative colitis severity (61;63). While the precise mechanism of the anti-inflammatory effects of nicotine is unproven in human disease, recent data from cell culture and animal studies indicate that the protection may have been achieved by decreasing cytokine release. 1. In Vitro Macrophages. The α7 subunit of the acetylcholine receptor is an essential regulator of inflammation in vivo, because transgenic animals devoid of α7 are exquisitely sensitive to inflammatory stimuli, and produce significantly higher levels of proinflammatory cytokines in response to endotoxin challenge than do their wild-type counterparts. In vitro, acetylcholine significantly and dose-dependently inhibits pro-inflammatory cytokine release from endotoxin-stimulated macrophages, but its in vivo use as an anti-inflammatory agent is limited by its extremely short extra-synaptic half-life. Nicotine, which has a half-maximal effective concentration (EC50 = 40 μM) on calcium influx that is three-fold lower than that of acetylcholine (EC50 = 150 μM) (64;65), is a suboptimal therapeutic because of its associated toxicity, short half-life, lack of specificity for α7, and undesirable pharmacokinetics. It is useful as a test agent in animal models of inflammation because it inhibits endotoxininduced release of TNF, IL-1, IL-18, and HMGB1 (and TNF-induced HMGB1 release) from the murine macrophage-like cell line RAW264.7 and differentiated human macrophages in culture. The antiinflammatory activity of nicotine is dependent upon α7 signaling, because transfection of macrophages with antisense oligonucleotides against α7, but not α1, α9, or α10, blocks the effects of nicotine and restores macrophage cytokine responses to endotoxin despite the presence of nicotine in the cell culture medium. Mecamylamine, a general nicotinic receptor antagonist, and α-conotoxin IMI, a selective α7 antagonist, also inhibit the anti-inflammatory effects of nicotine. In an alveolar macrophage cell line (MH-S), nicotine inhibited cytokine release in response to Legionella pneumophila infection and prevented macrophage clearance of the bacteria in vitro. Interestingly, this macrophage cell line does not express α7, and the immunosuppressive effects of nicotine appear instead to be dependent upon nicotinic receptors that include the α4 and β2 subunits. Although the intracellular sig-


naling cascades activated by α7 in response to ligand have not been fully characterized in macrophages, nicotine inhibits endotoxin- and peptidoglycaninduced activation of NF-κB, but not the MAP kinases Erk, JNK, or p38 (13). Endothelium. The endothelium is an important component of the inflammatory response. Endothelial cell expression of adhesion molecules (e.g., ICAM, VCAM, and E-selectin) and soluble chemokines (e.g., IL-8, MCP-1, and RANTES) recruits macrophages and neutrophils to the site of infection or injury. The expression of α7 on the surface of primary human microvascular endothelial cells in culture suggests that nicotine and other cholinergic agonists may attenuate endothelial cell inflammatory responses. Endothelial cells in culture do not respond to endotoxin, but TNF activates the surface expression of adhesion molecules, as well as the release of chemokines into the cell culture supernatant. Supplementing cell culture media with nicotine in primary human microvascular endothelial cells stimulated with TNF inhibited expression of ICAM, VCAM, and E-selectin on the cell surface; release of IL-8, MCP-1, and RANTES into the cell culture supernatants; and activation of NF-κB with an EC50∼10−7–10−8M. Similar anti-inflammatory effects were seen with a novel cholinergic agonist, CAP55. The activity of these compounds was inhibited with mecamylamine, a non-selective and non-competitive antagonist of nicotinic acetylcholine receptors (16). Microglia. The anti-inflammatory effect of α7 agonists has also been shown in microglial cells, bone marrow–derived cells with phagocytic function present in the central nervous system (66). In vitro, nicotine pretreatment of microglial cells reduces endotoxininduced TNF release, while levels of IL-1, IL-10, and NO remained unchanged. This effect may be mediated through α7, because microglial cells express α7 mRNA, and the TNF-suppressing effect of nicotine was negated by increasing concentrations of αBTx, a selective α7 antagonist. Interestingly, nicotine enhanced the mRNA levels of COX-2 and the synthesis of PGE2 in this neuronal cell type. Increased PGE2 levels may impair microglial activation and, as a consequence, modulate chronic inflammation. Also, since neurodegenerative pathologies are characterized by microglial activation and chronic inflammation, further understanding of α7 activation in these cells could contribute to potential pharmacological modulation of microglia in neurodegenerative disorders. 2. In Vivo: Animal Models Local inflammation. Localized endothelial cell inflammation can be activated in the Schwartzmann reaction,

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in which a small dose of endotoxin is introduced to the pinnae of the ear, followed by a much larger dose 24 hours later. Five hours after the second injection of endotoxin, ear tissue is harvested and analyzed histologically for expression of adhesion molecules on endothelial cells. Treatment with nicotine or CAP55 at each endotoxin injection significantly reduces the expression of VCAM, ICAM, and E-selectin on the surface of endothelial cells in vivo. To determine if cholinergic regulation of endothelial cell adhesion molecule expression leads to reduced macrophage and neutrophil tissue infiltration, animals were subjected to a carrageenan air-pouch model, in which air is injected subcutaneously to create an air pouch. Six days later, carrageenan, an inflammatory component of seaweed, is injected into the pouch. Pouch fluid collected 6 hours later can be analyzed for cell counts and inflammatory cytokines. Treatment with nicotine or CAP55 immediately prior to carrageenan injection significantly reduces inflammatory cell influx into the pouch; the anti-inflammatory effects of these compounds are dependent on nicotinic cholinergic receptors, because mecamylamine renders these compounds ineffective (16). Systemic inflammation (endotoxemia). Endotoxemia, induced by intraperitoneal injection of lipopolysaccharide, induces a lethal pro-inflammatory disease characterized by high blood levels of TNF, IL-1, IL-6, MIF, HMGB1, and other pro-inflammatory cytokines. Treatment with nicotine (400 μg/kg, i.p.) 30 minutes prior to, and 20 hours after, endotoxin administration significantly inhibited accumulation of HMGB1 in serum. In separate studies, continued dosing twice per day for 3 days conferred significant survival advantage (vehicle-treated survival = 44%, vs. 81% survival in nicotine-treated animals). Peritonitis/Sepsis. The therapeutic effects of nicotine also have been tested in a clinically relevant model of sepsis. Anesthetized animals undergo laparotomy; the cecum is isolated, ligated, and punctured; and the animals are sutured, closed, and returned to their cages. Over the following 24 hours, intestinal contents leaking from the punctured cecum cause peritonitis, and induce a systemic inflammatory response leading to organ failure, hypotension, and death. Treatment with nicotine, beginning 24 hours after surgery when animals have developed clinical signs of sepsis, confers significant survival advantage (vehicle-treated survival = 48% vs. 89% survival in nicotine-treated animals). Nicotine treatment was associated with reduced pro-inflammatory cytokines in the circulation, and improved clinical signs of sepsis. Vagus nerve control over inflammation and the anti-inflammatory effect of nicotine were also tested in

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a model of sepsis induced by direct injection of Escherichia coli into the peritoneum (45). Surgical vagotomy prior to bacterial injection was associated with increased TNF, IL-1, and IL-6 levels as compared to sham animals, indicating that the vagus nerve exerts a tonic anti-inflammatory effect during severe bacterial infection. Conversely, oral administration of nicotine prior to injection of E. coli decreased serum cytokine levels. Cholinergic signaling is important for preserving organ function during E. coli peritonitis, because vagotomy led to slight (but non-significant) increases in ALT and AST serum concentration, while nicotine treatment preserved liver function as assessed by these measures. These changes in hepatic injury were accompanied by increased or decreased neutrophil influx by vagotomy or nicotine, respectively. Interestingly, nicotine facilitated E. coli outgrowth in peritoneal lavage fluid, blood and liver, and accelerated mortality, suggesting that attenuation of macrophage function by cholinergic agonists may impair bacterial clearance (45).

C. Pharmacologic Vagus Nerve Stimulators We first identified the cholinergic anti-inflammatory pathway while working with the tetravalent guanylhydrazone CNI-1493, a potent macrophage deactivating agent (67;68). In macrophage cultures, CNI-1493 inhibits phosphorylation of p38 MAP kinase, an enzyme that occupies a critical role in regulating the synthesis of TNF and other cytokines (69). CNI1493 confers protection in lethal endotoxemia, and against the lethality of sepsis in a standardized model of cecal ligation and puncture (70). Following a Phase I trial (71) and a small-scale Phase II clinical trial (72) a large-scale, prospective, randomized clinical trial of CNI-1493 for Crohn’s disease is underway. In the early trials, CNI-1493 induced significant clinical and endoscopic improvement in 80% of the patients, and inhibited the expression of TNF in the colonic mucosa (72). While investigating the potential therapeutic utility of CNI-1493 as a TNF inhibitor in animal models of cerebral ischemia, we made a startling and significant discovery: The fundamental physiologic mechanism of action through which CNI-1493 inhibits inflammation in vivo is to stimulate efferent activity in the vagus nerve. It is now clear that CNI-1493 functions in vivo as a pharmacologic vagus nerve stimulator, and that enhanced vagus nerve activity is required for the macrophage deactivating effects of CNI-1493 when delivered systemically. The first direct experimental insight into the role of the CNS in mediating the anti-inflammatory effects of

CNI-1493 came from analysis of endotoxin-induced shock in animals receiving CNI-1493 via the intravenous or intracerebroventricular routes. While the potential for CNI-1493 as an experimental therapeutic for stroke was examined, animals subjected to middle cerebral artery occlusion (MCAO) received CNI-1493 through either intravenous (i.v.) or intracerebroventricular (ic.v.) routes. The controls for this study included animals that received an i.v. dose of endotoxin (11; 73). Vehicle-treated endotoxemic rats developed significant hypotension and increased serum TNF levels within 1 hour after exposure to a lethal dose of LPS. Pretreatment with intravenous CNI-1493 significantly and dose-dependently inhibited serum TNF release and prevented the development of LPSinduced hypotension, but the lowest i.v. CNI-1493 dose tested (100 mg/kg) failed to prevent TNF release or hypotension. Intracerebroventricular administration of a 100-fold dilution of this ineffective i.v. dose significantly attenuated serum TNF release, and protected against the development of hypotension. Much lower i.c.v. doses of CNI-1493 (10, 1.0, and 0.1 ng CNI1493/kg) conferred significant protection against the development of endotoxin-induced serum TNF and hypotension, suggesting that the CNS participates in the systemic anti-inflammatory action of CNI-1493 during endotoxemia (11). Indeed, i.v. dosing of radiolabeled CNI-1493 led to the accumulation of protective levels of the drug in the brain. Extremely low concentrations of CNI-1493 in the brain inhibited peripheral TNF responses to systemic endotoxin challenge, suggesting that CNI-1493 invoked second messengers to regulate innate immune responses in vivo. We found no evidence of increased ACTH or glucocorticoids following ic.v. administration of CNI-1493, and accordingly hypothesized that CNI-1493 might inhibit systemic TNF through activation of efferent neural signals. It seemed plausible that the autonomic nervous system could modulate immune function; and that of all autonomic nerves, the vagus nerve, which innervates all major reticuloendothelial organs, would be most likely to carry the CNI1493–induced signal. To determine whether an intact vagus nerve is required for inhibition of TNF and protection from endotoxin-induced shock by i.c.v. or i.v. CNI-1493, animals were subjected to either surgical or chemical vagotomy (11;73). Surgical vagotomy eliminated the protective effects of intracerebroventricular CNI-1493 against LPS-induced hypotension. Surgical vagotomy also eliminated the protective effect of intravenous CNI-1493 against endotoxin-induced shock and systemic TNF release, indicating that the protective effects of CNI-1493, whether administered into the brain or the peripheral circulation, require an


intact vagus nerve. CNI-1493 administered intravenously increased the discharge rate of the vagus nerve starting at 3–4 minutes after CNI-1493 administration, and lasted for 10–14 minutes. These results identified a previously unrecognized role of CNI-1493 in functioning as a pharmacological vagus nerve stimulator: to increase efferent vagus nerve activity and inhibit endotoxin-induced systemic TNF release and shock (11). Ligand-binding studies revealed that CNI-1493 can bind to muscarinic receptors, predominantly the M1 subtype. Therefore, we studied whether activation of central muscarinic receptors can inhibit TNF during lethal endotoxemia (PANS, in press). Administration of muscarine or the specific M1 agonist McN-A-343 prior to endotoxin exposure dose-dependently inhibits endotoxin-induced serum TNF levels, indicating that activation of central muscarinic receptors (M1) is sufficient to inhibit endotoxin-induced serum TNF. Systemic administration of muscarine, which does not cross the blood-brain barrier (BBB), had no statistically significant effect on serum TNF during endotoxemia, suggesting that peripheral muscarinic receptors do not regulate the inflammatory response to endotoxin. Together, our findings identify a central, muscarinic receptor-dependent mechanism that regulates systemic inflammation.

IV. PERSPECTIVES New insights into endogenous mechanisms that control inflammation have arisen from studies at the interface of immunology and neuroscience. The cholinergic anti-inflammatory pathway comprises the efferent arm of an inflammatory reflex, a neural feedback loop through which the central nervous system monitors and regulates immune function. In addition to providing several new therapeutic approaches to control inflammatory disease, the description of the inflammatory reflex suggests that it may be possible to study whether diseases presenting with an inflammatory component have an underlying neurological deficit. It is also possible to hypothesize that rational modulation of vagus nerve tone may have therapeutic benefit in inflammatory diseases. It may also be possible to study whether acupuncture, placebo effects, prayer, and meditation cause a functional response in neural-immune signaling via this pathway.

A. Autonomic Dysfunction in Disease It is now reasonable to suggest a hypothesis that dysfunction of the cholinergic anti-inflammatory

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pathway may predispose some individuals to excessive inflammatory responses, because the cholinergic anti-inflammatory pathway normally provides a brake on the immune system that restrains cytokine production. Animals with insufficient efferent signaling in the cholinergic anti-inflammatory pathway exhibit robust systemic responses to inflammatory disease, suggesting that this neural pathway provides tonic regulation of immunological homeostasis. Clinically, autonomic dysfunction has been associated with a number of inflammatory diseases characterized by increased proinflammatory cytokine activity, including rheumatoid arthritis, Crohn’s disease, lupus, diabetes, and sepsis. It is theoretically possible that autonomic dysfunction contributes to the dysregulated cytokine release in these diseases. In one large clinical trial, the fatality rate of septic patients with abnormal mental status was twice that of patients with normal mentation; loss of heart rate variability, a reflection of autonomic dysfunction, is also associated with increased sepsis mortality (74–77).

B. Biofeedback and Inflammation For centuries, patients and physicians have believed in the vague notion that an individual’s “state of mind” can influence somatic health. Folklore, art, and literature are replete with the themes that grief and depression are associated with increased disease susceptibility, and positive beliefs and expectations augur wellness. A familiar example is the death of a devoted spouse shortly after he or she buries his or her loved one. To many, this fundamental relationship between a sense of well-being and health is indisputable. One implication of the anti-inflammatory activity of the efferent vagus nerve is that subjects may be trained to rationally augment vagus nerve activity, and thus modulate peripheral inflammatory and immune responses. Via a computer interface, subjects can be trained to increase vagus nerve activity. This approach, known commonly as biofeedback, has been used in the treatment of headache (78), temporomandibular joint disorders (79), Raynaud disease (80), hypertension (81), diabetes (82), urinary (83) and fecal incontinence (84), asthma (85), and intermittent claudication (86) with varying success. Electronic sensors and graphic displays that monitor physiologic parameters such as heart rate, skin temperature, and muscle tension are measured. Subjects can learn to associate visual and auditory signals from a computer interface with changes in involuntary functions, and to recognize mental and physical states that induce desired physiologic changes regulated by the parasympathetic (vagus) nervous system (e.g., blood pres-

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sure reduction, warming of extremities, slowing of heart rate). The ability to rationally modulate vagus nerve activity through biofeedback techniques now makes it plausible to consider regulation of cytokine synthesis by voluntarily increasing vagus nerve activity. A treatment for diseases characterized by uncontrolled inflammation may now be at hand, by stimulating the cholinergic anti-inflammatory pathway to turn off cytokine synthesis.

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C H A P T E R

4 Significance of Sensory Neuropeptides and the Immune Response HANNEKE P. M. VAN DER KLEIJ AND JOHN BIENENSTOCK

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV.

INTRODUCTION 97 THE IMMUNE SYSTEM 98 THE NERVOUS SYSTEM 99 NEUROPEPTIDES 100 THE ENTERIC NERVOUS SYSTEM 105 THE CHOLINERGIC ANTI-INFLAMMATORY PATHWAY 105 NEUROTROPHINS 105 NEUROPEPTIDE COMMUNICATION: THE DIFFERENT IMMUNE CELLS 106 NERVES IN THE INFLAMMATORY PROCESS 110 NEUROGENIC INFLAMMATION 111 CENTRAL NERVOUS SYSTEM 111 STRESS RESPONSE 112 NERVE-IMMUNE COMMUNICATION IN THE VARIOUS TISSUES 113 SUMMARY 117

will show that local environmental effects may alter many of the functions of all cells present. As a consequence to this outline, the chapter contains some redundancy of information.

I. INTRODUCTION More and more studies are demonstrating interactions between the nervous system and immune system (1–3). It has been established that the central nervous system (CNS) can regulate immune functioning. For instance, lesions in the CNS lead to altered immune responses (4,5). Also, immune responses can be classically conditioned involving a CNS learning paradigm (6,7). Furthermore, it was found that peripheral immune responses could alter the firing rate of neurons in the CNS (8). Accordingly, information can flow not only from the CNS to the immune system, but also in the opposite direction. For instance, immune cells can produce neuropeptides such as substance P and βendorphin (9). Neuropeptides are made by the nervous system and by all types of other cells, e.g., substance P by eosinophils. Acetylcholine (ACh) is made by nonneuronal cells. On the other hand, neurons can make cytokines such as interleukin (IL) 1 (10). In addition, cells of the immune system possess receptors for a host of hormones and neurotransmitters, including receptors for catecholamines, ACTH, opioid peptides, substance P, and vasoactive intestinal peptide (VIP). These receptors have been shown to respond in vivo and/or in vitro to the neurotransmitter substances, and their

The nervous system and the immune system have been clearly shown to be closely related. This chapter will describe how the immune system and the nervous system interact, and address the overlap in compounds they contain and release plus the mutual expression of receptors. We will concentrate on the role of sensory neuropeptides in relation to cells involved in the inflammatory system. First, we will address the immune system and the nervous system from a more general point of view. Second, we will concentrate on the specific cell types and the way sensory neuropeptides interact with the different immune cells on this level. Last, since nerve fibers project into every organ of the body, we will focus on sensory neuropeptide interaction with immune cells in different tissues. We PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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manipulation can alter immune responses. (11,12). Additionally, the CNS shares receptors with the immune system for neuropeptides and neurotransmitters as well as for cytokines. Thus, the two systems can communicate in a bi-directional fashion as a result of a common set of signal molecules and their receptors (13). Nerves are closely associated with inflammatory cells, especially mast cells and lymphocytes (14,15). Nerve-immune associations were found in the intestine involving mast cells, as well as eosinophils and plasma cells (16). Cells of the immune system and their products have been shown to influence peripheral and central neurotransmission, leading to the conceptualization of a bi-directional neuroimmune communication system. In addition, neuropeptides from sensory nerves can directly modulate the function of Langerhans cells. Further work has demonstrated similar morphologic mast cell-nerve associations in the liver, mesentery, urinary bladder, and skin. These interactions influence a variety of physiologic and pathophysiologic functions including cellular development, growth and differentiation, immunity, leukocyte recruitment, inflammation, as well as tissue repair (17–19). Immune regulation is mediated via a cascade of events requiring cell–cell interactions and subsequent release of their contents, such as cytokines and antibodies, to regulate these responses. The complex interrelationships of the nervous system and the immune system have received increasing attention (a) on their bi-directional nature and (b) on the production of cytokines by the nervous system and neuropeptides by the immune/inflammatory system.

II. THE IMMUNE SYSTEM Immune molecules such as cytokines, chemokines, and growth factors can modulate functioning of the nervous system through multiple signaling pathways. Immunological stressors can engage cytokines and other immune molecules in bi-directional interactions with brain neuroendocrine, peptide, and neurotransmitter systems. There are two major defense responses of the body to immunological triggers. Innate immunity refers to the initial response by the body to defend against and eliminate microbes and prevent infection. Adaptive or acquired immunity refers to antigenspecific defense mechanisms that may take several days to become protective and are designed to react with, neutralize, and remove a specific antigen. Dendritic cells (DCs) and natural killer (NK) cells are the main cellular components of the innate immune

system. NK cells recognize and bind virus-infected host cells and tumor cells and induce their apoptosis (20). Upon activation, NK cells can produce a variety of cytokines and chemokines, which can have a direct effect on tumor growth and induce inflammatory and anti-viral responses (21). In disease states, the first contact between NK cells and DCs most likely occur at the site of infection as both cell types are recruited to tissues in response to infection. Many of the cytokines that are known to enhance NK cell function are produced by DCs in response to microbial infection (22). For instance, DCs are a major source of IL-15, a cytokine essential for NK cell development, that also promotes NK cell survival and proliferation (23). Following priming by pathogen-derived products, their reciprocal interactions result in a potent activating crosstalk regulating the innate immune responses. A second set of NK cells, the natural killer T-cells (NKT), has been more recently identified. NKT cells possess phenotypic characteristics of both NK and Tcells (24). NKT cells are considered to play an intermediary role bridging innate and acquired immunity. They respond to various external stimuli by an early burst of cytokines, especially IL-4 and IFN-γ (25). NKT cells play an important immunoregulatory role through the production of these Th1 (IFN-γ) and Th2 (IL-4) cytokines (24). It has been shown that the communication between NK and NKT cells is mediated primarily by IFN-γ. In addition, the production of IFN-γ has an important role in the cytotoxic response (25). Dysregulation in IL-4 production can result in chronic inflammation and autoimmune diseases (26). Impaired IFN-γ production by NKT cells results in inefficient NK cell cytotoxicity because NKT cells “cross-talk” via IFN-γ to activate NK cells (25). This pathway linking NKT and NK cell functions is important under conditions of low innate cytokine induction (i.e., of IL-12 and/or IFNα/β) during viral or bacterial infections (24). Innate immunity is designed to recognize a few highly conserved structures present in many different microorganisms. Infection by pathogenic microorganisms triggers in the host a set of immune, physiological, metabolic, and behavioral responses known as the acute phase reaction. These responses are mediated by the activation of innate immune cells that recognize bacterial and viral products via membrane Toll-like receptors (TLR). Studies have been showing an essential role for TLR in the activation of innate and adaptive immunity (27). Ligand recognition induces a host defense response, which includes the production of inflammatory cytokines, upregulation of co-stimulatory molecules, and the induction of anti-microbial defense. Activation of dendritic cells by TLR ligands is necessary for their maturation and con-

4. Significance of Sensory Neuropeptides and the Immune Response

sequent ability to initiate immune responses (27). Lipopolysaccharide (LPS), the active fragment of Gram negative bacteria, binds to Toll-like receptor-4 on monocytes and macrophages, which activates complex intracellular signaling pathways resulting in the activation of nuclear transcription factors. It has been shown that intraperitoneal LPS induced IL-1β protein within the vagus in a time frame consistent with signaling of immune activation. This suggests a novel mechanism by which IL-1β may serve as a molecular link between the immune system and vagus nerve, and hence the CNS (28). Furthermore, the systemic administration of LPS or recombinant pro-inflammatory cytokines such as IL-1 to healthy laboratory animals or human volunteers triggers the whole set of responses, including its central component in the form of fever, activation of the hypothalamic-pituitaryadrenal axis, and the behavioral symptoms of sickness (29,30). The same clinical signs can be induced by injection of LPS or interleukin-1 into the lateral ventricle of the brain, which indicates that the brain is able to recognize immune molecular signals. Cytokine-induced sickness behavior can be seen as a neuroimmune response to activation of innate immunity. In the brain, cytokines are produced that are the same as those expressed by innate immune cells, and they act on brain receptors that are identical to those characterized on immune cells. Primary afferent nerves represent the main communication pathway between peripheral and central cytokines (29,30). Recent reports identified and described neural pathways that control and adjust the peripheral immune response. For instance, the two immunomodulatory peptides VIP and the pituitary adenylate cyclase-activating polypeptide (PACAP) are present and released from both nerves and immune cells (31). VIP/PACAP have a general anti-inflammatory effect, both in innate and adaptive immunity. In innate immunity, VIP/PACAP inhibit the production of pro-inflammatory cytokines and chemokines from macrophages, microglia, and dendritic cells. In addition, VIP/PACAP reduce the expression of co-stimulatory molecules on antigen-presenting cells, and therefore reduce stimulation of antigen-specific CD4+ T-cells. In adaptive immunity, VIP/PACAP promote Th2-type responses, and reduce the pro-inflammatory Th1-type responses (31). The adaptive immune system can be divided into humoral immunity, mediated by antibody-producing B-cells, and cell-mediated immunity, mediated by Tlymphocytes. There are different types of T-cells: Thelper cells, Th0, Th1, Th2, T regulatory cells (Treg), and Natural Killer T (NKT) cells, which direct adaptive immune responses. Treg appear to be important in

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the prevention of autoimmune disease and in their response to incoming invaders. Th1 and Th2 cells are thought to derive from a naive Th0 precursor that makes a wide range of cytokines. In the presence of IL-12 and IL-18 (from dendritic cells), Th0 will differentiate into Th1 cells that secrete IL-2, interferon γ (IFN-γ), and TGF-β, or in the presence of IL-4 (from Bcells or lymphoid dendritic cells) they will differentiate into Th2 cells that secrete IL-4, IL-5, IL-6, IL-10, and IL-13. These types of responses are mutually inhibitory, and the switch to one or the other is directly linked with the cytokines generated by innate immune responses. The first step in immune responsiveness is the delivery of antigen in a form that DCs can acquire, process, and present to CD4+ T-cells, CD8+ T-cells, and/or Bcells (32). Immature tissue DCs sense invading organisms by their TLR (33). Upon receiving an activation signal, they undergo phenotypical and functional changes, including increased expression of surface molecules that aid and promote T-cell activation, a changed pattern of release of chemokines and cytokines leading to attraction of T-cells, promotion of Tcell activation, and direction of their ultimate phenotype (Th0, Th1, Th2, or T regulatory [Treg]) (34, 35). DCs treated with TNF, a pleiotropic stimulus of DC activation, and prostaglandin E produce a low level of IL-12 and induce a mixed population of Th0 and Th1 T-cells. DCs secrete high levels of IL-12 and IFN-α leading to a strong Th1 response. The nervous system releases neuropeptides at specific local sites of infection or challenge, which may then influence the direction of the Th1/Th2 response and therefore immune outcome. For instance, calcitonin gene-related peptide (CGRP) can inhibit IFN-γ production markedly in a dose-dependent manner, and substance P and VIP can suppress IL-4 production (36). Catecholamines may play an important role in regulating adaptive immune responses as Th1 cells express adrenergic receptors (37). Furthermore, adrenergic receptors on B-cells suggest that the sympathetic nervous system may also regulate antibody production (37).

III. THE NERVOUS SYSTEM The nervous system can regulate immune function and inflammation. Experimental evidence shows an important role of the autonomic nervous system in the bi-directional communication between the brain and the immune system, underlying the ability of the brain to monitor immune status and control inflammation. The central nervous system and immune system inter-

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act bi-directionally. Neuropeptides, neurohormones, and neurotransmitters of the CNS interact with the immune system which, in turn, feeds back to the brain inducing changes in both behavior such as sickness response and changes in the immune system (38–40). Cytokines produced in the periphery can affect the brain, producing illness behavior and activation of neuroendocrine and autonomic stress-related circuits (41,42). The CNS alerts the immune system to environmental changes using the shared neuropeptide, neurotransmitter, and cytokine receptors on immune cells. An example of this is the effects of stress to dampen immune function. Recognition of stimuli such as viruses and bacteria by the immune systems results in transmission of information to the CNS to cause a physiological response. Upon peritoneal infection with bacteria, changes occur in the body such as fever associated with sickness behavior. These responses are a direct result of immune cell–derived proinflammatory cytokines signaling the CNS via the subdiaphragmatic vagus nerve (13,43). In the autonomic nervous system, nerves are intimately associated with inflammatory cells, especially mast cells (2). The chemical transmitter at both preand post-ganglionic synapses in the parasympathetic system is ACh. Nerve fibers that release ACh from their endings are described as cholinergic fibers. ACh is the neurotransmitter via a nicotinic receptor at the pre-ganglionic synapse. Adrenaline and noradrenaline as well as norepinephrine (NE), the classical sympathetic neurotransmitter, are catecholamines. Noradrenergic nerve fibers of the sympathetic nervous system release NE and other co-localized peptides that signal cells bearing appropriate cell surface receptors. Cells of the immune system, including T and B lymphocytes, NK cells, and macrophages, express α- and β-adrenergic receptors, the receptors for NE (44,45). Activation of the sympathetic nervous system in vivo, or stimulation of lymphocytes with selective receptor agonists in vitro, alters proliferation, differentiation, and cytokine production by lymphocytes and macrophages. Furthermore, cytotoxicity of NK cells is probably suppressed in vivo through their β-adrenergic receptors in response to sympathetic nervous activity (46). The role of catecholamines in immune regulation will be further reviewed elsewhere. In addition to the classical neurotransmitters, acetylcholine and noradrenaline, a wide number of peptides with neurotransmitter activity have been identified in the past few years as being synthesized and released from both autonomic and parasympathetic cholinergic neurons—among them, CGRP and the tachykinins substance P, neurokinin A, neurokinin B, and the newly discovered hemokinin (47,48). The

tachykinins and CGRP also appear to act as mediators of non-adrenergic, non-cholinergic (NANC) excitatory neurotransmission. The excitatory and inhibitory non-adrenergic/non-cholinergic (e-NANC, i-NANC) systems have been extensively studied. The terms excitatory and inhibitory apply to airway smooth muscle, but the neurotransmitters also act on other targets such as blood vessels, glands, and the epithelium, where individual actions may vary. VIP and nitric oxide (NO) co-localize in vagal motor nerves, but they are also found in sympathetic and sensory nerves. In general they have similar actions on target tissues, and their relative importance may vary with the target tissue and the species. Substance P and neurokinin A are also released from sensory nerves, and belong to the family of tachykinins. Finally, it is important to recognize that ACh, substance P, NO, and even other neurotransmitters can be synthesized by non-neuronal cells (49,50). The regulation of synthesis and secretion of these mediators is largely undetermined. Therefore, any description of homeostasis, especially under conditions where immune stimulation or inflammation is present, must by necessity remain incomplete until this knowledge is available.

IV. NEUROPEPTIDES Neuropeptides are produced primarily in the brain, although almost every tissue in the body can produce and exchange neuropeptides. The immunological activity of neuropeptides is mediated through specific receptors. The presence of specific receptors for substance P (51,52), somatostatin (53), CGRP (54), Corticotrophin releasing hormone (CRH) (55), melanocortin peptides (56,57), and VIP and related peptides has been reported in immune cells. The existence of these specific neuropeptide receptors on immune cells represents the framework for neuropeptides functioning as mediators of neuroimmune interactions. Substance P, CGRP, and VIP represent the neuropeptides most involved in neuroimmune modulation. Somatostatin (SOM), CGRP, substance P, and neurokinin A are excitatory NANC neuropeptides. SOM exerts various inhibitory functions on immune responses via specific receptor activation (58). To date, five different receptor types have been cloned and characterized, sst1–5 (59,60). The main sst expressed in cells of the rat immune system were found to be sst3 and sst4 in contrast with the human and murine situations, in which sst2 appears to be the main subtype expressed in the immune system (61). Recently, ten Bokum et al. (61) demonstrated the localization of SOM receptors in inflammatory lesions in rheumatoid


arthritis. The SSt2 was expressed by endothelial cells of the synovial venules and a subset of synovial macrophages, which are thought to be important effector cells in rheumatoid arthritis. SOM affects the suppression of Ig production in B-cells, including IgE (62), modulation of lymphocyte proliferation, and reduction of eosinophil infiltration in hypereosinophilia. Sensory neurons are characterized by their expression of a certain group of neuropeptides, the tachykinins, and CGRP. For many years, SP, neurokinin A, neurokinin B, and two elongated versions of neurokinin A, neuropeptide γ (NPγ) and neuropeptide K (NPK), were thought to be the only members of the mammalian tachykinin family (63). This family is now expanded by the recent identification of hemokinin (47,48). The only functions so far ascribed to hemokinin are its possible role in lymphopoiesis. In mammals, tachykinins act as neurotransmitters, paracrine or endocrine factors, and neuroimmunomodulators. Important actions include vasodilatation, plasma extravasation, smooth muscle contraction, secretion, neuronal excitation, and processing of sensory information. Substance P, neurokinin A, and neurokinin B are the best described members of the tachykinin family and share a common C-terminal sequence of -Phe-X-GlyLeu-Met-NH2. Two distinct genes called the preprotachykinins A and B encode the tachykinins. Neurokinin B derives from preprotachykinin B, while the others are from preprotachykinin A. The preprotachykinin A has seven exons that can be alternatively spliced to produce α, β, γ, and δ-preprotachykinin A. Recently, an additional tachykinin gene, preprotachykinin C, has been identified that encodes the novel tachykinin-designated hemokinin (64). Neuropeptides exhibit a variety of proinflammatory effects. They are released in response to nociceptive stimulation by pain, mechanical, and chemical irritants to mediate skin responses to infection, injury, and wound healing (65). They are known to activate a variety of immune cells through highaffinity neuropeptide receptors or by direct activation of G-protein–signaling cascades without an intermediary receptor (66,67). Recent reports have also indicated the presence of neurokinin B precursor mRNA in the human and rat placenta (68) and uterus (69,70). These data argue for a broader distribution for these peptides that may not only act as neurotransmitters but also have an endocrine function.

A. Substance P Substance P is an 11 amino acid molecule derived from the preprotachykinin A gene. Substance P is widely distributed in the central, peripheral, and

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enteric nervous systems of many species. Substance P functions in the CNS as a neurotransmitter, and its neurokinin (NK) receptor 1 are localized in distinct areas of the brain important in affecting behavior and the neurochemical response to both psychological and somatic stress. It may also coordinate the response to stress by interacting with the HPA axis and the sympathetic nervous system (71,72). In addition, substance P is involved in sensory and, most notably, nociceptive pathways (73). In the periphery, substance P has been identified in C-type sensory nerve endings and autonomic afferents throughout the body (74). Substance P is present at sites of inflammation, and inflammation can enhance its expression (75–77). Substance P–containing nerves are abundant at mucosal sites and are in ganglia, spinal cord, brain, airways, and skin and around blood vessels (78,79). Some sensory neurons, both intrinsic and extrinsic, produce substance P. Neuronal substance P is stored in vesicles and released from sensory nerves in response to various stimuli such as leukotrienes, prostaglandins, and histamine (79). In addition, substance P can be synthesized and released from immune cells such as macrophages and eosinophils, particularly in disease (80,81). Neutral endopeptidase (NEP) or enkephalinase is a neuropeptide-degrading enzyme expressed on the cell surface of many cell types including neurons, leukocytes, epithelial cells, and smooth muscle cells. NEP has high specificity for substance P and degrades substance P, leaving no biologically active metabolites (82). Blocking NEP delays substance P degradation. NEP transgenic knockout mice develop a markedly increased form of colitis in response to dinitrobenzene sulfonic acid. The administration of substance P receptor antagonists or recombinant NEP prevents the exacerbated inflammation (83). This suggests that a defect in NEP expression with resulting over-expression of substance P worsens the level of inflammation. Guinea pigs are not affected by inhalation of substance P; however, even a small amount of substance P given together with an NEP inhibitor does cause bronchoconstriction in these animals, suggesting that the amount of local NEP may regulate the functional effects of substance P in vivo (84).

B. Hemokinin Recently, the tachykinin family has been extended by the discovery of a third tachykinin gene preprotachykinin-C mRNA encoding a tachykinin called hemokinin-1 (85,86). Like substance P, hemokinin-1 has a high affinity for the NK-1 receptor. An important observation that has been made is the fact that constitutive expression of hemokinin mRNA occurs prefer-

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entially in leukocytes and lymphoid tissue, not in the nervous system (87). Hemokinin-1 has been proposed as a regulator of B- and T-cell lymphopoiesis. PPT-C mRNA was expressed in B-cells from bone marrow, where it promoted the survival and proliferation of Bcell precursors. These observations suggest that hemokinin is an autocrine factor, playing a role in B-cell development. However, NK-1 knockout mice are not lymphopenic, which suggests that hemokinin is not essential for B-cell growth (88). Nelson et al. (87) demonstrated the expression of preprotachykinin-C mRNA by macrophages, dendritic cells, and in a microglial cell line. This implies that microglial cells, described as resident macrophages in the brain, are a potential source of hemokinin-1. There is the possibility that hemokinin-1 could play a significant role in communication between immune cells in the CNS (89), and between immune cells and neuronal cells expressing the NK-1 receptor. Studies have demonstrated the importance of the NK-1 receptor during bacterial (90), viral (91), and parasitic infections (92). The demonstration of preprotachykinin-C mRNA expression by macrophages and dendritic cells also has implications for tachykininmediated host responses against microbial pathogens. The fact that preprotachykinin-C mRNA is preferentially expressed in leukocytes suggests that hemokinin-1 may contribute to the development and/ or activation of the immune response.

C. Capsaicin Capsaicin is the spicy ingredient in red pepper fruits of the genus capsicum, including paprika, jalapeno, and cayenne (93). Capsaicin accomplishes its effects by evoking sharp burning pain sensation when it comes into contact with mucous membranes. Jancso and Porszasz (94) were the first to show that capsaicin selectively and specifically excites a subpopulation of primary sensory fibers. Later, Jancso et al.’s (95) finding that capsaicin injection into neonatal rats results in the loss of capsaicin-sensitive primary sensory neurons provided an excellent tool for studying this subpopulation of sensory neurons. The capsaicin receptor TRPV1 is a nociceptorspecific ion channel that serves as the molecular target of capsaicin (96). As the capsaicin-sensitive receptor also responds to other related molecules with vanilloid moiety, the receptor was named the vanilloid receptor (VR1 or TRPV1) (97). The structure and amino acid sequence of TRPV1 are similar to those of the transient receptor potential (TRP) family of cation channels (98). In addition to TRPV1, three more TRP receptors have been discovered (TRPV2, TRPV3, and TRPV4). There

is not much known so far on the role of these receptors in neurogenic inflammation. In this review we will therefore focus only on TRPV1. TRPV1 can be activated not only by capsaicin but also by noxious heat or protons, all of which are known to cause pain in vivo. TRPV1, in addition to a subpopulation of primary sensory neurons, is expressed by various neurons and non-neuronal cells. The great majority of TRPV1-expressing cells belong to nociceptive cells expressing substance P and CGRP. Both somatic and visceral primary afferents express TRPV1, and the molecule is expressed by peripheral terminals and in many areas of the CNS (99,100). Furthermore, TRPV1 protein and mRNA expression in cultured rat gastric epithelial cell and epithelial cells in the urinary bladder have also been shown to express TRPV1 both at mRNA and protein level (101). Alterations in the sensitivity of VR1 to agonists or its level of expression would be expected to markedly affect the sensitivity of sensory neurons with important implications for inflammatory diseases such as asthma and IBD. Most of the neurogenic effects in the airways, gut, or urinary tract are mediated by a direct action on sensory nerve terminals via the stimulation of VR1 (102). Thus, antagonists of VR1 may be useful for the treatment of a wide range of diseases that affect the sensory nervous system. TRPV1 appears to be upregulated during inflammatory conditions. Nerve growth factor (NGF), bradykinin, and proteases can upregulate the function of VR1 (103). For instance, tryptase, the major protease released from mast cells, excites VR1-positive sensory neurons (104). Factors that downregulate TRPV1 expression include vanilloid treatment and NGF deprivation (105). These findings imply an important role for TRPV1 expression in the development of neuropathic pain and hyperalgesia. Disease-related changes in TRPV1 expression have already been described in IBD and IBS although the mechanisms that regulate TRPV1 gene expression under pathological conditions are unknown.

D. Neurokinin Receptors Tachykinins can bind with different affinities to their potential neurokinin receptors. Three distinct subtypes of mammalian receptors have been identified and denoted as NK-1, NK-2, and NK-3. The NK-1 receptor has the highest affinity for substance P and hemokinin. The NK-1 and NK-2 receptors bind with the highest affinity to neurokinin A and B, respectively (106–108). Tachykinin receptors belong to the Gprotein–coupled receptor superfamily with the structural characteristics of seven transmembrane helices.


All three neurokinin receptors share a high degree of homology (109,110). Substance P is believed to act as one of the most significant neurotransmitters (111). The NK-1 receptor is widely distributed in both central and peripheral nervous systems and is localized on smooth muscle cells, submucosal glands, blood vessels, and inflammatory cells (108,112). Tachykinin NK-1 receptors have been proposed to be involved in many physiological and pathological conditions such as noxious stimuli, neurogenic inflammation, intestinal motility, vasodilatation, smooth muscle contraction, as well as immune response (77,113). NK-2 receptors have been detected in the central nervous system but are more widely distributed in the periphery such as respiratory, gastrointestinal, and urinary tract. Activation of this receptor subtype results in facilitation of transmitter release, neuronal excitation, and stimulation of certain immune cells (113). The NK-2 receptor is also found on smooth muscle. Animal studies have shown that NK-2 receptors are involved in bronchoconstriction (65,113). However, the exact function of this receptor in the central nervous system has yet to be delineated. Compared with the NK-1 and NK-2 receptors, much less is known about the biological function of the NK-3 receptor. The tachykinin NK-3 receptors are predominantly present in the central and peripheral nervous system (114,115) and have been detected only in certain peripheral tissues, such as the human uterus and skeletal muscle, brain, and certain enteric neurons from the gut of different species (111,116,117). Studies using selective agonists or antagonists point to a central role of the tachykinin NK-3 receptor, which might be involved in depression and anxiety (118). On the contrary, the role of this tachykinin receptor type at the peripheral level remains elusive. Cells of the immune system have also shown to express neurokinin receptors, especially the NK-1 receptor. Moreover, macrophages (119,120), dendritic cells (121), T lymphocytes (51,122), and B lymphocytes (123,124) all have the ability to express NK-1 receptors.

E. Inhibitory NANC Neuropeptides The main transmitters that mediate NANC inhibitory neural transmission are VIP and NO. VIP is found in the CNS and in peripheral nerves, particularly in peptidergic nerves (125). Peptidergic nerves are abundant in mucosal tissues such as the lungs, the upper respiratory and nasal mucosa, and the small and large intestine (126,127). Furthermore, VIP can be found together with substance P in the enteric neurons of the myenteric plexus and submucosal plexus, the lym-

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phoid tissue including the thymus, spleen, and Peyer’s patches (128). The general consensus is that VIP is an important inhibitory NANC neurotransmitter that relaxes airway smooth muscle, as does NO. It can be broken down by local enzymes such as neutral endopeptidase (NEP), and mast cell tryptase and chymase. Three receptors for VIP have been identified belonging to the family of G-protein–coupled, seventransmembrane receptors. Mouse T lymphocytes have been shown to express VIP receptors (129) both on CD4+ and CD8+ T-cells (130). Lymphocytes have been shown to produce VIP upon their activation (131). VIP and its receptors will be discussed in more detail elsewhere. Nitric oxide (NO) is also present in airway nerves and relaxes airway smooth muscle. NO is a signaling molecule with important regulatory functions such as regulation of blood pressure, neurotransmission, and host and immune defense (132). NO, co-released with CGRP, seems to also play a role in the UVB-induced immunosuppression through a yet undiscovered mechanism of action (133). In the respiratory tract, NO is formed and released by various sources including endothelial and epithelial cells, nerves, airway smooth muscle, and inflammatory cells. NO is found in vascular endothelium, in the respiratory epithelium of guinea pigs (134) and asthmatic (135) and possibly healthy humans, in sympathetic motor nerves to the lungs, and even in epithelial nerves, emphasizing the importance of NO as a mucosal mediator (134). The mechanism of release of NO from nerves is unclear. The evidence points to NO being an important or the main inhibitory NANC transmitter, at least in humans. In other species it is present and may play a cooperative role with VIP. Most VIP-containing nerves are also cholinergic, containing choline acetyltransferase, and VIP has also been shown to co-localize with SP or CGRP in mucosal nerves, including probably the epithelium. NO is present in cholinergic motor nerves together with VIP. PACAP belongs to the VIP family of peptides. The two neuropeptides are structurally related and are released within the lymphoid organs following antigenic stimulation, and modulate the function of inflammatory cells through specific receptors (31). So far, three different receptors have been recognized and defined as PAC1–R, VPAC1–R, and VPAC2–R. PACAP, but not VIP, binds to PAC1–R, whereas PACAP and VIP both bind to VPAC1–R and VPAC2–R (136). VIP/ PACAP receptors are expressed in various immune cell populations. VPAC1 seems to be constitutively expressed in both unstimulated and stimulated lymphocytes and macrophages (137). VPAC2, on the other hand, is expressed in lymphocytes and macrophages

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only following stimulation (130, 138). VIP and PACAP are co-expressed in single- and double-positive (CD4+CD8+) thymocytes and in T and B lymphocytes from spleen and lymph nodes (139). They promote Th2-type and inhibit Th1-type responses in vivo and in vitro. Macrophages treated in vitro with PACAP gain the ability to induce Th2-type cytokines (IL-4 and IL-5) and inhibit Th1-type cytokines (IFN-γ, IL-2) in antigenprimed CD4 T-cells. Furthermore, VIP and PACAP inhibit TNF, IL-1β, IL-6, and NO production by lipopolysaccharide (LPS)-activated microglia (140).

F. Calcitonin Gene-related Peptide CGRP is a 37 amino acid neuropeptide. CGRP is synthesized in the body of sensory nerves and transported to the nerve endings, where it is released by voltage-dependent calcium uptake. Sensory nerve fibers are stimulated by noxious stimuli and certain inflammatory mediators like IL-1 and PGE2 (141). CGRP is present in all body surfaces, around all blood vessels and at all internal and external surfaces, where it can influence inflammatory and immune responses. Release of CGRP is often accompanied by release of substance P (142), but CGRP can be found by itself in some motor and enteric neurons (142). There are two isoforms of CGRP, α and β, derived from two distinct genes, one of which also encodes the hormone calcitonin. The CGRP receptors are seventransmembrane domains, G-protein–associated receptors. CGRP receptors have been classified in two major classes, namely the CGRP1 and CGRP2 subtypes. CGRP receptors were identified several years ago in lymphoid tissue. Receptors have been identified on mature T- and B-cells and macrophages, and on developing B-cells in the bone marrow (143–145). CGRP1 is found throughout the body including the nervous and endocrine systems (146–148). CGRP inhibits T-cell proliferation by inhibiting IL-2 production. In macrophages, it inhibits antigen presentation and other macrophage functions including phagocytosis (149). It also stimulates production of a number of cytokines such as IL-6, IL-10, and TNF. In vivo injection of CGRP was shown to inhibit the onset of delayed type hypersensitivity reactions (150) supporting the role of CGRP in systemic regulation of immune activation. Indeed, Hosoi et al. (151) previously showed conclusively that CGRP could inhibit Langerhans cell (LC) activity and the initiation of antigen-induced immune responses. Furthermore, it was shown that intracutaneously injected CGRP participates in the pathogenesis of failed contact hypersensitivity induction after acute, low-dose ultraviolet B radiation (152). These authors showed that CGRP

has the capacity to promote cutaneous tolerance and that the mechanism contributes to the tolerance promoted by acute, low-dose ultraviolet B radiation. Endogenous CGRP may generally modulate immune function, presumably in part, through an inhibitory effect on LC function. This indicates that CGRP is capable of inhibiting both the induction and expression of fundamental cellular immune functions and suggests an interaction between the nervous system and immunological function. These observations imply the likelihood that the CNS may be capable of suppressing the initiation of immune responses, and also be effective in their modulation. Overall, there is substantial experimental evidence demonstrating that CGRP can influence the function and development of inflammatory and immune cells in local microenvironments by specific receptor-mediated mechanisms.

G. Non-neuronal Neurotransmitters Although classically neuropeptides are released from autonomic and sensory nerves, there is increasing evidence that they may also be synthesized and released from inflammatory cells, particularly in disease. Over the last two decades there have been several reports suggesting that lymphocytes produce many different neuropeptides. Substance P has been shown to be a product of human, rat, and mouse leukocytes that can be released at sites of inflammation (50,153,154). Human and mouse eosinophils produce and secrete substance P (153,155), and it is known that T-cells in granulomata in the spleen in murine schistosomiasis express preprotachykinin A mRNA. Furthermore, LPS induces preprotachykinin A mRNA and substance P production from rat peritoneal macrophages (156) as well as rat and human alveolar macrophages (80). Collectively, these studies clearly demonstrate that stimulated leukocytes can express PPT mRNA and the products of PPT genes, although the concentrations are significantly less than those reported for neuronally derived tachykinins. This alternative source of immune cell-derived neuropeptides could represent an additional source of tachykinins in inflamed tissues, providing a non-neurogenic tachykininergic contribution to the local inflammatory process (157). It is so far not clear why leukocytes might express tachykinins. It is likely that the stimuli that induce neuronal production and secretion of tachykinins will be different from the stimuli that would evoke substance P secretion by leukocytes. Furthermore, it is possible that leukocyte-derived neuropeptide production occurs in areas of limited innervation by sensory


neurons and thus may locally compensate for the relative paucity of the modulatory response. The neurotransmitter Ach has been found in epithelial cells, endothelial cells, muscle and immune cells such as mononuclear cells, granulocytes, alveolar macrophages, and mast cells (49,158,159). The expression of ACh is accompanied by the presence of cholinesterase and nicotinic and muscarinic receptors (49,158,160). This suggests that non-neuronal Ach acts as a local cellular signaling molecule rather than a neurotransmitter. Thus, non-neuronal Ach has to be discriminated from neuronal ACh especially since there are apparent functional differences between the two.

V. THE ENTERIC NERVOUS SYSTEM The enteric nervous system is an independent, though connected network of nerve fibers that innervate the viscera (gastrointestinal tract, pancreas, gall bladder). The enteric nervous system is essential for regulating motility and secretion. It consists of ganglionated plexuses embedded in the wall of the intestine divided into submucosal ganglia and myenteric ganglia distributed between the longitudinal and circular muscles. Neurons within submucosal ganglia can be classified as intrinsic primary afferent neurons (IPANs), interneurons, secretomotor and vasodilator neurons (161,162). Myenteric ganglia also contain motor neurons, innervating the longitudinal and circular muscle layers, and secretomotor neurons, which project to the mucosa (163). With these neural components the enteric nervous system (ENS) can elicit neural reflexes that control and coordinate motility, secretion, and blood flow. The nervous system of the gut is capable of integrative functions independent of the central nervous system. The human GI system, deprived of CNS innervation, is capable of coordinated digestion, motility, secretion, and absorption. The neurons that control bowel functions are located entirely within the gut, and for this reason the ENS is sometimes called the “little brain” (164). Substance P is found predominantly in submucosal IPANs. The presence of NK-1 receptors on isolated colonocytes suggests that appropriate elements are present for axon reflex activation of intestinal epithelial cells (165). MacNaughton et al. (166) characterized tachykinin-evoked secretomotor responses in in vitro submucosal and mucosal-submucosal preparations of the guinea pig ileum using combined intracellular and Ussing chamber recording techniques. Their findings suggest that tachykinin-evoked secretion in guinea pig ileum is mediated by NK-1 and NK-3 receptors on submucosal secretomotor neurons and that capsaicin-

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sensitive nerves release tachykinin(s) that activate the NK-1 receptors.

VI. THE CHOLINERGIC ANTI-INFLAMMATORY PATHWAY Afferent signals to the brain can generate a reflex causing an anti-inflammatory response, which is partly mediated by the efferent branch of the vagus nerve (167,168). This cholinergic anti-inflammatory pathway is mediated primarily by nicotinic ACh receptors expressed upon tissue macrophages and blood monocytes. ACh can interact specifically with macrophage alpha7 subunits of nicotinic ACh receptors, leading to cellular deactivation and inhibition of cytokine release (169). Endotoxin (or LPS) is a product of all gram-negative bacteria, which can cause shock (hypotension) and, ultimately, death. This occurs as a result of LPS activation of macrophages to release TNF, which is a principal mediator of acute LPS-induced shock (169,170). Wang et al. (169) showed the ability of ACh to suppress macrophage TNF production in vitro. Stimulation of efferent vagus nerve activity inhibited the systemic inflammatory response to endotoxin. These findings were the first to demonstrate a previously unrecognized, parasympathetic anti-inflammatory pathway by which the CNS modulates systemic inflammatory responses. The Tracey group termed this the “cholinergic anti-inflammatory pathway.”

VII. NEUROTROPHINS Classical mediators of inflammation are not alone in their ability to influence the interaction between cells of the immune system and nerves. Based on their expression profile, neurotrophins are good candidates for mediating immune-nerve cell interactions. NGF was the first discovered member of the family of neurotrophins in the 1950s, now including brainderived neurotrophic factor (BDNF) and neurotrophins 3/5. NGF is the best characterized neurotrophic protein and is required for survival and differentiation of neuronal cell types in both the peripheral and central nervous system. Functional properties of neurons are also affected by NGF. For instance, NGF was shown to upregulate neuropeptide production in sensory neurons and to contribute to inflammatory hypersensitivity (171). In addition, in cultured nodose ganglion neurons, substance P production is regulated by NGF (172). NGF and BDNF are some of the most effective

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mediators involved in inflammatory hyperalgesia (171). The biological effects of neurotrophins are mediated by binding either to the specific high affinity receptors trkA (for NGF), trkB (for BDNF), and trkC (for NT-3) or the low affinity receptor pan-neurotrophin receptor p75 (NTR). Neurotrophin receptors are widely expressed in the peripheral and the CNS as well as on cells of the immune system (169,173). NGF receptors on mast cells act as autoreceptors, regulating mast cell NGF synthesis and release, while at the same time being sensitive to NGF from the environment. Many immune cells express the high affinity NGF receptor. This allows NGF to promote the release of inflammatory mediators. Several of these mediators such as IL-1, IL-4, IL-5, TNF, and IFN can, in turn, induce the release of NGF (169,174). Therefore, NGF seems to be a mediator with functions on both immune cells and nerve cells and is likely an important factor integrating communication between the nervous and immune systems (175). There is increasing evidence that NGF acts on cells of the immune system, apart from its neurotrophic effects. NGF promotes differentiation, activation, and cytokine production of mast cells and macrophages (176,177), and activates eosinophils (178). Furthermore, NGF is a chemoattractant, thereby causing an increase in the number of mast cells as well as their degranulation (179–181). Injection of NGF causes mast cell proliferation in part by mast cell degranulation (182). NGF also influences activity of basophils, eosinophils, neurophils, macrophages, and T-cells. Both murine and recombinant human NGF enhance histamine release and strongly modulate the formation of lipid mediators by basophils in response to various stimuli (183). This priming effect of NGF on basophils occurs rapidly, is dose-dependent, and requires low concentrations of NGF. In addition, BDNF can trigger histamine release from rat brain mast cells, suggesting that these mast cells contain the TrkA and TrkB receptors (184). The mammalian skin expresses a variety of neurotrophic growth factors that are essential for growth, proliferation, and maintenance of nerves such as NGF. Cutaneous neurotrophins are expressed by sensory and sympathetic neurons and non-neuronal cells, thereby regulating nociception, mechanoreception, epidermal homeostasis, inflammation, and hair growth (185,186). During inflammation, NGF is markedly upregulated in nerves associated with the inflamed area (171). During the inflammatory process, NGF is also produced by a wide range of immune cells. Mast cells (187), T-cells (188) and B-cells (189), eosinophils, lymphocytes (190), and epithelial cells (191) can synthesize

NGF. In vitro, allergen stimulation of mononuclear cells from sensitized mice resulted in enhanced NGF synthesis (192). In addition, NGF production was enhanced by antigen stimulation in murine and human Th2 cells (193,194). BDNF synthesis has been detected in human T-cells, B-cells, macrophages, and mast cells (195,196). There is also evidence for enhanced neurotrophin production in inflammation (192). Activated T-cells and macrophages produce BDNF during allergic inflammation (192). NGF can have pro-inflammatory as well as antiinflammatory effects depending on the situation and concentration of the compound. In mice, Braun et al. have recently shown that nasal treatment of mice with NGF induced airway hyperresponsiveness measured by electrical field stimulation (197). Another study by Braun et al. showed that nasal treatment of mice with anti-NGF prevented the development of airway hyperresponsiveness (192). On the other hand, the expression of NGF is increased after brain injury. There is evidence indicating that the increased production of NGF in the central nervous system (CNS) during brain diseases such as multiple sclerosis can suppress inflammation by switching the immune response to an anti-inflammatory, suppressive mechanism (198). In a compelling study, the injection of CD4+ lymphocytes transfected with the NGF gene either before or after the induction of allergic encephalomyelitis inhibited the onset of demyelination (199). This powerful inhibition of an auto-immune process showed that local expression of NGF prevented the migration of inflammatory cells across the vascular endothelium. Furthermore, the functional role of NGF and NT-3 was studied in a model for experimental colitis. The pretreatment with anti-NGF or anti-NT-3 caused a significant increase in the severity of the experimental inflammation (200). These findings suggest a regulatory role for NGF and NT-3 in experimental inflammation of the gut. The fact that NGF may under different circumstances be either pro-inflammatory or antiinflammatory is an important example of the complexity of interactions between neuroactive molecules and the immune and inflammatory systems.

VIII. NEUROPEPTIDE COMMUNICATION: THE DIFFERENT IMMUNE CELLS A. T and B Lymphocytes Peptidergic nerve fibers are in close approximation to lymphocytes in many lymphoid tissues (201). Indeed, mononuclear cells are modified by neuropep-


tides. The presence of neuropeptide receptors on lymphocyte surfaces supports this interaction. Substance P may enhance the mitogen-induced proliferation of human (202) and murine lymphocytes and IgA synthesis by cells from the spleen, Peyer’s patches, and mesenteric lymph nodes (203). VIP increased the IgA response in mesenteric lymph nodes and spleens, but inhibited IgA synthesis in lymphocytes from Peyer’s patches. Furthermore, substance P is chemotactic for human peripheral blood T- and B-cells (204). It is also reported that substance P is a growth and differentiation factor for B-cells (192) and it stimulates cytokine production, chemotaxis (78), and a Th1/Th2 phenotype switch in T-cells (205). Murine and human T-cells can express NK-1 receptors, but their expression on B-cells remains controversial. Some of the biological activity of substance P occurs at only high concentration, suggesting pathways independent of NK-1 receptors, whereas some activity could be blocked by NK-1 receptor antagonists. Clear evidence for the importance of the NK-1 receptor on T-cells comes from studies by Blum et al. (206) using a murine model for schistosomiasis. In NK-1 receptor knockout mice, they observed significant reductions in the size of granulomas in comparison with disease in wild-type animals. The limited IFN-γ production by infected knockout mice suggests that T-cells may be an important target for substance P during schistosomiasis. Substance P and VIP have been shown to affect the ability of mature B-cells to secrete immunoglobulins. Braun et al. (192) showed that tachykinins could augment Ig secretion in cytokine-stimulated cultures. NK-1 receptors have been demonstrated on B-cells, and their biological impact was evidenced by the ability of substance P to enhance Ig synthesis in the presence of a second signal. The addition of LPS to varying concentrations of substance P resulted in optimal IgM production at subnanomolar substance P concentrations, whereas elevated concentrations (100 nM) were not effective (207). These events were NK-1 receptor mediated. VIP stimulation of lymphocytes, like substance P, requires a co-stimulatory signal (208). Substance P is found in the highest concentration in the GI tract (209) and in the lungs (210). In Peyer’s patches, evidence suggests that substance P–containing nerve fibers infiltrate T-cell zones and associate with macrophages. For example, in patients with ulcerative colitis, an increased level of T lymphocytes is found together with an increase in substance P– containing neurons. In human inflammatory bowel disease, increased substance P, as well as NK-1 receptor mRNA, in the gut has been shown, which associates with mucosal T-cells (77). Various studies have

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reported increased expression of substance P derived from neurons following inflammation. Several animal models of inflammation and human disease provide evidence that the NK-1 receptor, located on leukocytes, and its ligand, substance P, influence immune responses. For instance, trichinella spiralis, a parasite that induces a Th2-immune response in rat intestine, induces a T-cell–dependent increase in substance P in the muscle-mesenteric plexus, and blocking of the NK-1 receptor reduces the intestinal inflammation (211). In murine schistosoma inflammation, it has been shown that NK-1 and substance P play an important part in the regulatory response. Substance P regulates T-cell IFN-γ production through interaction with the NK-1 receptors expressed on these cells (122). Another recently discovered molecule, hemokinin, belonging to the family of tachykinins, is a selective NK-1 agonist (212). High concentrations of this neuropeptide were shown to stimulate the proliferation of B lymphocytes (85). It has been suggested that hemokinin is an autocrine factor that contributes to the survival of B-cell precursors in the bone marrow (85).

B. Dendritic Cells DCs, the major antigen-presenting cells, play an important role in the immune response against infections. Their function is crucial in both the early innate responses and subsequent adaptive response (213). In the course of inflammation in peripheral tissue, various cytokines and chemokines are released by resident DCs. DCs are reported to contain substance P or the preprotachykinin mRNA which encodes for substance P and neurokinin A (50). As well, Nelson et al. (87) have clearly shown constitutive expression of PPT-C mRNA by cultured DCs. Myeloid DCs express the highest levels of the message encoding hemokinin-1. Similarly, NK-1 or NK-1 mRNA has been detected in DCs (50) as well as VPAC-1 and VPAC-2 receptor expression (149). CGRP, VIP, and somatostatin are being released from DCs (214). Therefore, neuropeptides should be included in any examination of the immune regulation of T-cell function by DCs. Since DCs are the most efficient antigen-presenting cells that stimulate naive T-cells, thus promoting adaptive immunity, it is not surprising that interactions between neuropeptides and dendritic cells take place. DCs are found in peripheral and CNS (215). Using neuropeptide and DC immunostaining, anatomical and functional connections between the two have been shown (151). In the liver, contacts between nerve fiber staining for substance P, VIP, and CGRP were observed (216). In the lung, being a rich source of neuropeptides

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significantly increasing upon inflammation, DCs are located in close proximity to unmyelinated nerve fibers (217). Functionally, pulmonary DCs bound to substance P and displayed increased motility in vitro in response to graded concentrations of substance P. To examine the role of neural influences on the pulmonary immune response to inhaled antigen, rats were pretreated with capsaicin, and challenged with antigen. Neonatal capsaicin treatment inhibited the accumulation of DCs around small pulmonary vessels (217). Peripheral neuropeptides can directly attract immature DCs, and at the same time they may capture mature DCs at sites of neurogenic inflammation. Low neuropeptide concentrations can improve the motility of DCs to the source of the neuropeptides, primarily sensory nerve fibers. Cells that reach these fibers may undergo functional and phenotypical maturation, which will keep them arrested there. This opposite behavior is probably due to changes in signal transduction pathways of neuropeptide receptors in immature and mature DCs (218). There is no difference in neuropeptide receptor expression on immature and mature DCs (219), but differences have been found for several chemokines (220). Thus, the effects are mediated by specific receptors, which switch signal transduction pathways in DCs of distinct maturation stages. These findings provide evidence for a connection between adaptive immunity and the nervous system, as do the observations of Hosoi et al. (151) who showed the importance of CGRP as a potent downregulator of DC function.

C. Macrophages Macrophages have various important functions involving host defense, immunomodulation, and tissue repair. They produce many types of mediators including interleukins, chemokines, and nitric oxide. There is growing evidence that macrophages can produce substance P and express NK-1 receptors (119,221). Substance P can stimulate human macrophages to release TNF, IL-6, and IL-1 (222,223). Substance P inhibits IFN-γ and LPS-induced TGF-β production (224). In addition, murine macrophages produce somatostatin in response to LPS, IL-10, IFN-γ, and TNF. Other reports suggest that substance P works as a priming factor rather than a direct stimulator. Substance P should sensitize macrophages, making them more responsive to LPS (225,226). Macrophages from stressed mice released IL-6 upon exposure to LPS in vitro (227). Substance P was responsible for the activation of the macrophages and primed them for LPSinduced IL-6 release. Macrophages, stimulated by substance P, produce the inflammatory mediators

PGE2, thromboxane B2, and superoxide ions (228,229). Macrophages also have receptors for CRF, and CRF can induce the production of IL-1, IL-6, and TNF (230). The innate immune response can be influenced by neuropeptides, especially since NK-1 receptors have been found on macrophages (119,222) and dendritic cells (121). In vivo studies have demonstrated a role for the NK-1 receptor in the initiation on the host response against bacterial and viral pathogens. Upregulation of NK-1 receptor expression by salmonella may significantly increase the substance P–mediated macrophage response, directly killing the bacteria. In addition, substance P can enhance IL-1, IL-6, and TNF secretion by macrophages (222). Mice deficient in NK-1 receptor expression have also been used to investigate the importance of this neuropeptide receptor in the host response against pathogens. For instance, the significance of the NK-1 receptor during schistosomiasis has been discussed earlier (206).

D. Neutrophils and Eosinophils There are some reports on direct effects of substance P on neutrophils and eosinophils, but the concentrations of substance P are usually not of physiological relevance. Most of the effects require high concentrations of neuropeptides, whereas at low concentrations, neuropeptides prime the response to other stimuli that otherwise would be ineffective (231). CGRP and substance P have a degranulating effect on eosinophils, and substance P, CGRP, and VIP were found to stimulate eosinophil migration (232). In an in vivo study with allergic rhinitis patients, it was shown that substance P enhanced the recruitment of eosinophils after repeated allergen challenge. Furthermore, substance P can induce IL-8 production from human peripheral blood neutrophils (233) and stimulate guinea pig eosinophil peroxidase secretion (234). CGRP is capable of causing eosinophilia in the lung in vivo and may contribute to airway inflammation in patients with asthma (235). A study on eosinophil chemotaxis showed that neuropeptides alone did not have an effect. However, when eosinophils were pre-treated with peptides, chemotactic response to platelet-activating factor (PAF) or leukotriene B4 was significantly enhanced in allergic patients (236). Potentiating effect of substance P and CGRP on PAF-induced eosinophil chemotaxis in allergic subjects was significantly attenuated by a substance P antagonist and by a human CGRP receptor antagonist, respectively. These results suggest that neuropeptides may play a significant role in eosinophil infiltration by priming cells in allergic inflammation.


Aside from the direct effects, the indirect effects of substance P are more common and better described. Neutrophils migrate into tissue due to neurogenic inflammation resulting from the interaction of substance P with the NK-1 receptor on endothelial cells. In addition, substance P can induce the production of neutrophil chemotactic factor from bovine bronchial epithelial cells (237). Moreover, it stimulates adhesion between cells via induction of adhesion molecules (238).

E. Mast Cells 1. Mast Cell-Nerve Communication Histological studies reveal an intimate association between mast cells and neurons in both the peripheral and central nervous system (2,175). A close anatomical relationship between mast cells and substance P and CGRP-containing sensory nerve endings has been reported in various tissues including skin (239,240), intestine (1), dura mater (241,242), and airway mucosa (16,240). Other than an anatomical link, mast cells also form a functional link between the immune and nervous systems, and mast cells appear to act as bidirectional carriers of information. Neuronal mechanisms are involved in mast cell activation, and mast cells act as principal transducers of information between peripheral nerves and local inflammatory events (243). In an in vitro model of co-culture of mast cells and superior cervical ganglion neurites, selective activation of neurites by scorpion venom has been shown to cause increased calcium uptake in associated mast cells through substance P release and interaction with NK-1 receptors (76), with subsequent focused membrane ruffling at the points of contact (244). Peripheral nerves are highly populated with mast cells, and manipulation of these peripheral nerves causes changes in mast cell densities (245). Mast cells and nerves in many tissues are in constant contact with each other and share a number of activating signals, for some of which both cells express receptors (such as vanilloids) (104). Additionally, both mast cells and nerves respond to stimulation by secretion of preformed mediators, many of which are produced by both cells (NGF, neuropeptides, and endothelin-1). Moreover, a large proportion of primary spinal afferent neurons which contain CGRP and substance P express the proteinase-activated receptor 2 (PAR-2). Proteases such as tryptase from degranulated mast cells have recently been shown to cleave PAR-2 on primary spinal afferent neurons, which causes release of substance P, activation of the NK-1 receptor and amplification of inflammation, and thermal and

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mechanical hyperalgesia (246). This mechanism of protease-induced neurogenic inflammation may contribute to the pro-inflammatory effects of mast cells in human disease. In addition, PAR-2 is also highly expressed in keratinocytes and endothelial cells of inflamed skin (247). Tryptase may in turn activate PAR-2 on keratinocytes and endothelial cells during inflammation. Many studies have shown that mast cell–derived mediators such as histamine, serotonin, and cytokines modulate NANC neurotransmission (248,249). NANC nerve endings express receptors for histamine (H1 and H3) and serotonin (5-HT2a) (250,251), and histamine H1 receptor expression at least is upregulated on primary NANC nerves in inflammation (252). Mast cell mediators like TNF can sensitize afferent C fibers by lowering their threshold (249,253) but can also cause direct release of substance P, neurokinin A, and CGRP from unmyelinated fibers (248). 2. Mast Cell Stimulation In addition to IgE and antigen, cytokines, hormones, and neuropeptides can trigger mast cell secretion (254,255). The latter include somatostatin (256), neurotensin (257), PACAP (258), CGRP (259), and substance P (260). Stem cell factor (SCF) and NGF can promote mast cell growth and can also trigger mast cell degranulation (261). SCF has also been reported to induce mast cells to become responsive to PACAP (262). SCF and NGF are also secreted by mast cells, while substance P has been localized in human skin mast cells, indicating autocrine actions (263,264). Keratinocytes, Langerhans cells, fibroblasts, mast cells, and endothelial cells express functional neurokinin receptors, while G proteins on mast cells can in addition be activated by substance P in a nonreceptormediated fashion (265). Stimulation of G-proteins will eventually lead to mast cell mediator production and release. It has been shown that functional NK-1 receptors are expressed on murine mast cells in vitro in the presence of IL-4 and SCF (266). Very recently, Bischoff et al. examined the expression of tachykinin receptors on human mast cells and found that human mast cells derived from intestinal mucosa do not constitutively express NK-1, NK-2, or NK-3 receptors (267). However, when stimulated by IgE receptor crosslinking, these mast cells started to express NK-1, but not NK-2 or NK-3 receptors, suggesting that specific tissue conditions such as allergic inflammation may lead to mast cell expression of NK-1 receptors. Nerve-mast cell interaction is involved in both homeostatic and pathologic regulations. Because the synaptic cell adhesion molecule (SynCAM), alterna-

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tively named spermatogenic Ig superfamily (SgIGSF), is expressed on both nerves and mast cells and because it binds homophilically, this molecule may be the best candidate to sustain this association (268). In vitro, mast cells with SgIGSF/SynCAM attached to neurites; this was inhibited dose-dependently by blocking Ab to SgIGSF/SynCAM. Mast cells without SgIGSF/ SynCAM were defective in attachment to neurites, and transfection with SgIGSF/SynCAM normalized this, showing that SgIGSF/SynCAM on mast cells predominantly mediated attachment and also promoted communication with associated nerves. The activity of mast cells is clearly more complicated than being an all-or-nothing condition. Janiszewski et al. (393) reported in a patch clamp study that mast cells do not respond to very low concentrations of substance P (in the picomolar range), but that activation and delayed degranulation occurred after a second exposure. Therefore, mast cells can be primed when exposed to physiologically relevant low concentrations of substance P, and lower their thresholds to subsequent activation. It has been proposed that this initial stimulation is the functional correlate of priming. If mast cells are indeed primed by exposure to substance P, it would enable a subthreshold stimulus to initiate mast cell activation. Furthermore, secretion can occur without evidence of degranulation, and even molecules stored within the same granules can be released and secreted in a discriminatory pattern (269). Mast cells have been increasingly implicated in inflammatory processes where explosive degranulation is not commonly observed. Ultrastructurally, a study by Ratliff et al. (270) showed mast cells in close proximity to unmyelinated nerve fibers. These mast cells contained granules showing ultrastructural features of activation or piecemeal degranulation, which have been associated with differential secretion. Marshall et al. (179) showed that NGF at doses as low as 10 ng/ml will induce IL-6 production but inhibit TNF release from rat peritoneal mast cells, indicating that even a single molecular signal can give rise to a balanced proand anti-inflammatory outcome in a single cell type. These data support the potential for mast cell regulation by the central nervous system and suggest that modulation of mast cells can occur in vivo without degranulation.

IX. NERVES IN THE INFLAMMATORY PROCESS Nervous innervation of an organ is a requirement for establishing certain inflammatory reactions (271). Substance P and the related tachykinin peptides are

involved in inflammatory processes and in the transmission of sensory nociceptive information. Organs with a high density of neuropeptide receptors, such as the intestines and the lung, have been proposed to be more susceptible to inflammation (272). For instance, infection of rats with the nematode Nippostrongylus brasiliensis results in mast cell hyperplasia and neuroplasticity of intestinal mucosal nerves during inflammation. This was shown to include early neurodegenerative and late regenerative phases that appeared to correlate with mast cell densities (1). During inflammation, hyperalgesic mediators are produced by invading immune cells or by resident cells (cytokines, chemokines, NGF, leukotrienes, prostaglandins, ATP). It is less well known that analgesic mediators are also produced in inflamed tissue, which counteract pain. These include opioid peptides, somatostatin, and anti-inflammatory cytokines (273). While these mediators contribute to the body’s ability to counteract infection and the destruction of tissue integrity, they also elicit pain by activation of specialized receptors localized on primary afferent neurons “nociceptors.” Opioid peptides are produced in peripheral inflamed tissue by immune cells and can be released upon certain types of stimulation. They bind to peripheral opioid receptors and thereby elicit potent endogenous analgesia (274). Peripheral tissue injury causes a migration of opioid peptide-containing immune cells to the inflamed site. The subsequent release and action of these peptides on opioid receptors localized on peripheral sensory nerve terminals cause endogenous analgesia (275). Opioid peptides secreted from immune cells are so far the best studied peptides in peripheral inflammatory pain control. Immigration of opioidcontaining immune cells is also dependent on neuroimmune interactions (275), since the central inhibition of pain by intrathecal application of morphine reduces the number of opioid-containing immune cells at the site of inflammation and also impairs endogenous peripheral opioid analgesia. Rheumatoid arthritis is an autoimmune disease characterized by inflammation of the synovial membrane of multiple joints. Experimentally, adjuvant arthritis is most severe in joints which are most densely innervated (276). Clinically, it was shown that hemiplegic patients do not have arthritis on their paralyzed side (277). Moreover, if the nerve innervating a joint is cut, arthritis cannot be induced in the denervated joint (276, 278). Miao et al. (279) have shown that impulse activity in vagal afferents has an inhibitory effect on the modulation of bradykinin-induced plasma extravasation by nicotine. Spinal pathways seem to be important in mediating this effect. In rheumatoid arthritis fibroblast-like synoviocytes are at the interface between


the immune and the nervous systems (280). There is evidence implying that substance P and the NK-1 receptor play a role in arthritic disease. Synoviocytes of patients with rheumatoid arthritis express NK-1 receptor mRNA (281). Substance P potentiates the effect of pro-inflammatory cytokines on the expression of VCAM-1 on these cells, suggesting that substance P increase of cytokine-induced VCAM-1 expression acts via this specific substance P receptor (282). The relationship between nervous innervation and inflammation is observed in other clinical situations such as diabetes in which the inflammatory response is inadequate because the nervous innervation is affected by the disease (diabetic neuropathy).

X. NEUROGENIC INFLAMMATION Neuropeptides released from sensory nerves induce a wide range of inflammatory reactions including vasodilatation, increased plasma extravasation, tissue swelling, and adherence of inflammatory cells to the endothelium, thereby facilitating cellular infiltration (106,283). The effects produced by tachykinins (substance P, neurokinin A, and neurokinin B) and CGRP released from peripheral endings of capsaicinsensitive primary sensory neurons are collectively referred to as neurogenic inflammation (284). Several studies have shown a role for neurogenic inflammation in the airways (285–287), skin (288,289), pancreas (290), urinary tract, and the digestive system (291). Neurogenic inflammation involves a change in function of sensory neurons due to inflammatory mediators, inducing an enhanced release of neuropeptides from the sensory nerve endings (292,293). Sensory neurons play a role in neurogenic inflammation (292). It has become apparent that the mast cell and its mediators also are important in neurogenic inflammation (293). In various studies, tissue mast cells invariably show ultrastructural evidence of activation even in normal healthy conditions, suggesting that these cells are constantly providing information to the nervous system (241). The fact that they are located at sites under constant exposure to the external environment, such as the skin, respiratory and gastrointestinal tract, emphasizes the significance of these associations. Aside from the generation of action potentials, the C-fiber terminal is a secretory system, releasing tachykinins to cause neurogenic inflammation. There is also histological evidence of the presence of cytokines, especially IL-6, within both sensory and autonomic nerves (294). The release of these pro-inflammatory cytokines would cause inflammation in the affected

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area, and indicates a direct link between neurogenic stimulation and the release of pro-inflammatory cytokines (294). Stimulation of these C-fibers by a range of chemical and physical factors results in afferent neuronal condition eliciting parasympathetic reflexes and antidromic impulses traveling along the peripheral nerve terminal. Such communication from one nerve to another, without passing through a cell body, is called the axon reflex and results in local release of tachykinins and CGRP from C-fiber terminals (107). Axon reflexes account for many of the local physiological responses to antigen, for instance, in sensitized lung (295,296) and gut tissues (297,298), and have long been recognized to be involved in local vasodilatation in the skin (299). Antidromic stimulation of guinea pig vagal sensory fibers results in contractions of the isolated airway smooth muscle mediated by tachykinins (300). Further studies indicate that neuropeptide release can also be induced via direct depolarization of the terminal.

XI. CENTRAL NERVOUS SYSTEM The brain, nervous, and immune systems are the major adaptive systems of the body (301). Several pathways have been shown to link the brain and the immune system, such as the autonomic nervous system via direct neural influences and, second, the neuroendocrine humoral outflow via the pituitary. CRH, secreted by the pituitary gland, is a major regulator of the HPA axis and cortisone synthesis, and acts as a coordinator of the stress response (302). CRH is also thought to be involved peripherally in tissue responses to stress in the skin, respiratory tract, and intestine. The central nervous system can and does activate or even inhibit immune and inflammatory events. Stress activation of the hypothalamic-pituitary-adrenal (HPA) axis has such a bimodal effect. CRH activates peripheral mast cells (303), corticosterone may inhibit inflammatory events, and catecholamines have complex effects. Hypnosis has been shown to inhibit the expression of skin reactivity to allergen in classical tests of allergic hypersensitivity (304,305), and we have shown that classical Pavlovian conditioning could cause mast cell secretion (306). In the central nervous system, mast cells may participate in the regulation of inflammatory responses through interactions with the HPA axis. Matsumoto et al. (307) showed that in the dog, degranulation of CNS mast cells evoked HPA activation in response to histamine release and CRH. In this study, dogs were passively sensitized with IgE and challenged with specific antigen centrally or peripherally. Both routes

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resulted in cortisone release from the adrenal glands. The effect could be mimicked by intracranial injection of the mast cell secretagogue compound 48/80, and blocked by CRH antibodies or histamine H1 blockers, but not H2 blockers. These results suggest that intracranial mast cells may act as allergen sensors, and that the activated adrenocortical response may represent a host defense reaction to prevent anaphylaxis. CRH is also thought to be involved peripherally in tissue responses to stress in the skin, respiratory tract, intestine, and bladder. Many, if not all, of the recorded changes have involved mast cells and neuronal activation, the latter being often mediated by neurotensin and/or substance P. Central neuropeptides initiate a systemic stress response by activation of neuroendocrinological pathways such as the sympathetic nervous system, hypothalamic pituitary axis, and the renin angiotensin system, with the release of the stress hormones (i.e., catecholamines, corticosteroids, growth hormone, glucagons, and renin)(308). These effects have been found in a variety of stress models including cold, restraint stress, and water avoidance stress (309–311). The neuropeptide CGRP is present in central structures like the pituitary and is involved in the modulation of inflammation (312). The presence of dense CGRP-like immunoreactivity in vagal nuclei (313) suggests that CGRP may have a functional role in the protective effect in vagal afferents in inflammatory processes. Studies have shown that exposure of experimental animals to immobilization stress increases colonic motility and that these effects are mediated by release

After Dickerson et al., J. Leuk. Biol., 1998 Maier et al., Ann. N.Y. Acad. Sci., 1998

FIGURE 1 Schematic outline after Maier et al. (43) with modifications. This work has shown that if endotoxin is introduced into the peritoneal cavity in vagotomized animals, the fever response of the brain is markedly diminished with consequent downstream effects on cortisol production. This response appears to involve both mast cells and nerves in a complex set of interactions. Reprinted with permission.

of CRH. Pothoulakis et al. (314) demonstrated that CRH released during immobilization stress increases colonic transit via a neuronal pathway and stimulates colonic mucin release via activation of neurons and colonic mast cells. These results provide support for an important role for CRH in stress-mediated colonic responses and a link between the nervous and the immune systems.

XII. STRESS RESPONSE The CNS seems to have the capacity to produce as well as modulate general inflammatory reactions, not only in response to infection, trauma, and tissue damage, but in response to stress as well (38,40). Psychological stress results in the release of chemical mediators including norepinephrine (NE), serotonin, and acetylcholine, which activate cells of the paraventricular nucleus (PVN) of the hypothalamus producing CRH (308). CRH is a major regulator of the HPA axis and principal coordinator of the stress response. Peripheral CRH is thought to have pro-inflammatory effects. CRH also stimulates the locus coeruleus, a dense collection of autonomic cells in the brainstem, to secrete NE at sympathetic nerve endings (315). Although CRH is the major coordinator of the stress response, substance P may also participate in the stress response by activating the HPA axis and the sensory nervous system. Substance P becomes elevated in the brain in response to many different types of psychological stress such as handling (316), restraint (317), and anxiety (318). In the amygdala, there is a marked increase in substance P and its precursor preprotachykinin after various psychological stressors in rats. Two other areas, involved in behavior, innervated with substance P are the substantia nigra and the median raphe. Substance P may stimulate the HPA axis directly or indirectly by increasing arginine vasopressin (AVP), a potent stimulator of HPA activity (319). Immune cells such as macrophages are also involved in certain types of stress. The cold water swim test resulted in the increase of substance P and its receptor in peritoneal macrophages as well as an increase in peritoneal substance P.

A. Acute versus Chronic Stress A general pattern of immunological changes is emerging as part of a result of examination of the effects of stress. As a generalized observation in animal models of stress, acute stress appears to enhance and chronic stress appears to diminish cell-mediated


immune responses and other immune reactivity (320). The generalized pattern of deficits seen with chronic stressors includes diminished lymphocyte proliferation and diminished production of cell-mediated immune cytokines as IL-2 and IFN-γ (321). These immunological alterations were often accompanied by evidence of elevated production of stress mediators, such as epinephrine and cortisol. Active immune responses themselves represent a source of systemic stress which impacts the brain and modifies various neuroendocrine and behavioral functions. Therefore, the immune system has been conceived of as a potential contributor to stress-related behavioral abnormalities, such as clinical depression (29,322). It is suggested that immunologically induced changes in the brain activate neuropeptides, thereby sustaining an adaptive state of arousal that promotes appropriate behavioral adjustments during infectious illness.

XIII. NERVE-IMMUNE COMMUNICATION IN THE VARIOUS TISSUES A. Skin Both nerve fibers and inflammatory cells are able to release neuromediators and thereby activate specific receptors on target cells in the skin. The dermis is richly innervated by primary efferent sensory nerves, post-ganglionic cholinergic parasympathetic nerves and post-ganglionic adrenergic and cholinergic sympathetic nerves (311). Cutaneous neuromediators include classical neurotransmitters such as catecholamines and acetylcholine being released from the autonomic nervous system. Neuropeptides including substance P, VIP, and CRGP may be released from sensory or autonomic nerve fibers and several epidermal as well as dermal cells. Neuropeptides have been shown to activate a number of target cells including Langerhans cells, endothelial cells, and mast cells (323). Observations of Egan et al. (324) showed that especially unmyelinated axons were associated with mast cells as well as Langerhans cells in primate as well as murine skin.

1. Neuropeptides and Mast Cells Skin mast cells can respond to trauma, releasing a variety of inflammatory mediators through an immediate sensory nerve-stimulated response or via an axon reflex, inducing the release of neuropeptides from peripheral nerve endings, in turn leading to more mast cell activation. Neuropeptides are released in response to nociceptive stimulation by pain, mechanical and

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chemical irritants to mediate skin responses to infection, injury, and wound healing. Substance P effects have been described on keratinocytes including hair follicles, mast cells, fibroblasts, and endothelial cells. Increased epidermal SPimmunoreactive nerve fibers have been observed in certain inflammatory human skin diseases (325). Substance P is one of the main neuropeptides responsible for the skin reaction characterized by erythema, pain, and swelling (239). In addition, substance P can cause the release of histamine (326) and TNF (327) from skin mast cells, which in turn lead to vasodilatation. Capsaicin (which releases neuropeptides from nerves) applied to human skin induces the release of chymase within 6 hours and induction of E-selectin in adjacent microvascular endothelium. These events are consistent with release of substance P from axons and subsequent stimulation of cytokine-mediated mast cell-endothelial interaction. However, identical application of capsaicin to human skin grafted onto immunodeficient mice, and thus experimentally lacking in unmyelinated axons, failed to show similar findings (324). These results demonstrate that unmyelinated axons connect Langerhans cells and dermal mast cells. Immune-nerve interaction has been shown to be involved in the skin during stress. Stress can significantly increase the number of hair follicles containing apoptotic cells and also significantly increase the number of activated perifollicular macrophage clusters and the number of degranulated mast cells, whereas it downregulated the number of intraepithelial T lymphocytes. Substance P seems to be a key mediator of stress-induced hair growth inhibition in vivo. These stress-induced immune changes could be mimicked by injection of the neuropeptide substance P in non-stressed mice (328,329). Increased numbers of substance P-immunoreactive sensory fibers, as seen in the dermis of stressed mice, are a result of transient high levels of NGF (330), suggesting that NGF is a central element in the perifollicular neurogenic inflammation that develops during the murine skin response to stress. So, stress can inhibit hair growth in vivo, probably via a substance P and NGF-dependent activation of macrophages and/or mast cells. Recent studies have suggested that mast cells play a crucial role in the downregulation of immune responses and induction of tolerance after exposure of skin to ultraviolet B (UVB) radiation. Interactions between mast cells and the nervous system appear to be involved in UVB-mediated immune suppression. TNF, reported to be derived from mast cells, has been shown to be a major cytokine implicated in signaling

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Alard, Niizeki, Hanninen et al., 1999

FIGURE 2 Schematic drawing of skin responses to UVB radiation and the consequent immune suppression dependent on mast cells, Langerhans cells, and nerves (331). Modified with permission.

the immunosuppressive effects of UVB (331). Evidence indicates that mast cells are triggered to release TNF in response to the neuropeptide CGRP, which is also released from UVB-damaged cutaneous nerve endings (332). Several inflammatory skin conditions, including atopic dermatitis and psoriasis, are exacerbated by stress. Recent evidence suggests that crosstalk between mast cells, neurons, and keratinocytes might be involved in such exacerbation. CRH and its receptor are present in the skin, and their levels are increased following stress (333). Human mast cells synthesize and secrete CRH in response to immunoglobulin E receptor crosslinking. Mast cells also express CRH receptors, activation of which leads to the selective release of cytokines and other pro-inflammatory mediators (334). CRH receptor antagonists could be used to inhibit stress-induced mast cell activation and provide new therapeutic options for chronic inflammatory conditions exacerbated by stress. 2. Neuropeptides in Nerve-Dendritic Cell Communication Nerve cells may regulate Langerhans cell (LC) function by elaboration of certain neuropeptides, whereas LC may promote the differentiation of nerves by elaboration of interleukin-6 and, possibly, other factors (149). LC functions are modulated by both substance P and CGRP, because these two neuropeptides are often co-localized and co-released by the same cutaneous nerve fibers (335,336). Brain and Williams (336) have shown that injection of CGRP in the human skin together with substance P converts the long-lasting vasodilatation induced by CGRP into a temporary

response. Thus, it may be hypothesized that biological effects occur through the combined action of substance P and CGRP. Furthermore, Brain and Williams (336) showed that this phenomenon is dependent on the action of proteases released from mast cells by substance P, revealing a new mechanism for the inactivation of a neuropeptide due to a direct consequence of mast cell activation. Although substance P seems to be involved in local inflammatory reactions in the skin, it has been shown that substance P may be involved in the inhibition of antigen presentation (265). CGRP is a neuropeptide and vasodilator that modulates some macrophage functions, including antigen presentation in vitro. Many, if not most, epidermal LC appeared to be closely associated anatomically with CGRP-containing nerves as determined by confocal laser scanning microscopy (337). CGRP inhibits the Ag-presenting function of macrophages (338) and Langerhans cells in various systems (151). Nong et al. (338) found that CGRP inhibits macrophage activation induced by IFN-γ. In addition, pretreatment of LC by CGRP before antigen sensitization prevents the ability of the cells to elicit a delayed-type hypersensitivity reaction (151). Furthermore, when injected intradermally, CGRP inhibits the induction of hypersensitivity contact reactions (150), supporting the concept that endogenous CGRP may modulate immune function, presumably in part, through an inhibitory effect on LC function. These findings indicate that CGRP is capable of inhibiting both the induction and expression of fundamental cellular immune functions and suggest at least one inhibitory pathway of interaction between the nervous system and immunological function.

B. Airways Various studies have demonstrated the presence of SP and NKA in the respiratory tract in mammalian nerve fibers. The majority of nerve fibers reach the lung via the vagus nerve. The vagal innervation is divided into vagal afferent (sensory) nerves and autonomic (parasympathetic) nerves. The interaction between the nervous and immune systems in the lung, as in other organs, is bi-directional. Some immune cells in the airways are in intimate contact with nerves. They possess cell-surface receptors for a variety of neurotransmitters. The function of monocytes and macrophages, for example, can be modified by adrenergic and cholinergic receptor activation (339). In addition, neuropeptides including tachykinins and VIP can interact with receptors on macrophages to modify their function (340,341). In the trachea and


bronchi, SP-immunoreactive fibers have been found in the smooth muscle layer and around local ganglion cells. In the bronchial tree, most of the SP-positive fibers are of vagal origin; but in the lung, the fibers are both of vagal and thoracic spinal origin (342–344). Another important neural network in the lung is the non-adrenergic non-cholinergic (NANC) nervous system. Inhibitory NANC nerves contain vasoactive intestinal peptide (VIP) and nitric oxide (NO). Under normal conditions in the mouse lung, VIP receptors are localized on alveolar macrophages. Immunized and intratracheally challenged mice demonstrated increased levels of VIP and NO receptor expression on mononuclear cells and neutrophils in inflammatory infiltrates (126). VIP is a potent relaxant of the airways and has also been suggested to inhibit mediator release from mast cells (345). In cats, Miura et al. (346) demonstrated that bilateral electrical nerve (vagus) stimulation of i-NANC in atropinized and propanolol-treated animals inhibited antigen-induced bronchoconstriction and histamine secretion from histamine-containing cells. Other direct effects of VIP include inhibition of T-cell proliferation; IL-2, IL-4, and IL-10 cytokine production; and inhibition of IgE release by B-cells (347). A further role of NO is skewing T-cells towards a Th2 phenotype by inhibition of Th1 cells and IFN-γ (348). In addition to inhibitory NANC systems in the airways, there is also an NANC afferent nervous system that protects the airways against inhaled irritants and chemical particles. These excitatory NANC nerves or sensory nerves play a regulatory role in airways. Excitatory NANC system-associated neuropeptides with immunomodulatory functions are SOM, CGRP, the members of the tachykinin family substance P and NKA, present in the airways of various mammals including man (78,106,349). These sensory neuropeptides contract smooth muscle, dilate bronchial arteries, increase vascular permeability, increase mucus production, and modulate ganglionic transmission. Many animal studies show that capsaicin-induced depletion of excitatory NANC neuropeptides prevents airway hyperresponsiveness and pulmonary inflammation. For instance, airway hyperresponsiveness induced by a respiratory viral infection, ovalbumin (350), plateletactivating factor (351), or toluene diisocyanate (352) can be attenuated by interruption or blockade of the excitatory NANC system. Sensory neuropeptides can be released into the airways as a consequence of action potentials (axonreflex pathways)(353). Once released in the airways, neuropeptides participate in the inflammatory response via various mechanisms collectively referred to as neurogenic inflammation (as described above). In addition,

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they are certain to influence the recruitment, proliferation, and activation of inflammatory cells. Specific receptors for CGRP and SOM have been demonstrated on macrophages, monocytes, B-cells, and T-cells (58,354). SOM has various inhibitory effects on immune responses via specific receptor activation (58) and affects the suppression of Ig production in B-cells, including IgE (62). CGRP inhibits substance P–induced super oxide production in neutrophils and the proliferation as well as antigen presentation by peripheral mononuclear cells (355). In allergic rhinitis patients, it was shown that substance P administered after antigen challenge enhanced the recruitment of eosinophils. Forsythe et al. (356) demonstrated that substance P and NKA induce histamine release from human airway mast cells. This is underlined by an in vitro model using trachea from Fischer 344 rats, in which substance P stimulation of mast cells represented a major factor leading to bronchoconstriction (357).

C. Gastrointestinal Tract The main sources of the neuronal tachykinins in the gut are (a) the intrinsic enteric neurons of the myenteric plexus, (b) the intrinsic enteric neurons of the submucosal plexus, and (c) the extrinsic primary afferent fibers. The most quantitatively important source of tachykinins in the gut is the enteric nervous system, which has its cells in the wall of the intestine and supplies all gastrointestinal effector systems. The mammalian gastrointestinal tract contains both substance P and neurokinin A and various extended forms of these tachykinins. The gastrointestinal tract is characterized by a unique accumulation of immune and inflammatory cells. Close approximations between peptidergic nerves and mast cells, lymphocytes, eosinophils, and plasma cells have been reported in the lamina propria (1,16,358,359). Neurotransmitters, neuropeptides, and neuroendocrine hormones can affect immune function, and conversely cytokines can affect neuronal function. As many leukocytes express receptors for neuropeptides and neuroendocrine hormones, this strongly implies the importance of a neuroimmune interaction in the intestine. Neurally mediated epithelial ion secretion is well established, and immunomodulation of epithelial barrier function has become evident (14,360). Epithelial cells can also produce neuroregulatory factors, indicating the communication between nerves, immune cells, and epithelium (361). An example of a neuropeptide-induced inflammatory process is diarrhea induced in rats by clostridium difficile toxin (362–364). Substance P, CGRP, and neurotensin were involved in this inflammatory process.

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The toxin stimulated the release of these neuropeptides from primary C afferents in the lamina propria. This was likely followed by the production of substance P by macrophages and the release of greater amounts of TNF. Furthermore, mast cells were degranulated in the mucosa by substance P, their mediators also contributing to the inflammatory process. Pothoulakis et al. (314) studied the intestinal responses to stress. Increased motility, mucus hypersecretion and intestinal chloride ion secretion, was mediated by CRH. Both mast cells and nerves are involved since the effects were inhibited by NK-1 receptor antagonists and mast cell stabilizers (365). Structural as well as functional alterations of enteric nerves have been reported in both ulcerative colitis and Crohn’s disease. An altered expression of substance P (366) and VIP (367) has been found in both diseases. An increased expression of CRH has been demonstrated in mucosal inflammatory cells in ulcerative colitis (368). Furthermore, NGF immunostaining is significantly increased in Crohn’s disease tissue (369). The consequences for these neuronal changes for mucosal function in IBD have yet to be fully clarified. 1. Mast Cells Intestinal mast cells have been repeatedly reported to communicate with the enteric nervous system. Stead et al. (370) reported an anatomical association between mast cells and nerves in the human intestinal mucosa based on electron microscopy studies. Nerve stimulation has been reported to cause mast cell degranulation in the intestine. First, Shanahan et al. (371) showed that substance P caused mediator release from intestinal mucosal mast cells. Subsequently, substance P and CGRP fibers have been reported to activate peptidergic mast cells in intestinal mucosa of healthy and infected rats as well as in patients with inflammatory bowel disease (1). Mast cell mediators have an effect on the nerves in the intestine. Intestinal mast cell infiltration may perturb nerve function leading to abdominal pain perception in irritable bowel syndrome (372). Recent evidence for activated mast cells associated with enteric nerves in irritable bowel syndrome strongly imply that mast cells are involved in this symptom complex (372). A study by Jiang et al. (373) in an intestinal model for anaphylaxis showed that serotonin and histamine, released from the mast cells after intestinal anaphylaxis, stimulate mesenteric afferents via 5HT3 and histamine H1 receptors. Mesenteric afferent nerve discharge increased approximately 1 minute after luminal antigen challenge and was atten-

uated by serotonin and histamine receptor antagonists. Mast cell-nerve association appears to function as a homeostatic unit in the regulation of gut physiology and in response to antigen (14). Perdue et al. (298) determined the existence of an integral nerve-to-mast cell and mast cell-to-nerve connection during intestinal anaphylaxis. A role for the mast cell-to-nerve connection was also established by increases in the short-circuit current after antigen challenge, in Ussing chamber experiments. The response to antigenic stimulation was reduced in mast cell– deficient mice compared to their littermates and was inhibited by different mast cell antagonists in +/+ but not in W/Wv mice, pointing to mast cell-to-nerve connection. Furthermore, reconstitution of the mast cell deficiency was followed by a restoration of the neural response. In sensitized guinea pig intestine, the short-circuit current secretory response to antigen occurred simultaneously to acetylcholine release and could be blocked by atropine (374). This showed conclusively that nerve excitation and the secretion of the main cholinergic neurotransmitter could be induced by antigen via mast cells through an immunemediated response. It can reasonably be concluded that nerves and mast cells form a physiological unit which presumably maintains and regulates homeostasis of the mucosal epithelial secretory response. This unit is involved in health, in response to stress,

FIGURE 3 Representation of neuroimmune interactions involved in stress-induced functional changes of intestinal mucosa. Activation of mucosal nerves, possibly releasing CRH and/or Ach, leads to the stimulation of mast cells. Mast cells (and possibly neurons) can release bioactive chemicals that enhance epithelial ion secretion and permeability of both the transcellular and paracellular pathway (Modified with permission from Soderholm and Perdue, 2001) (394).


and also in response to injuries and environmental pathogens.

D. Urinary Tract Bladder function is regulated by complex and interrelated neural circuits involving the peripheral and CNS (375). In addition to the classic neurotransmitters Ach and NE, neuropeptides released from the different innervating nerves and cytokines liberated from mast cells and leukocytes profoundly affect bladder function (376). Dysfunction of these interactions could lead to bladder hyperreactivity and systemic inflammatory symptoms. The distribution of SP and NKA has been extensively studied in the urinary bladder of several species (377,378). Capsaicin treatment results in an almost complete disappearance of the tachykinin-immunoreactive fibers, suggesting that the major sources of tachykinins in the urinary bladder are sensory fibers (379,380). Emotional or physical stress can release CRF or its analogue urocortin (Ucn) from the hypothalamus, which then stimulates the NTS to release Ach or neuropeptides in the bladder, inducing hyperreactivity. CRF/Ucn can also be released from the dorsal root ganglia and can stimulate bladder mast cells, directly or via neuropeptides (376). For instance, stress-induced bladder mast cell activation is inhibited by neonatal capsaicin treatment, which depletes sensory neurons of their substance P (381). Bladder afferents synapsing at the sacral DRG contain CGRP, glutamate, NO, VIP, and substance P (382). Depletion of these neuropeptides by capsaicin reduces bladder pain and irritation (383–385). Moreover, selective NK-1 receptor antagonists block substance P–induced bladder muscle contractions and plasma extravasation (386). An intimate relationship has been demonstrated between mast cells and peptidergic sensory nerves in the bladder. As shown by Keith and Saban (387), mast cells of mucosal and connective tissue type were found within nerves and ganglia and were in close contact with individual nerve fibers displaying substance Pand CGRP-like immunoreactivity in healthy tissue. In the bladder, axon reflexes can cause noninfectious neurogenic inflammation (376,381,388) via the influx of inflammatory cells from the vascular system. Mast cell immigration has been described in urinary bladder disorders such as interstitial cystitis. Interstitial cystitis (IC) has continued to be an unresolved problem in clinical urology. Mast cells were in close contact with nerve fibers displaying substance P- and CGRP-like immunoreactivity (389). Elbadawi demon-

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strated the presence of mast cells, undergoing piecemeal degranulation, in close proximity to intrinsic nerves in the bladder wall (375). Bladder neuronal proliferation has been demonstrated in IC patients, and an increase in nerve fibers containing NPY and CGRP has also been noted (390). In addition, increased mast cell numbers have been reported in a subset of IC patients (391) together with increased concentrations of histamine (389). The data provide evidence for the involvement of neurogenic inflammation in the pathogenesis of interstitial cystitis. Saban et al. (392) showed that NK-1 receptor −/− mice were protected from inflammation, failing to present bladder inflammatory cell infiltrate or edema in response to antigen challenge. This work supports evidence for the role of substance P and the NK-1 receptor in the development of interstitial cystitis.

XIV. SUMMARY The brain and nervous system have a bi-directional communication with the immune system. Over the last decade especially, it has become apparent that many of the cells in these different systems possess the same receptors and can make the same molecules. Thus, the nervous system can make a major pro-inflammatory cytokine, IL-6, and cells of the immune system can make substance P and acetylcholine. Similarly, cells in both systems can synthesize neurotrophins such as nerve growth factor and brain-derived neurotrophic factor. Behavioral changes can be initiated as a result of peripheral inflammation (illness behavior), and pyrexia can be caused by peripheral infections and inflammation as a result of communication with and through the vagus nerve to the brain. In turn the brain routinely receives communication signals from peripheral immune events and can coordinate responses through the HPA axis and the autonomic nervous system. The intimate nature of these connections is exemplified by the consistent finding of associations between non-myelinated nerves and lymphocytes, mast cells, eosinophils, and Langerhans cells, at which locations bi-directional communication clearly occurs. How these events are orchestrated under different circumstances of emotion or inflammation and injury are only just beginning to be worked out. Much remains to be done, but scientific progress promises much greater complexity as well as understanding. What is clear, however, is that immune mechanisms cannot be ignored in dealing with a functional understanding of the nervous system or even behavior, and the reverse is also true.

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C H A P T E R

5 Vasoactive Intestinal Peptide: An Anti-inflammatory Neuropeptide DOINA GANEA AND MARIO DELGADO

I. INTRODUCTION 131 II. VIP PRESENCE IN PRIMARY AND SECONDARY LYMPHOID ORGANS 132 III. VIP RECEPTORS IN IMMUNE CELLS 133 IV. EFFECTS OF VIP ON INNATE IMMUNE RESPONSES 135 V. EFFECTS OF VIP ON T-CELL ACTIVATION, DIFFERENTIATION, AND FUNCTION 139 VI. VIP IN MODELS OF AUTOIMMUNITY, INFLAMMATION, AND BONE MARROW TRANSPLANTATION 143 VII. CONCLUSIONS AND PERSPECTIVES 149 VIII. ABBREVIATIONS 150

in various models of autoimmune diseases and inflammatory conditions both in the CNS and the periphery.

I. INTRODUCTION In addition to the hypothalamus-pituitary-adrenal axis, neuroendocrine-immune interactions occur through peripheral nerve fibers innervating the lymphoid organs, and are mediated by neurotransmitters and neuropeptides. In addition, immune cells express and release various neuropeptides and hormones. Although the factors controlling neuropeptide release from neuronal or immune sources during an inflammatory response remain to be identified, it has been shown that functionally relevant amounts of vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) are released during an immune response in patients and animal models (Delgado et al., 1999b; Kaltreider et al., 1997; Lee et al., 2002; Mosimann et al., 1993; Nieber et al., 1992). Immune cells and cells of the nervous system share the same specific receptors for neuropeptides, and the presence of both neuropeptides and neuropeptide receptors in lymphoid tissues creates the framework for the immunomodulatory function of neuropeptides. VIP, one of the best studied immunoregulatory neuropeptides, affects both innate and adaptive immune responses, and acts as a major anti-inflammatory factor

ABSTRACT The vasoactive intestinal peptide (VIP) discovered by Said and Mutt more than 30 years ago and characterized as a widely distributed neuropeptide also functions as a potent immunomodulator. Here, we review the evidence supporting the role of VIP as an endogenous anti-inflammatory agent, released by the innervation and by activated immune cells and acting through specific receptors present on macrophages, dendritic cells, microglia, and T lymphocytes. VIP prevents the activation of macrophages, microglia, and dendritic cells, and promotes the generation of Th2 effectors and regulatory T-cells. Due to its pleiotropic effects on both innate and adaptive immunity, VIP is an attractive potential therapeutic agent for the treatment of autoimmune and inflammatory diseases. This review presents recent evidence on the effect of VIP PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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in animal models of autoimmune diseases. In this chapter we propose to review the role of VIP in immunity, and to discuss its potential use in the treatment of inflammatory, autoimmune, and neurodegenerative disorders.

peptide synthesis was described initially in neuronal cells, prohormone convertases required for neuropeptide processing have been recently identified in immune cells as well (Decroly et al., 1996).

B. VIP Sources in Lymphoid Organs II. VIP PRESENCE IN PRIMARY AND SECONDARY LYMPHOID ORGANS A. VIP Gene and Protein VIP, a 28 amino acid peptide, belongs to a family of structurally related hormones and neuropeptides, which includes PACAP27, PACAP38, secretin, glucagon, gastric inhibitory peptide, peptide histidine methionine (in humans), and peptide histidine isoleucine (its counterpart in other mammalian species) (Said, 1986) (Figure 1A). The human VIP gene contains seven exons, with exon five coding for VIP (Figure 1B). The mRNA is processed through differential splicing, and the translated preproVIP undergoes enzymatic processing resulting in the mature C-terminus amidated VIP peptide (Bloom et al., 1983; Itoh et al., 1983). Although the biochemical process required for neuro-

1. Neuronal VIP VIP-ergic nerve fibers have been identified in both central and peripheral lymphoid organs. In the thymus, the VIP+ nerve endings are most abundant in the capsule, enter the cortex and occasionally the medulla (Bellinger et al., 1996). In the spleen, VIP+ nerve endings are prominent in the red pulp, extending as free fibers in the parenchyma and the white pulp, as well as in the periarteriolar lymphatic sheath among T lymphocytes (Bellinger et al., 1996). In lymph nodes, the VIP innervation is relatively sparse, being present beneath the capsule and along the vasculature (Bellinger et al., 1996; Fink and Weihe, 1988). VIP+ fibers occasionally leave the vascular plexus to extend among T-cells in the adjacent cortex (Bellinger et al., 1990). In the mucosal-associated lymphoid tissue, an extensive nerve plexus containing various neuropeptides such

cac tet gat gcc gtc ttc aca gat aac tac acc gcg ctc aga aag cee etg gct gtg aag aaa tac ctg aac tcc atc ctg aat gga VIP mRNA His Ser Asp AiA Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gin Met Aia Val Lys Lys TyrLeu Asn Ser Iie Leu Asn Gty VIP

FIGURE 1 VIP structure: protein and gene. (A) sequence of VIP family peptides. (B) VIP gene structure.

5. Vasoactive Intestinal Peptide: An Anti-inflammatory Neuropeptide

as VIP, SP, CGRP, cholecystokinin, and somatostatin is present in the intestinal lamina propria (Bellinger et al., 1996). In the Peyer’s patches, the VIP+ nerve fibers are predominantly along the lymphatics and high endothelial venules. The major VIP sources in the gut are the intrinsic enteric VIP-containing neurons found in the myenteric and submucosal ganglionic plexuses, and the extrinsic parasympathetic and sensory fibers (Furness and Costa, 1980). In the bronchus-associated lymphoid tissue, VIP+ fibers are present in the walls of bronchia and bronchioles (Dey et al., 1981). The VIPergic innervation in the lung arises presumably from the vagus and from microganglia present in the walls of the bronchia (Dey et al., 1981; Lundberg et al., 1979). The nature of the signals leading to neuronal VIP release during an inflammatory reaction is not known. However, a strong candidate is nitric oxide (NO), produced at high levels in inflammatory responses and shown to release VIP from enteric ganglia (Grider and Jin, 1993; Matsuyama et al., 2002). In addition, in a murine model of pulmonary delayed–type hypersensitivity, intratracheal antigenic challenge resulted in an increased secretion of VIP, recruitment of T-cells bearing VIP receptors, and upregulation of VIP receptors on the responding T-cells, suggesting that inflammatory signals result in VIP release (Kaltreider et al., 1997). 2. Immune VIP The first evidence for the production of VIP by immune cells was the identification of VIP-immunoreactivity (VIP-ir) in rat mast cells (Cutz et al., 1978). However, the VIP identified in mast cells corresponded mostly to the truncated VIP10–28 fragment, and to a mixture of amino-terminally extended VIPs (Goetzl et al., 1988; Wershil et al., 1993). Further studies showed VIP-ir in blood polymorphonuclear and mononuclear cells, basophils, and eosinophils (Aliakbari et al., 1987; Lygren et al., 1984; O’Dorisio et al., 1980; Weinstock and Blum, 1990). Using different experimental approaches, several studies demonstrated that lymphocytes synthesize and secrete functional VIP. Both thymocytes and peripheral T-cells express VIP mRNA as determined by RT-PCR (Gomariz et al., 1994; Leceta et al., 1994). Northern blot analysis indicated that the 1 kb predominant VIP transcript in lymphocytes resulting from the use of the proximal polyadenylation site in the VIP gene (Chew et al., 1994) differs from the preferentially expressed 1.7 kb transcript found in most tissues (Leceta et al., 1996). Use of HPLC followed by Elisa identified VIP1–28 and two higher molecular weight

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precursors in lysates obtained from lymphocyte cultures (Gomariz et al., 1992). In addition to VIP expression and accumulation of intracellular VIP1–28, T lymphocytes activated through the T-cell receptor were shown to secrete VIP (Martinez et al., 1999). We reported recently that Th2, but not Th1, effectors express and secrete functional VIP following antigenic stimulation (Delgado and Ganea, 2001a; Vassiliou et al., 2001). Since VIP acts as a Th2 differentiation and survival factor (see Section V.A.2 and 3), VIP synthesis and release by activated Th2 cells suggests a positive feedback mechanism leading to the alteration of the Th1/Th2 balance in favor of Th2 immune responses. The role of endogenous, T-cell–derived VIP in T-cell differentiation was confirmed recently through the elimination of VIP with VIPase IgG from cultured TCR-stimulated T-cells, leading to the restoration of the Th1/Th2 balance (Voice et al., 2003).

III. VIP RECEPTORS IN IMMUNE CELLS A. Classification of VIP/PACAP Receptors Following the discovery of PACAP, it became apparent that VIP and PACAP act as endogenous ligands for the same receptors. So far, three VIP/ PACAP receptors have been cloned and classified as VPAC1 and VPAC2 (with high affinity for both VIP and PACAP) and PAC1 (preferred PACAP receptor) (Harmar et al., 1998). The VIP/PACAP receptors are seven transmembrane domain G-protein linked receptors, with highly conserved amino acid residues in the amino-terminal extracellular region, the transmembrane domains, and the first intracellular loop. Recently, a natural VIP deletion variant of the VPAC2 receptor was identified in immune cells (Grinninger et al., 2004). This receptor binds VIP but does not signal. In contrast to VPAC1 and VPAC2, there are multiple variants of PAC1 resulting from alternative splicing (Pisegna et al., 1996; Spengler et al., 1993). Signaling through VIP/PACAP receptors results in the activation of adenylyl cyclase (AC) and of phospholipase C (PLC) and D (PLD) (Delporte et al., 1995; Harmar et al., 1998; McCulloch et al., 2000; McCulloch et al., 2001; McCulloch et al., 2002; Van Rampelbergh et al., 1997). VPAC1 and VPAC2 receptors stimulate PLD less potently than AC (McCulloch et al., 2001). The rat isoform PAC1-TM4, which differs from the canonical PAC1 receptor only by discrete sequences located in transmembrane domains II and IV and in the extracellular domain, is not coupled to AC, PLC, or PLD but mediates increases [Ca2+] via L-type Ca+2 channels (Chatterjee et al., 1996).

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B. VIP/PACAP Receptors in Immune Cells VIP binding sites have been described initially for human, rat, and mouse lymphocytes (Calvo et al., 1986; Guerrero et al., 1981; O’Dorisio et al., 1981; Ottaway et al., 1983; Ottaway and Greenberg, 1984). Following cloning of VPAC1, VPAC2, and PAC1, the expression of these receptors was investigated in various immune cell subpopulations. Due to lack of appropriate specific antibodies, the expression of VPAC1/2 and PAC1 receptors has been followed at the mRNA level. Discrepancies between the results reported by different laboratories can be attributed to the use of different primers and of different culture conditions. In addition, the expression of the three receptors appears to be modulated during activation and differentiation of immune cells, which introduces a new experimental variable. In most immune cells, VPAC1 appears to be constitutively expressed. For example, macrophages and macrophage cell lines such as THP-1 and RAW cells express PAC1 and VPAC1 mRNA constitutively, whereas VPAC2 is induced upon activation (Delgado et al., 1998). In bone marrow–derived dendritic cells, VPAC1 is expressed at higher levels than VPAC2, with no PAC1 expression (Delgado et al., 2004b). Functional VPAC1 and VPAC2 receptors coupled to adenylyl cyclase have been also reported in murine Langerhans cells (Asahina et al., 1995). Similar to macrophages, murine microglia express VPAC1 and PAC1 constitutively (Delgado et al., 2002b; Kim et al., 2000). The best studied immune cells in terms of VIP/ PACAP receptors are the T lymphocytes. Initial localization of VIP and PACAP binding sites in immune organs revealed a high density of receptors in the Tcell areas without identifying the receptor (Reubi, 2000). There is general agreement that VPAC1 is constitutively expressed in murine and human naïve Tcells, and that VPAC2 is expressed primarily in CD4+

TABLE 1

T lymphocytes following TCR-stimulation (Delgado et al., 1996; Lara-Marquez et al., 2001; Pankhaniya et al., 1998). In contrast to VPAC2, VPAC1 transcription after TCR cross-linking and CD28 co-stimulation was reported to be downregulated in human peripheral T-cells and thymocytes (Lara-Marquez et al., 2000; Lara-Marquez et al., 2001). This observation led to a model of opposite regulation for VPAC1 and VPAC2, with VPAC1 being downregulated and VPAC2 upregulated following CD4+ T-cell activation. However, recent studies from our laboratory suggest that this is a rather simplified model that does not take into account the differentiation of CD4+ T-cells into Th1 and Th2 effectors following activation and proliferation. We found that naïve CD4+ T-cells express VPAC1 and very little, if any, VPAC2; however, following several rounds of proliferation and differentiation into Th1 and Th2 effectors, Th2 cells express significantly higher levels of both VPAC1 and VPAC2 receptors, compared to Th1 effectors (Sharma et al., 2006). A final resolution for the regulation of VPAC1 and VPAC2 expression during Tcell activation, proliferation, and differentiation will require quantitative measurements at both message and protein level during the various phases of T-cell activation. The question of the biological role of the various VIP/PACAP receptors has been addressed in functional assays. With the exception of IL-6 inhibition, VPAC1 mediates the inhibition of pro-inflammatory cytokine and chemokine production in LPS-stimulated macrophages and microglia (Delgado et al., 1998; Delgado et al., 1999d; Delgado et al., 1999e; Delgado et al., 1999f; Delgado et al., 2002b; Delgado and Ganea, 1999; Delgado and Ganea, 2001d), the VIP-induced inhibition of hematopoiesis (Rameshwar et al., 2002), and the induction of tolerogenic dendritic cells (Delgado et al., 2005b) (Table 1). In contrast, VPAC2 acts as the major biological receptor in T-cells, promoting Th2-type responses in vivo and in vitro (Goetzl et al., 2001; Voice et al., 2001; Voice et al., 2003).

Receptors, Intracellular Pathways, and Transcription Factors Involved in the Anti-inflammatory Activity of VIP and PACAP

IL-6 IL-10 IL-12 TNF NO CK Hematopoiesis Tolerogenic DCs

Action

Receptor

Intracellular pathways

Transcription factors

Inhibition Stimulation Inhibition Inhibition Inhibition Inhibition Inhibition Induction

PAC1 VPAC1 VPAC1/VPAC2 VPAC1 VPAC1 VPAC1 VPAC1 VPAC1

PKC cAMP cAMP/cAMP indep cAMP/cAMP indep cAMP/cAMP indep cAMP ? cAMP/PKA

ND CREB IRF-1/NF-κB CREB/NF-κB IRF-1/NF-κB NF-κB ? NF-κB


IV. EFFECTS OF VIP ON INNATE IMMUNE RESPONSES

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A. VIP Inhibits the Expression and Release of Pro-inflammatory Mediators from Activated Macrophages and Microglia

To eliminate invading pathogens, the immune system mounts two different but interconnected responses: the innate and adaptive immunity. The innate immune response occurs upon the recognition through Toll-like receptors (TLRs) of foreign patterns characteristic for groups of pathogens. The innate immune response, characterized by phagocytosis, release of oxygen and nitrogen radicals, and production of pro-inflammatory cytokines and chemokines, involves cells such as macrophages, neutrophils, dendritic cells in the periphery, and microglia in the CNS. In addition to their immediate function in killing pathogens, macrophages, microglia, and particularly dendritic cells have the capacity to process antigen and initiate an adaptive immune response, through stimulatory and co-stimulatory contacts with naïve T-cells bearing the appropriate TCR. Although both innate and adaptive immune responses are required for the successful elimination of pathogens, an uncontrolled inflammatory response can lead to serious tissue damage, organ failure, and ultimately to death. Therefore, endogenous anti-inflammatory factors such as glucocorticoids, lipid mediators, anti-inflammatory cytokines, and neuropeptides such as VIP play an important role in the successful resolution of the inflammatory response. The various mechanisms that promote the anti-inflammatory activity of VIP are summarized in Figure 2.

Following signaling through TLRs, macrophages secrete several pro-inflammatory mediators, such as TNF-α, IL-12, IL-1β, IL-6, and NO, followed by secretion of the anti-inflammatory cytokines IL-10 and transforming growth factor β (TGFβ) (Laskin and Pendino, 1995). Despite the beneficial role of proinflammatory factors in host defense, their sustained production can lead to serious pathological conditions (Evans, 1995; Van Snick, 1990; Vassalli, 1992). As one of the endogenous anti-inflammatory factors, VIP inhibits the production of TNF-α, IL-6, IL-12, and the induction of iNOS, and stimulates the production of the anti-inflammatory cytokines IL-10 and IL-1Ra in LPS-stimulated macrophages and macrophage cell lines (Delgado et al., 1999c; Delgado et al., 1999d; Delgado et al., 1999e; Delgado et al., 1999f; Dewit et al., 1998; Martinez et al., 1998; Xin and Sriram, 1998) (Figure 3). The use of specific agonists and antagonists established that VPAC1 is the major receptor involved in the effect on macrophages with the exception of IL6, which is downregulated following signaling through PAC1 (Table 1). Indeed, in vivo experiments in an inflammation disease model using PAC1-deficient mice suggested that VPAC2 and PAC1 are also involved, although minimally, in the anti-inflammatory effect of VIP (Delgado et al., 2000; Martinez et al., 2005).

FIGURE 2 General immunoregulatory effects of VIP. VIP is released from the lymphoid innervation and from activated Th2 effectors and acts on bone marrow cells inhibiting hematopoiesis; deactivates stimulated macrophages, dendritic cells, and microglia; reduces antigen presentation; promotes Th2 responses at the expense of Th1 proinflammatory responses; and induces the generation and/or activation of regulatory T-cells.

FIGURE 3 Effects of VIP on LPS-stimulated macrophages. VIP inhibits the expression of the pro-inflammatory genes TNF-α, IL-6, iNOS, IL-12p40, and chemokines, and stimulates production of the anti-inflammatory factors IL-10 and IL1Ra. Through its effect on IL-12 production, VIP inhibits the release of IFNγ from activated CD4+ T-cells.

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Accumulation of immune cells at the site of pathogen invasion is mediated through inflammatory chemokines released primarily by cells involved in innate immunity, i.e., macrophages/monocytes, dendritic cells, and microglia. Although chemokines usually attract several types of immune cells, there is a certain degree of specificity. For example, CXCL1 (IL-8) and CXCL2 (MIP-2) activate and induce the directional migration of neutrophils, whereas CCL3 (MIP-1α), CCL4 (MIP-1β), CCL2 (MCP-1), and CCL5 (RANTES) are chemotactic for monocytes/macrophages and Tcells (Luster, 1998; Rollins, 1997; Rossi and Zlotnik, 2000). Although several studies have addressed the effects of VIP on lymphocyte adhesion and traffic, there is little agreement beyond the fact that VIP affects lymphocyte traffic. At the present time, there are few reports regarding the effects of VIP on inflammatory chemokine production and only one report showing that VIP prevents the migration of mononuclear cells by phosphorylating and deactivating the chemokine receptor CCR5 (Grimm et al., 2003). We reported that VIP inhibits the expression of two CXC chemokines (CXCL1/KC and CXCL2/MIP2) and four CC chemokines (CCL2/MCP1, CCL3/MIP1α, CCL4/MIP1β, and CCL5/RANTES) in LPS-stimulated mouse macrophages (Delgado and Ganea, 2001b). Similarly, VIP inhibits endotoxin-induced expression of IL-8 (CXCL1) in human peripheral blood monocytes (Delgado and Ganea, 2003b). The inhibitory effect is mediated by VPAC1 and correlates with a reduction in NFκB binding and transactivating activity (Delgado et al., 2003; Delgado and Ganea, 2001f). In agreement with the reported chemoattraction of neutrophils by CXCL1 and CXCL2, and of monocytes/macrophages and Tcells by CCL2, CCL3, CCL4, and CCL5, the VIP treatment of human monocytes resulted in a reduced in vitro chemotactic activity for peripheral blood neutrophils, and the in vivo administration of VIP in a murine model of acute peritonitis led to a significant reduction in the recruitment of neutrophils, macrophages, and lymphocytes to the peritoneal cavity (Delgado and Ganea, 2001c). Microglia, the CNS functional equivalent of mononuclear phagocytes in the periphery, are considered to play a central role in the regulation of immune and inflammatory activities and tissue remodeling in the CNS (Gonzalez-Scarano and Baltuch, 1999; Streit et al., 1988). In response to brain injury, infection or inflammation, microglia are activated similar to peripheral tissue macrophages. Activation of microglia is a histopathological hallmark of several neurodegenerative diseases where inflammation plays a major role. It has been proposed that in response to pro-inflammatory cytokines or antigens, activated microglia secrete

inflammatory mediators such as NO and pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, which contribute to neuronal damage and cell death (Bartholdi and Schwab, 1997; Chao et al., 1995; Ding et al., 1997; Lee et al., 1993; Lipton et al., 1994; McManus et al., 1998; Peterson et al., 1997; Streit et al., 1998; Taupin et al., 1993). Since the intensity and duration of inflammatory processes in the CNS depend on the local balance between pro- and anti-inflammatory factors, a number of regulatory molecules termed microglia-deactivating factors have been the focus of considerable research. Recently, VIP was identified as a microgliadeactivating factor. VIP inhibits in a dose-dependent manner the production of TNF-α, IL-1β, IL-6, NO, and of the pro-inflammatory chemokines RANTES, MCP1, MIP-1α, MIP-1β, MIP-2, and KC by LPS-stimulated microglia (Delgado et al., 2002b; Delgado et al., 2003; Kim et al., 2000). At the same time, VIP stimulates the expression of the anti-inflammatory factors IL-10 and IL-1Ra (Delgado et al., 2003). The VIP-mediated inhibition of chemokine production correlates with a decreased microglia-induced chemotaxis for macrophages, lymphocytes, and neutrophils (Delgado et al., 2002b). Similar to macrophages/monocytes, the effects of VIP on activated microglia are mediated primarily through VPAC1 (Delgado et al., 2002b; Delgado et al., 2003; Kim et al., 2000). In addition, VIP also inhibits the expression of several genes involved in the IFNγinduced inflammatory response in microglia, including CD40, CXCL10 (IP-10), and iNOS (Delgado et al., 2003; Kim et al., 2002). The anti-inflammatory effect of VIP has also been described in vivo. I.c.v. administration of LPS caused neurodegeneration accompanied by massive microglia activation and by increases in the microglial expression of TNF-α, IL-1β, and iNOS. Local i.c.v. VIP administration prevented LPS-induced neurodegeneration, microglia activation, and TNF-α, IL-1β, and iNOS expression (Delgado and Ganea, 2003a; Delgado and Ganea, 2003c; Kim et al., 2002).

B. Signaling Pathways Involved in the Inhibitory Effect of VIP VIP affects the expression of pro- and antiinflammatory factors in LPS- and LPS+IFNγstimulated macrophages and microglia by regulating the expression and/or transactivating activity of transcription factors such as IRF-1, NFκB, CREB, and AP-1. LPS binding to TRL4 results in the activation of NFκB and of the three MAPK pathways, i.e., the p38 MAPK, the extracellular signal-regulated kinases, and the cJun N-terminal kinase (JNK) (Pyo et al., 1998; Zhang


et al., 1998). JNK phosphorylation and activation are mediated through the MEKK1/MKK4/JNK pathway, and phosphorylation of c-Jun by JNK increases its transactivating activity. Activation of the MEKK1/ MKK4/JNK pathway by LPS is involved in the upregulation of several pro-inflammatory factors (Cobb, 1999).We showed that VIP treatment of LPS-stimulated macrophages and microglia results in the inhibition of MEKK1/MKK4/JNK phosphorylation and the upregulation and nuclear translocation of JunB (Figure 4A) (Delgado, 2002; Delgado and Ganea, 2000b). Subsequently there is a change in the composition of AP-1 with c-Jun being replaced by JunB and loss of transactivating activity for TNF-α expression (Delgado et al., 1998). The pleiotropic transcription factor NFκB plays an important role in the transcriptional regulation of all pro-inflammatory factors. The most common transcriptionally active NFκB is a p50/p65 heterodimer. In unstimulated cells, NFκB is localized in the cytosol bound to the inhibitor IκB. LPS treatment results in IκB phosphorylation by specific kinases (IKK), ubiquitination, and proteosomal degradation. This is followed by the rapid nuclear translocation of NFκB and binding to specific κB elements within cytokine and chemokine promoters. The transactivating activity of NFκB requires interaction with co-activators that bridge transcriptional activators and components of the basal transcriptional machinery. The CREB-binding protein (CBP) is an ubiquitously expressed nuclear coactivator present in limiting amounts (Goldman et al., 1997). CBP serves to interconnect various transcription factors, including NFκB with TFIIB, a member of the basal transcriptional machinery, which in turn makes contact with the TATA box-binding protein (TBP) (Janknecht and Nordheim, 1996; Kamei et al., 1996). The interaction of p65 with CBP is essential for NFκB transcriptional activity (Gerritsen et al., 1997; Zhong et al., 1998), and this interaction is prevented by competition from other CBP-binding factors such as CREB (Chrivia et al., 1993; Kwok et al., 1994). Our studies indicate that VIP affects NFκB-transactivating activity through a cAMP-independent pathway, which results in the inhibition of IκB phosphorylation by IKK, a cAMP-dependent pathway which prevents MEKK1/ MEK3-6/p38 phosphorylation and subsequent TBP phosphorylation and nuclear translocation, and the cAMP-dependent phosphorylation of CREB, which becomes a high-affinity competitor for CBP (Figure 4B) (Delgado and Ganea, 2001f). Since NFκB is involved in the expression of TNF-α, IL-12, and iNOS, and of the pro-inflammatory chemokines, the VIP-induced reduction in NFκB-transactivating activity affects multiple target genes.

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In addition to LPS, optimal expression of IL-12 and iNOS in macrophages and microglia requires IFNγ signaling. Binding of IFNγ to its receptor induces the assembly of an active receptor complex with subsequent transphosphorylation of Jak1 and Jak2, followed by recruitment and phosphorylation of STAT1 (Darnell, Jr. et al., 1994). Upon phosphorylation, STAT1 forms homodimers which translocate to the nucleus, where they bind to the IFNγ-activated site (GAS) found in the promoter of many IFNγ-induced genes including IRF1 (Sims et al., 1993). Many of the regulatory effects of IFNγ in macrophages and microglia appear to be mediated by IRF-1, which transactivates multiple effector genes including CXCL10 (IP-10), CD40, IL-12, and iNOS (Ma et al., 1997; Martin et al., 1994; Nguyen and Benveniste, 2002; Ohmori and Hamilton, 1993). Our studies indicate that VIP inhibits Jak1/STAT1 phosphorylation in a cAMP-dependent manner, and therefore prevents IRF-1 expression (Figure 4C) (Delgado and Ganea, 2000a). Although the link between cAMP induction and inhibition of Jak1/STAT1 phosphorylation has not been elucidated, preliminary studies in T-cells suggest that VIP inhibits IL-12 signaling through the phosphorylation and activation of the phosphatase SHIP-1, which in turn dephosphorylates some of the Jaks (unpublished data). Therefore, the family of SHIP phosphatases represents an attractive target for the VIP effect on the Jak/STAT signaling pathways.

C. VIP Inhibits the Capacity of Antigen-presenting Cells to Initiate Adaptive Immunity In vertebrates, innate immunity is followed by an antigen-specific immune response which involves B and T lymphocytes. The link between innate and adaptive immunity is built on the capacity of antigenpresenting cells (APCs), such as dendritic cells, macrophages, and microglia, to process antigen, present it to naïve CD4+ T-cells, and induce T-cell proliferation and differentiation. Optimal T-cell stimulation requires co-stimulatory signals aside from signaling through the TCR. In addition, the APCs provide the cytokine microenvironment necessary for the initial stimulation of CD4+ T-cells. Among the co-stimulatory signals involved in the APC/T-cell interactions, the B7 family, primarily CD80 and CD86, as well as CD40, play major roles. Dendritic cells (DCs) express CD80, CD86, and CD40 constitutively, and upregulate their expression following TLR signaling. In macrophages, the co-stimulatory molecules are expressed only following activation. We reported previously that VIP is among the endogenous factors regulating CD80 and CD86 expres-

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FIGURE 4 Signaling pathways involved in the VIP effect on activated macrophages. (A) LPS induces c-Jun phosphorylation through the MEKK1/MEK4/JNK pathway. VIP acts through the VPAC1 receptor, activates PKA, and phosphorylates JunB. VIP also inhibits the MEKK1/MEK4/JNK pathway and subsequently reduces c-Jun phosphorylation. This results in the replacement of c-Jun with JunB in the AP-1 transcription complex and inhibition of TNF-α transcription. (B) LPS induces TATA-box binding protein (TBP) phosphorylation through the MEKK1/MEK3-6/p38 pathway and induces NFkB (p65/p50) nuclear translocation by activating IKK and inducing IkB phosphorylation and degradation. VIP inhibits the MEKK1/ MEK3-6/p38 pathway in a cAMP-dependent manner, resulting in a reduction in TBP phosphorylation. VIP also inhibits IKK activation in a cAMP-independent manner, preventing IkB degradation and NFkB nuclear translocation. In addition, VIP induces CREB phosphorylation in a cAMP-dependent manner, and phosphorylated CREB competes with p65 for the coactivator CBP. All these effects contribute to reduce transcription of NFkB-dependent genes, such as TNF-α, iNOS, IL-12p40, and chemokines. (C) IFNγ signals through the Jak12/STAT1 pathway to induce transcription of IFNγ-activated genes (GAS), including IRF-1. IRF-1 is an essential transcription factor for IL-12p40 and iNOS. VIP inhibits in a cAMPdependent manner Jak1/STAT1 phosphorylation and subsequently inhibits IRF-1 transcription. The effect on Jak/STAT phosphorylation might be mediated through the activation of the phosphatase SHIP1 (see text).


sion in macrophages (Delgado et al., 1999g). Interestingly, VIP affects the expression of co-stimulatory molecules in an opposite manner in resting and activated macrophages. In resting macrophages, VIP upregulates CD86, but not CD80 expression both in vivo and in vitro. In contrast, in LPS/IFNγ-activated macrophages, VIP downregulates both CD80 and CD86 expression. Similar results were obtained recently with bone marrow–derived DCs (Delgado et al., 2004b). The effect of VIP on the expression of co-stimulatory molecules correlates with the effects on the macrophage/DC stimulatory activity for T-cells. The inhibition of CD80/CD86 expression in activated macrophages and DCs results in a significantly reduced capacity to stimulate allogeneic or antigen-specific syngeneic CD4+ T-cells, in vivo and in vitro (Delgado et al., 1999g; Delgado et al., 2004b). This is in perfect agreement with the accepted role of VIP as an endogenous anti-inflammatory agent. In contrast, the upregulation of CD86 in immature DCs and resting macrophages correlates with an increase in the stimulatory activity for T-cells, and with a pronounced bias in the generation of Th2 effectors (see Section V.A.2).

D. Effects of VIP on Hematopoiesis In mammals the immune cells are generated by hematopoiesis in the bone marrow. During an active immune response, hematopoiesis is enhanced mainly through the effect of cytokines. Once the infectious agent is eliminated, immune cell homeostasis is reestablished, in part through a reduction in hematopoiesis. Although peptidergic nerve fibers were identified in the bone marrow (Felten, 1993; Tabarowski et al., 1996), there are few reports regarding the effects of neuropeptides on hematopoiesis. A recent study demonstrated that VIP reduces the number of granulocytic and erythroid colonies, mostly through direct effects on the CD34+ bone marrow progenitors (Rameshwar et al., 2002). The inhibitory effect of VIP on hematopoiesis could contribute to the re-establishment of homeostasis in vivo following an immune response, supporting the general anti-inflammatory role of VIP.

V. EFFECTS OF VIP ON T-CELL ACTIVATION, DIFFERENTIATION, AND FUNCTION Following recognition of antigenic peptides complexed to MHC class I or II, T lymphocytes proliferate and differentiate into effector cells. CD4+ T-cells recognize antigen complexed to MHC class II presented by APCs and differentiate into Th1 and Th2 effectors.

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CD8+ T-cells recognize antigen complexed to MHC class I and become fully functional cytotoxic T-cells. T lymphocytes, particularly CD4+ T-cells, represent major targets for VIP regulation. Both the activation of naïve T-cells and their differentiation into effector and memory cells are affected by VIP.

A. VIP Effects on CD4+ T Helper Cells 1. Effect on CD4+ T-Cell Activation Proliferation of naïve CD4+ T-cells is initiated following contact with APCs, with both stimulatory and co-stimulatory signals required for T-cell activation. Initial reports indicated that VIP reduces T-cell proliferation (Ganea, 1996). The inhibitory effect of VIP can be attributed to the reduction in the expression of costimulatory molecules on APCs and to direct effects on CD4+ T-cells. Indeed, we reported that VIP downregulates CD80/CD86 in LPS/IFNγ-activated macrophages and DCs, and the reduction in B7 expression correlates with a reduction in the proliferation of antigen-specific T-cells (Delgado et al., 1999g; Delgado et al., 2004b; Ganea et al., 2003). VIP was also reported to directly affect IL-2 gene expression in CD4+ T-cells and to inhibit T-cell proliferation (Ganea, 1996; Gomariz et al., 2001). Both VPAC1 and VPAC2 mediate the inhibitory effect of VIP/ PACAP on IL-2 expression, primarily through the induction of intracellular cAMP and subsequent effects on the transcription factors NFAT and AP-1 (Wang et al., 1999; Wang et al., 2000). 2. VIP Promotes Differentiation into Th2 Effectors Following antigenic stimulation and several rounds of proliferation, CD4+ T-cells differentiate into Th1 and Th2 effectors, characterized by different cytokine profiles. The differentiation into Th1 or Th2 effectors is controlled by the cytokine microenvironment, the nature of the APCs, the nature and amount of antigen, and the genetic background of the host (O’Garra, 1998). In addition, endogenous factors such as progesterone and glucocorticoids have been reported to favor Th2 differentiation (Miyaura and Iwata, 2002). Recent studies indicate that neuropeptides, particularly VIP, also favor Th2 differentiation. Macrophages treated in vitro with VIP gain the ability to induce Th2-type cytokine (IL-4 and IL-5) and to inhibit Th1-type cytokine (IFNγ and IL-2) production in primed CD4+ T-cells (Delgado et al., 1999a). Similar results were obtained recently with bone marrow–derived dendritic cells (Delgado et al., 2004b; Ganea et al., 2003). In vivo, administration of VIP to immunized mice results in a reduction in the number of IFNγ-secreting cells and an

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increase in the number of IL-4 secreting cells (Delgado et al., 1999a). Two recent studies confirmed the prevalence of Th2-type responses in transgenic mice overexpressing the human VPAC2 receptor in CD4+ T-cells (Voice et al., 2001) and of Th1-type responses in VPAC2 knockout mice (Goetzl et al., 2001). The studies in mice genetically altered for VPAC2 expression indicate the importance of endogenous VIP for the regulation of Th1/Th2 responses in vivo and the prominent role of the VPAC2 receptor in CD4+ T-cell differentiation and/ or responses. The role of endogenous, Th2-derived VIP in maintaining the Th2 bias was confirmed recently in studies in which elimination of VIP from TCR-stimulated T-cells with VIPase antibodies resulted in the readjustment of the Th1/Th2 balance (Voice et al., 2003). These studies confirm the concept that VIP affects the Th1/Th2 balance in vivo and indicate the prevalent role of VPAC2 in this process. A number of different, non-excluding mechanisms contribute to the VIP-induced Th2 bias (Figure 5). The

neuropeptide may affect Th1/Th2 generation directly or through effects on APCs, or could act on the alreadygenerated effectors by preferentially promoting Th2 proliferation and/or survival. VIP affects APCs in ways relevant to Th1/Th2 differentiation. First, VIP inhibits the production of IL-12, the major Th1inducing cytokine (Delgado et al., 1999e; Xin and Sriram, 1998). This results in the preferential generation of Th2 effectors. Second, the level of co-stimulatory molecule expression on APCs is more important for Th2, compared with Th1 development (Ranger et al., 1996). VIP has been shown to induce CD86 expression in resting macrophages and immature DCs, and the capacity of the VIP-treated macrophages/DCs to induce Th2 cytokines in antigen-primed T-cells was abolished by treatment with neutralizing anti-CD86 Abs (Delgado et al., 1999a; Delgado et al., 2004b; Ganea et al., 2003). VIP also affects differentiating T-cells directly. Indeed, addition of VIP to TCR-transgenic T-cells cul-

FIGURE 5 Effects of VIP on CD4+ T-cell differentiation and survival. VIP promotes Th2 differentiation through effects on APCs (inhibition of IL-12 release and of CD80/CD86 expression by activated macrophages, induction of CD86 in resting macrophages and DCs), and through direct effects on the differentiating CD4+ T-cells such as induction of c-Maf and JunB expression. VIP promotes the survival of Th2 effectors through the reduction in both FasL and granzyme B expression (see text), and their differentiation into memory Th2 cells.


tured with irradiated APCs and antigenic peptide or stimulated with anti-CD3 and anti-CD28 in the absence of APCs, with or without polarizing cytokines (IL-12 for Th1 and IL-4 for Th2 differentiation), leads to increased levels of IL-4 and decreased levels of IFNγ. This suggests that VIP promotes the development of Th2 cells directly by acting on the differentiating CD4+ T-cells (Ganea et al., 2003). Recent experiments indicate that VIP blocks IL-12 signaling in differentiating CD4+ T-cells, preventing Jak2/STAT4 phosphorylation, presumably through the activation of the SHIP-1 phosphatase (unpublished data). A recent report also indicated that VIP induces the expression of the Th2 master transcription factors c-Maf and JunB in differentiating CD4+ T-cells (Voice et al., 2004). Finally, VIP could act on the already-generated Th1/Th2 effectors. Both in vitro and in vivo experiments indicate that VIP supports the survival and possibly the proliferation of Th2, but not Th1, effectors (Delgado et al., 2002c). The in vivo surviving Th2 cells following inoculation with antigen and VIP exhibit memory cell markers (Delgado et al., 2002c) (see Section V.A.3). In addition to promoting Th2 survival, VIP also affects Th1/Th2 migration in a differential manner. Recent studies indicate that VIP promotes Th2 and inhibits Th1 migration in vitro and in vivo, through effects on the production of chemokines. VIP promotes the expression and release of CCL22 (MDC), a Th2-attracting chemokine and inhibits the release of the Th1-attracting chemokine CXCL10 (IP-10) (Delgado et al., 2004a) (see Section V.A.4). The preferential recruitment of Th2 effectors could further contribute to the VIP induction of Th2, as opposed to Th1-type immunity by VIP.

3. VIP Promotes the Survival of Th2 Effectors The majority of Th1/Th2 effectors become apoptotic following a relatively short period of intense activity. The elimination of effector T-cells occurs through either active or passive apoptosis, depending on whether the antigen persists or is eliminated. The few surviving effector T-cells become antigen-specific memory cells and differ from naïve T-cells in terms of homing and activation requirements. Although VIP acts as a survival factor in the CNS, we expected VIP to promote T lymphocyte apoptosis in the peripheral immune system, based on its antiinflammatory function. Surprisingly, VIP was shown to protect activated CD4+ T-cells against active (antigeninduced) cell death (AICD) both in vitro and in vivo. This protection is associated with an inhibitory effect on FasL ligand (FasL) expression (Delgado and Ganea,

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2000c). FasL expression requires a number of transcription factors, including NFκB and Egr2 and 3. VIP inhibits NFκB binding through the stabilization of the IκB inhibitor and retention of p65/p50 in the cytoplasm. In addition, VIP prevents NF-ATp nuclear translocation in a calcineurin-independent manner, inhibiting the NF-ATp dependent expression of Egr2 and 3 (Delgado and Ganea, 2001e). At first glance, the protective effect against apoptosis, resulting in a higher number of surviving effector CD4+ T-cells, could lead to a more intense inflammatory response, in apparent contradiction to the general anti-inflammatory effect of VIP. However, VIP might specifically protect Th2 effectors, to the detriment of Th1 cells, which are the major players in acute inflammation through the mobilization and activation of neutrophils and macrophages. Therefore, the VIPinduced development of Th2 immunity, with its subsequent reduction in Th1 effectors, would have an anti-inflammatory effect. The function of VIP as a specific Th2 survival factor is in agreement with the Th2-type immunity observed in transgenic mice overexpressing the VPAC2 receptor or following VIP administration in wild-type mice (Delgado et al., 1999a; Voice et al., 2001). We confirmed the preferential effect of VIP on the survival of Th2 effectors in a recent report (Delgado et al., 2002c). When thymectomized hosts were reconstituted with Th1 and Th2 cell lines derived from TCR transgenic mice, followed by immunization with the specific antigen and VIP, transgenic CD4+ T-cells could be recovered 62 days later only from hosts that received Th2 cells. When the hosts received transgenic CD4+ Tcells instead of Th1 or Th2 cell lines, the recovered transgenic T-cells exhibited a phenotype typical of memory Th2 cells (CD44hi, L-selectinlo, CD45RBlo, and IL-4 and IL-5, but not IFNγ or IL-2 producers). The preferential survival of Th2 cells in the presence of VIP was also confirmed in vitro, with VIP/PACAP supporting the survival of Th2, but not Th1, cell lines (Delgado et al., 2002c). Mechanistic studies established that AICD in Th1 and Th2 effectors occurs through two apparently independent pathways, initiated by FasL/Fas interactions and through the induction of granzyme B (GrB), respectively (Sharma et al., 2006). Both Th1 and Th2 effectors upregulate FasL and GrB expression following TCR restimulation. There is a significant difference between Th1 and Th2 cells in the expression of VPAC1/2 receptors, with Th2 cells expressing higher levels of VIP receptors. VIP prevents FasL expression in both Th1 and Th2 cells, but inhibits GrB expression selectively in Th2 effectors (Sharma et al., 2006).

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4. VIP Favors the Directional Migration of Th2 Cells The selective recruitment of effector T-cells to an inflammatory site is mediated through chemokines and chemokine receptors. Although chemokines are largely redundant, the homing of Th1 and Th2 cells depends on the expression of different chemokine receptors (Bonecchi et al., 1998; Sallusto et al., 1998). Th1 cells express CXCR3 and CCR5, whereas Th2 cells preferentially express CCR3, CCR4, and CCR8 (Bonecchi et al., 1998; Loetscher et al., 1998; Qin et al., 1998). Among chemokines, IP-10 (CXCL10), MIG (CXCL9), and I-TAC (CXCL11) bind only to CXCR3, whereas MDC (CCL22), TARC (CCL17), and TCA-3 (CCL1) bind to CCR4 and/or CCR8 (Zlotnik and Yoshie, 2000). Activated Th1 and Th2 cells were reported to migrate in response to IP-10 (CXCL10) and MDC (CCL22), respectively (Andrew et al., 1998; Bonecchi et al., 1998; Imai et al., 1999). In a recent study we showed that VIP downregulates CXCL10 and upregulates CCL22 release in spleen cell cultures (Jiang et al., 2002). Similar results were obtained with bone marrow–derived DCs (Delgado et al., 2004a; Ganea et al., 2003). VIP induces CCL22 release in immature and mature DCs and inhibits CXCL10 release from mature DCs. There is a good correlation between the VIP effects on CCL22 and CXCL10 production and chemotaxis for Th2 and Th1 cells, both in vitro and in vivo, where VIP-treated DCs loaded with Ag and inoculated i.p. lead to the preferential accumulation of Th2 effectors in the peritoneal cavity (Delgado et al., 2004a). The selective effect of VIP on Th1/Th2-attracting chemokines represents a new additional mechanism for its Th2-promoting activity.

B. VIP Effects on CD8+ and CD4+ Cytotoxic T-Cells Cytotoxic CD8+ T-cells kill targets through a major, calcium-dependent, perforin/granzyme-mediated mechanism, with a minor contribution from the calcium-independent, FasL-mediated pathway. VIP does not affect the perforin/granzyme-mediated cytotoxicity but inhibits drastically the FasL-mediated lysis of both allogeneic and syngeneic Fas-bearing targets. This results from the VIP-induced inhibition of FasL expression in CD8+ T-cells (Delgado and Ganea, 2000d). Since VIP affects only the minor FasL/Fas-mediated lysis in CD8+ cytotoxic T-cells, their overall effect on CD8+ T-cell cytotoxicity does not have major consequences. In contrast, CD4+ cytotoxic T-cells kill targets solely through the FasL/Fas-initiated pathway (Stalder et al.,

1994), and VIP has a major impact on their cytotoxic function. Cytotoxic CD4+ T-cells are activated by APCs in an MHC II–restricted manner, leading to FasL expression. Subsequently, the activated CD4+ T-cells lyse both cognate APCs (direct targets) and neighboring Fas-bearing cells (bystander targets) (Smyth, 1997; Wang et al., 1996). VIP inhibits FasL expression in CD4+ effectors generated in vivo and reduces the cytotoxicity for both direct and bystander targets (Delgado and Ganea, 2001g). Killing of bystander targets results in collateral damage, which is detrimental particularly in tissues with limited MHC II expression such as the brain. In this context, the fact that endogenous agents such as VIP, a neuropeptide highly expressed in the brain, controls Fas-mediated lysis of innocent bystanders might be highly significant.

C. VIP Contributes to the Generation of Antigen-specific Regulatory T-Cells Dendritic cells are a heterogeneous population of antigen-presenting cells that besides, contributing to innate immunity and initiating adaptive immunity, also play an important role in immune homeostasis, by inducing and maintaining tolerance (Banchereau et al., 2000; Steinman, 2003). Regulatory T-cells (Treg) generated in the thymus and in the periphery are major tolerance enforcers. Although questions regarding Treg generation remain to be answered, recent reports indicate that immature or semi-mature DCs contribute to the induction of Treg. Treg-inducing DCs, called tolerogenic DCs, appear to be generated in a variety of ways. For example, human monocytederived immature DCs secreting IL-10 act as tolerogenic DCs (Levings et al., 2005). Tolerogenic DCs have been generated from human monocyte and murine bone marrow in the presence of 1,25(OH)2D3, the active form of vitamin D3 and of vitamin D3 analogs (Gregori et al., 2001; Piemonti et al., 2000). Also, DCs grown in the presence of TNF and IL-10 function as tolerogenic, and are characterized by a specific phenotype, i.e., CD11clowCD45RBhigh (Menges et al., 2002; Wakkach et al., 2003). We evaluated the capacity of VIP to induce tolerogenic DCs in vitro and in vivo. Bone marrow– derived DCs generated in the presence of VIP are CD11clowCD45RBhigh and do not upregulate the costimulatory molecules CD40, CD80, and CD86 following LPS stimulation. In contrast to control DCs (generated in the absence of VIP), the LPS-stimulated VIP-DCs secrete low levels of pro-inflammatory cytokines and high levels of IL-10. Antigen-pulsed VIPDCs induce IL-10/TGFβ secreting T-cells that suppress


the response of freshly activated CD4+ T-cells in an antigen-specific manner (Delgado et al., 2005b). The antigen-loaded VIP-DCs induce antigen-specific tolerance in vivo, and generate antigen-specific Treg capable of transferring tolerance to naïve hosts (Delgado et al., 2005b). The in vivo administration of VIP together with low doses of antigen results in a significant increase in the number of CD4+CD25+ Foxp3-expressing Treg. The in vivo VIP-induced Treg transfer antigen-specific suppression, inhibit delayed-type hypersensitivity, and prevent graft-versus-host disease in irradiated hosts reconstituted with allogeneic bone marrow (Delgado et al., 2005a; see Section VI.D). The capacity of VIP to generate tolerogenic DCs and antigen-specific Treg suggests new and exciting therapeutic avenues for the treatment of autoimmune diseases and the potential use of VIP in bone marrow transplantation.

VI. VIP IN MODELS OF AUTOIMMUNITY, INFLAMMATION, AND BONE MARROW TRANSPLANTATION Due to its pleiotropic functions, VIP and VIP analogs are considered for the development of multiple therapies. Here, we review the potential VIP therapeutic applications in immunological disorders, such as autoimmunity, inflammatory conditions, and bone marrow transplantation. Due to the inhibition of macrophage/ microglia activation, and to the ability to promote Th2 immunity and to generate and/or activate antigenspecific regulatory T-cells, VIP is as an attractive therapeutic factor for inflammatory disorders and/or Th1-type autoimmune diseases.

A. Autoimmune Disorders 1. Rheumatoid Arthritis Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive joint inflammation and subsequent cartilage and bone destruction. RA treatments are focused on the decrease of joint inflammation, primarily by inhibiting the secretion or activity of pro-inflammatory cytokines and chemokines. Agents that inhibit TNF-α secretion or block receptor binding are increasingly viewed as therapeutic agents with increased specificity in comparison to traditional drugs (Feldmann, 1996). Since animal models indicated a predominant Th1 role in RA (Joosten et al., 1997; Mauri et al., 1996; Walmsley et al., 1996), alternative therapeutic strategies focus on the alteration of the Th1/Th2 balance.

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VIP is a potent inhibitor of macrophage activation (including secretion of TNF-α, IL-6, and NO; discussed in Section IV.A), and a Th2-promoting factor (discussed in Section V.A), and therefore, an excellent prospect for RA therapy. In fact, several recent reports using the animal model of collagen-induced arthritis (CIA), which shares a number of clinical, histological, and immunological features with human RA, demonstrated that VIP administration to arthritic mice or rats decreases the frequency, delays the onset, and reduces the severity of the disease (Figure 6A) (Delgado et al., 2001; Williams, 2002; Yin et al., 2005). VIP reduces the proliferative response of collagen-specific T-cells, induces a shift towards Th2 cytokines, and reduces the titer of autoreactive anti-collagen antibodies (particularly IgG2a) (Delgado et al., 2001). VIP reduces the inflammatory response by downregulating the production of several pro-inflammatory agents in the inflamed joints and synovial cells, including TNF-α, IL-6, IL-1β, iNOS, IL-12, and IL-18, and of various chemokines (RANTES, MCP-1, MIP-1α, MIP-1β, and MIP2) (Delgado et al., 2001). In addition, VIP increases the production of the anti-inflammatory cytokines IL-10 and IL-1Ra, which have been reported to ameliorate arthritis symptoms (Feldmann, 1996). A similar VIP effect on cytokine/chemokine release by human synovial cells from RA patients was reported (Juarranz et al., 2004; Takeba et al., 1999). A recent study from our laboratory indicates that VIP inoculation in CIA mice after disease onset results in the generation of CD4+CD25+ Treg, which significantly ameliorate the clinical score upon transfer to arthritic hosts. This is associated with a decrease in the expression of IL-12, IFNγ, and TNF-α in the affected joints, and with a decrease in the proliferation and IFNγ and IL-2 production in spleen cells of the arthritic hosts (Gonzalez-Rey et al., 2006a). The capacity of VIP to regulate a wide spectrum of macrophage-derived inflammatory mediators, to switch the Th1/Th2 balance in favor of Th2 immunity, and to contribute to the generation and/or activation of Treg offers an advantage over treatments with neutralizing antibodies and receptor antagonists directed against a single cytokine. Of obvious biological significance is the fact that VIP levels, similar to those of other described antiarthritic neuropeptides and hormones such as CGRP and αMSH (Lipton and Catania, 1997; Takeba et al., 1999), are increased in serum and joints of arthritic mice (Delgado et al., 2001; Delgado et al., 2002a). This suggests that endogenous neuroimmune factors act as natural anti-arthritic agents. Nevertheless, arthritis-induced endogenous VIP levels are two to three orders of magnitude lower

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than the required protective concentrations for exogenous VIP. 2. Crohn’s Disease Crohn’s disease (CD) is an autoimmune inflammatory bowel disease characterized by chronic inflammation in the gastrointestinal tract leading to abdominal pain, rectal bleeding, diarrhea, weight loss, skin and eye disorders, and delayed growth and sexual maturation in children (Hanauer and Present, 2003). The abnormal inflammatory process results in a prolonged and severe inflammation of the intestinal mucosa, uncontrolled production of pro-inflammatory cytoA

kines and oligoclonal expansion, and activation of CD4+ Th1 cells (Blumberg and Strober, 2001). Classical therapeutic agents used in Crohn’s disease are mostly non-specific and exhibit adverse side effects (Hanauer and Present, 2003). Novel, more specific therapeutic strategies include the blockage of some of the pro-inflammatory cytokines (such as TNF-α, IL-6, and IL-12) (Atreya et al., 2000; Fuss et al., 1999; Neurath et al., 2002; Nikolaus et al., 2000; Yamamoto et al., 2000). Since both macrophage-derived inflammatory agents and Th1-derived cytokines are involved in CD, the ideal treatment should address both. In a recent report, Abad et al. (2003) used the murine IBD model induced by intrarectal administration of trinitrobenB

C

FIGURE 6 Effects of VIP in disease models. (A) Collagen-induced arthritis. DBA/J mice were immunized with bovine collagen and treated with VIP (1 and 5 nmol) at different times after immunization (day 28, or at the time of the secondary antigen administration—day 34). Open circles—VIP treatment; closed circles—control (no VIP). (B) Inflammatory bowel disease. IBD was induced in Balb/c mice with TNBS in 50% ethanol. Mice were treated with VIP (5 nmol) 12 hours after TNBS administration, and body weight was measured daily. Open circles—VIP treatment; closed circles—control, no VIP. Numbers in parentheses indicate mortality. (C) Septic shock. Balb/c mice were injected i.p. with a high dose of LPS and VIP or PACAP at the time of LPS administration. Viability was determined at 12-hour intervals. Positive control (D) represents mice injected with LPS and anti-TNF-α Abs.


D

E

FIGURE 6 cont’d. (D) EAE. SJL/J mice were immunized with PLP and injected with VIP either at the time of immunization (left panel) or at different times after immunization (right panel). The mean clinical score was determined at different times after immunization. (E) Graft-versusleukemia. Irradiated Balc/c mice were injected with syngeneic leukemic cells and reconstituted with allogeneic T-cell–depleted bone marrow cells (BM)—closed circles. Open circles—in addition to leukemic and BM cells, the mice received CD4+ T-cells from allogeneic TCR-transgenic mice inoculated with the specific Ag. Closed triangles—in addition to leukemic and BM cells, the mice received CD4+ T-cells from allogeneic TCR-transgenic mice inoculated with specific Ag and VIP. Survival was monitored daily after bone marrow transfer (BMT) (left panel). Leukemic cells (A20) H-2Kd+B220+ cells were monitored in blood as (right panels).

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zene sulfonic acid (TNBS) and demonstrated that treatment with VIP reduced the clinical severity (Figure 6B) and the disease-related histopathology, preventing or reversing body weight loss, diarrhea, and intestinal inflammation. VIP was effective both when administered together with TNBS and when given several days after colitis onset. VIP significantly reduced the inflammatory response by downregulating the expression of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-12, and IL-18 and chemokines MIP-1α, MCP-1, MIP-2, KC, CXCL12, CXCL13, and CXCL14 (Abad et al., 2003; Abad et al., 2005). As expected from the effect on chemokine expression, the VIP treatment reduced the number of infiltrating immune cells, including neutrophils, macrophages, and CD4+ T-cells in the lamina propria. Spleen and lamina propria CD4+ Tcells from mice treated with VIP showed a preferential Th2 response, with increased production of IL-4 and IL-10 (Abad et al., 2003; Abad et al., 2005). In addition to the described mechanisms for VIP-induced deactivation of macrophages and preferential development of Th2 responses (see Sections IV.A and V.A), a recent report indicates that VIP downregulates the expression of TLR2 and 4 in the murine IBD model (Gomariz et al., 2005). Since GI immune cells are presumed to be chronically activated by local microorganisms in Crohn’s disease, the VIP-induced downregulation of receptors recognizing various groups of microorganisms is certainly relevant. From a therapeutic point of view, it is important to point out that VIP reduced disease severity even when given after the onset of clinical symptoms, and that VIP dramatically reduced disease recurrence upon administration of a second dose of TNBS (Abad et al., 2003). Therefore, VIP could function as a possible multi-step therapeutic agent for Crohn’s disease.

B. Peripheral Inflammatory Disorders 1. Septic Shock Syndrome Septic shock in humans is caused primarily by endotoxins released during severe Gram-negative bacterial infections and carries a staggering 30–50% mortality rate (Danner et al., 1991). High endotoxemia following LPS administration to experimental animals leads to pathological changes similar to human septic shock syndrome. In the last decade our understanding of the pathophysiology of septic shock has increased markedly (Baumgartner and Calandra, 1999; Glauser et al., 1994; Parrillo, 1993). The septic shock follows massive bacterial infections, resulting in the release of bacterial products and the activation of host immune cells and of soluble factors such as complement and clotting molecules. It is generally recognized that the

severe pathology associated with septic shock results from a hyperactive and out-of-balance network of endogenous pro-inflammatory cytokines, including TNF-α, IL-12, IL-6, and IFNγ (van Deuren et al., 1992). The overproduction of inflammatory cytokines generates systemic activation, which affects vascular permeability, cardiac function, and induces metabolic changes that can lead to tissue necrosis and eventually to multiple organ failure and death. Since VIP inhibits the production of pro-inflammatory macrophage-derived cytokines, it was expected to protect against endotoxemia. Indeed, VIP is protective in high endotoxemic models for septic shock (Figure 6C) (Delgado et al., 1999b; Delgado et al., 2000; Martinez et al., 2005; Tuncel et al., 2000). Endotoxemic animals suffer from generalized intravascular coagulation with multiple organ failure as evidenced by severe congestion, hemorrhage, fibrin deposits, edema, thrombosis, massive accumulation of leukocytes in lungs and the intestinal tract, severe congestion of the medullar sinusoids in the spleen, and segmental ischemia of the bowel with regions of hemorrhage or necrosis and an infracted caecum. In contrast, VIPtreated individuals did not present any of the histopathological alterations associated with septic shock (Delgado et al., 1999b). The protective effect of VIP is associated with the downregulation of pro-inflammatory agents released from activated macrophages, i.e., TNF-α, IL-6, IL-12, and NO and immune cell recruitment in target organs (Delgado et al., 1999b; Delgado et al., 2000; Martinez et al., 2005). Studies with specific VIP receptor agonists and the use of PAC1-deficient mice showed that although VPAC1 is the main receptor involved in the protective effect of VIP in septic shock, all three receptors participate in the VIP-induced downregulation of cytokine production and the inhibition of neutrophil recruitment (Delgado et al., 2000; Martinez et al., 2002; Martinez et al., 2005). Whether VIP or VIP analogs might provide a successful therapy in septic shock syndrome remains to be ascertained. The current clinical trials for various therapies have been disappointing. There are several reasons for the lack of success in applying the results obtained in animal models to patients with septic shock syndrome. In animal models, the events starting with the initial stimulus and resulting in the release of the pro-inflammatory cytokine cascade follow a predictable time course. Thus, experimental protocols designed to block the cytokine cascade are relatively straightforward. In contrast, in human septic shock syndrome, the sequence of events is more complex, and the course of the disease generally lasts days rather than hours, as in most animal models. The design of a clinical trial is based essentially on experimental


models using parenteral injections of LPS/bacteria. In contrast, in humans the organisms and site of infections are diverse, and the patients have a variety of underlying diseases. Most important, interventions in animal models have been successful only when applied early, whereas therapy in patients starts during fullblown disease. Since VIP has pleiotropic effects on inflammation, and since it remains protective in the high endotoxemic murine model even when administered 2 hours after LPS inoculation, treatment with VIP or VIP analogs may represent a better biological therapeutic alternative than anti-inflammatory cytokines or anti–TNF-α antibodies, which are effective only at an early stage.

C. Neuroinflammation In addition to multiple sclerosis, the best characterized autoimmune disease in the CNS, recent evidence points towards inflammation as a player in neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. Also, inflammation is the central player in secondary neuronal cell death occurring in brain trauma and spinal cord injury. Microglia, the CNS equivalent of peripheral mononuclear phagocytes, play a major role in CNS inflammation through the release of pro-inflammatory cytokines and chemokines and the capacity to efficiently present antigen to CD4+ T-cells (Kim and de Vellis, 2005; Minghetti et al., 2005; Nelson et al., 2002). Microglia activation is a hallmark of several neurodegenerative diseases, and microgliareleased inflammatory mediators including TNF-α, IL-6, IL-1β, and NO contribute to neuronal damage (Lipton et al., 1994; Taupin et al., 1993). Since VIP was recently identified as a microgliadeactivating factor (see Section IV.A), we assessed its potential role in models of Parkinson’s, brain trauma, and multiple sclerosis. 1. Parkinson’s Disease In vitro VIP has been shown to prevent LPS-induced neuronal cell death in mixed glia-neuronal cell cultures (Delgado and Ganea, 2003a). This neuroprotective effect was especially relevant for dopaminergic neurons (tyrosine hydroxylase, TH-positive neurons). The LPS-induced neurodegeneration is associated with and probably mediated by increased microglial activation, characterized by morphological changes, increased Mac-1 expression, release of free radicals, TNF-α, and NO (Delgado and Ganea, 2003a). The involvement of activated microglia is supported by the lack of LPS effect on pure neuronal cultures and by the fact that cell-free supernatants from LPS-stimulated microglia induce significant cell death in neuro-

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nal cultures. VIP inhibited the LPS-induced microglial activation and the production of TNF-α and NO, suggesting that the neuroprotective effect of VIP is mediated through its inhibitory effect on microglia activation. In Parkinson’s disease (PD) there is progressive degeneration of the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and subsequent loss of their projecting fibers to the striatum. The etiology of PD remains unknown, and present therapies are successful only short term. Several mechanisms, including mitochondrial dysfunction, oxidative damage, excitotoxicity, and α-synuclein deposition, have been proposed to initiate neuronal cell death (Goedert, 2001; Jenner and Olanow, 1996; Mizuno et al., 1995). In addition, neuronal cell death occurs subsequent to inflammatory processes (Fahrig et al., 2005; Jackson-Lewis and Smeyne, 2005; Nagatsu and Sawada, 2005). Pro-inflammatory cytokines are known to participate in mitochondrial impairment and oxidative stress, and therefore, the inflammatory response may serve as an integral feature in PD pathogenesis. Insights into the pathogenesis of PD have been achieved experimentally by using the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model. MPTP produces irreversible clinical, biochemical, and neuropathological effects that mimic those observed in idiopathic PD, including the dramatic neurodegeneration of the nigrostriatal dopaminergic neurons (Bloem et al., 1990; Grunblatt et al., 2000; Przedborski et al., 2000). We have recently demonstrated that the stereotaxic SNpc administration of VIP in the MPTP mouse model of PD is beneficial, reducing SNpc dopaminergic neuronal degeneration and nigrostriatal nerve fiber loss (Delgado and Ganea, 2003a). VIP prevents MPTPinduced activation of microglia in SNpc and striatum and the expression of the cytotoxic mediators iNOS, IL-1β, TNF-α, and NADPH-oxidase. The VIP effect is mediated through VPAC1 and is PKA-dependent. Based on the correlation between the neuroprotective effect of VIP and its inhibition of MPTP-induced microglial activation, we propose that VIP exerts its neuroprotective activity as a “microglial-deactivating factor.” Interestingly, systemic i.p. administration of VIP also inhibited MPTP-induced loss of TH immunoreactivity, although it required 15-fold higher doses and was 50% less efficient than cerebral VIP administration. 2. Brain Trauma Although there is agreement that the increase in the levels of pro-inflammatory cytokines is an early feature in CNS trauma (Bartholdi and Schwab, 1997b; Streit

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et al., 1998; Taupin et al., 1993), whether inflammation in the injured CNS serves a beneficial or detrimental purpose remains controversial. There is evidence that the administration of inflammatory cytokines to injured areas is neuroprotective and/or promotes axonal regeneration (Schwartz et al., 1999). Bloodderived macrophages were shown to change the nonpermissive environment of the CNS white matter to an axonal growth-promoting environment (David et al., 1990). In contrast, other studies support the involvement of pro-inflammatory mediators such as TNF-α, IL-1β, and NO in neuronal and oligodendroglial cell death (Gonzalez-Scarano and Baltuch, 1999; MorgantiKossmann et al., 2002; Stoll et al., 2002). Whether inflammation plays a beneficial or detrimental role in neuronal degeneration might depend on the type of inflammatory cells, the CNS region involved, or the stage of neuronal degeneration. The source of inflammatory cytokines in the CNS following trauma remains unresolved. Infiltrating leukocytes are obvious candidates, but the CNS influx of leukocytes is delayed compared to other tissues. Thus, neutrophils do not infiltrate appreciably until about 6 hours after lesion, whereas T lymphocytes and monocytes appear 12 to 24 hours later (Frank and Wolburg, 1996; Giulian et al., 1989; Moreno-Flores et al., 1993). This suggests that resident cells in the CNS mount the early inflammatory response in trauma. Microglia have been proposed as the source of the early increase in CNS inflammatory cytokines following injury (Bartholdi and Schwab, 1997; Streit et al., 1988). In a model of murine stab wound brain trauma in the periventricular area, VIP was shown to significantly reduce neurodegeneration, the recruitment of mononuclear phagocytes, and microglia activation (Delgado and Ganea, 2003c). We propose that VIP reduces neuronal cell loss through the inhibition of microglial release of pro-inflammatory mediators such as TNF-α, IL-1β, and of chemokines responsible for the recruitment of blood-derived leukocytes into the brain parenchyma. Whether VIP also acts directly on neurons as a neuroprotective agent, or affects neuronal survival through secondary effects on astrocytes, remains to be established. 3. Experimental Autoimmune Encephalomyelitis Experimental autoimmune encephalomyelitis (EAE) is an inflammatory, autoimmune demyelinating disease of the CNS in rodents, with pathologic and clinical similarities to human multiple sclerosis (MS). EAE is used as a model to study the basic mechanisms of autoimmune demyelination and to test potential therapeutic agents (Steinman, 1999). Development of

EAE involves two main phases, i.e., initiation and establishment of autoimmunity to myelin sheath components, and later events leading to the destruction of myelin sheaths. The autoimmune response involves the development of reactive Th1 cells with encephalitogenic potential, their entry into the CNS, microglia activation and recruitment, and activation of peripheral inflammatory cells (Behi et al., 2005; Owens, 2003; Segal, 2005; Wheeler and Owens, 2005). Various therapeutic approaches address the autoimmune components of EAE and MS, complementing existing anti-inflammatory therapies (Calabresi et al., 2002; Howard and Miller, 2004; Killestein et al., 2005; Mezzapesa et al., 2005; Murdoch and Lyseng-Williamson, 2005; Pease and Horuk, 2005; Steinman, 2005; Yong, 2002). Although available therapies reduce the relapse rate and/or delay disease onset, they do not suppress progressive clinical disability. This illustrates the need for novel therapeutic approaches to prevent the inflammatory and autoimmune components of the disease and to promote repair and regeneration mechanisms. Since VIP acts as a potent “deactivating factor” for both macrophages and microglia, promotes Th2 differentiation, and generates antigen-specific regulatory T-cells (see Sections IV.A and V.A and C) we investigated the potential therapeutic effect of VIP in the chronic C57BL/6 and the remitting-relapsing SJL/J EAE models. A recent report indicated that the structurally related neuropeptide PACAP ameliorates EAE in C57BL/6 mice primarily through its effect on antigen-presenting cells (Kato et al., 2004). Our results indicate that VIP treatment prevents the clinical symptoms when administered at the time of immunization and significantly reduces disease severity when administered post-immunization (Figure 6D) (Gonzalez-Rey et al., 2006b). The therapeutic effect correlates with a significant reduction in the spinal cord expression of the pro-inflammatory mediators TNF-α, IL-6, IL-1β, IL-18, IL-12, iNOS; and of various chemokines including CCL2 (MCP-1); an increase in IL-10, TGFβ1, and IL-1Ra expression; and a significant decrease in the number of encephalitogenic Th1 cells both in the periphery and the CNS (Gonzalez-Rey et al., 2006b). We also observed a bias towards Th2 responses in mononuclear cells isolated from both CNS and periphery. Most important, VIP treatment post-immunization was also effective. Since our results indicate that VIP administration induces the generation and/or activation of efficient CD4+CD25+ Treg, including the appearance of CD4+CD25+ cells with a Treg phenotype in the CNS, we propose that the beneficial effect of VIP is mediated, at least partially, through regulatory T-cells


(Chorny et al., 2005; Fernandez-Martin et al., 2006). The Treg induced by VIP in the periphery apparently cross the blood-brain barrier, accumulate in the CNS, and induce suppression of the encephalitogenic response. Indeed, CD4+CD25+ Treg cells have been reported to play a critical role in the regulation of autoimmune diseases, including EAE and MS (Bynoe et al., 2003; Kohm et al., 2002; Reddy et al., 2004; Viglietta et al., 2004). Attention has been focused lately on repair and regeneration mechanisms as targets for MS therapy, especially in the secondary progressive phase of the disease. Interestingly, the delayed administration of VIP in EAE not only prevented the progression of the disease, but induced significant recovery, suggesting a role of VIP in repair and/or neuroregeneration. In fact, in our study, VIP inhibited oligodendrocyte cell loss and axonal damage. VIP stimulates the release of Tcell–derived brain-derived neurotrophic factor (BDNF) (unpublished data), which has been reported to promote axonal outgrowth and rescue of the degenerating neurons (Murray et al., 2002; Yin et al., 1998). VIP has been also reported to induce the production of astrocyte-derived neurotrophic factors (Gozes and Brenneman, 2000). Therefore, in addition to its antiinflammatory activity, VIP might also contribute to neuroprotection and neuroregeneration.

D. Bone Marrow Transplantation Allogeneic bone marrow transplantation is the treatment of choice in hematopoietic malignancies. Following high dose chemotherapy or irradiation, the host is reconstituted with bone marrow cells, and the donor T-cells are responsible for the graft-versus-tumor (GVT) effects that eliminate the remaining malignant host cells. However, the same donor T-cells can initiate a graft-versus-host reaction (GVHR) that represents the major complication following allogeneic bone marrow transplantation. Therefore, the ideal therapy would allow a graft-versus-tumor response without a graft-versus-host reaction. In mice, regulatory T-cells have been shown to prevent lethal GVHR in lethally irradiated hosts reconstituted with allogeneic bone marrow (Cohen et al., 2002; Hoffmann et al., 2002). Recent studies have also demonstrated that, while controlling GVHR, CD4+CD25+ Treg maintain the graftversus-tumor (GVT) response (Trenado et al., 2003). In recent experiments we demonstrated that the in vivo administration of VIP together with low doses of soluble antigen results in the development/activation of antigen-specific CD4+CD25+ Treg (Delgado et al., 2005a; see Section V.C). Using a similar experimental system, we addressed the question whether VIP-

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induced Treg control GVHR while still allowing the beneficial graft-versus-leukemia response. The potential use of VIP-induced Treg in a model of GVHR was assessed by reconstituting lethally irradiated H-2d recipients with allogeneic (H-2k) bone marrow cells depleted of T-cells. The recipients also received CD4+ splenic T-cells obtained from H-2k TCR-transgenic mice previously inoculated with Ag (CD4-control) or with Ag+VIP (CD4-VIP). Mice receiving CD4-control developed severe GVHR, including weight loss, reduced mobility, hunched posture, diarrhea and ruffled fur, and died within 30 days. In contrast, mice receiving CD4-VIP were protected, and more than 60% survived for more than 75 days. The protective effect was even more pronounced upon use of purified CD25+ T-cells from the Ag+VIP-injected donors (Delgado et al., 2005a). While these experiments proved that VIP-induced CD4+CD25+ Treg prevented GVHR, the question remained whether a beneficial GVT effect would be preserved. Irradiated H-2d recipients were reconstituted with T-cell–depleted allogeneic bone marrow cells (H-2k) plus A20 leukemic cells (H-2d) and either CD4-control or CD4-VIP T-cells. The hosts receiving only bone marrow cells died between days 20 and 30 of leukemia (Figure 6E). The hosts receiving bone marrow and CD4-control died of GVHR between days 5 and 24, although the A20 tumor cells had been eliminated. In contrast, 80% of the mice that received bone marrow and CD4-VIP survived, with no evidence of A20 leukemic cells in blood (Figure 6E) (Delgado et al., 2005a). These studies indicate that the VIPgenerated Treg protect against GVHR, while still allowing GVT to occur, and suggest the possible use of VIP-induced Treg in various pathological conditions, including hematopoietic cell transplantation.

VII. CONCLUSIONS AND PERSPECTIVES Macrophages and dendritic cells in the periphery and microglia in the CNS are major players in innate immunity and serve as a link to adaptive immunity by functioning as antigen-presenting cells. Locally activated macrophages, dendritic cells, and microglia decrease the pathogen load through the release of cytotoxic cytokines, oxygen radicals, and nitric oxide, and recruit additional immune cells through the release of pro-inflammatory chemokines. In response to stimulatory and co-stimulatory signals delivered by APCs, CD4+ T-cells proliferate and differentiate into effector Th1 and Th2 cells. At the conclusion of an immune response, both activated dendritic cells/macrophages/ microglia and T-cells have to be deactivated and/or

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eliminated to avoid excessive tissue and organ damage. VIP, released in the lymphoid organs by the innervation and activated immune cells, modulates the function of inflammatory cells through specific receptors, and affects both innate and adaptive immunity functioning primarily as an endogenous anti-inflammatory agent. VIP exerts its anti-inflammatory function through several different mechanisms: (1) direct inhibition of pro-inflammatory cytokine production (TNF-α, IL-6, and IL-12) by activated macrophages and microglia; (2) upregulation of IL-10 production (a potent antiinflammatory cytokine); (3) inhibition of expression and release of pro-inflammatory chemokines from activated macrophages and microglia; (4) inhibition of CD80/CD86 expression in activated macrophages and dendritic cells, and subsequent inhibition of their stimulatory activity for antigen-specific T-cells; (5) alteration of the Th1/Th2 balance by promoting Th2 responses and inhibiting the pro-inflammatory Th1type response; and (6) generation of antigen-specific regulatory T-cells that inhibit proliferation and cytokine production by effector T-cells. From the point of view of a normal physiological role, the fact that VIP downregulates innate immunity and promotes the generation of Th2 effectors and regulatory T-cells is particularly relevant in view of the concept that immune privilege in organs such as brain and eye, where VIP is abundant, is an active process of immune deviation. From a pathological point of view, these findings open up the possibility of using VIP and its analogs as therapeutic agents. Since it interferes with major signaling pathways, i.e., NFkB, MAPK, and Jak/STAT, VIP represents an attractive target for future drug development strategies, particularly for therapies directed at autoimmune and inflammatory conditions.

Acknowledgments This work was made possible by the following grants: 2RO1A047325 (DG) and AI52306(DG and MD), Spanish Ministry of Health (PI03/0526, MD), and La Caixa Foundation (NE03-009, MD).

VIII. ABBREVIATIONS AC, adenylate cyclase; APC, antigen-presenting cell; CBP, CREB-binding protein; CD, Crohn’s disease; CGRP, calcitonin gene-related protein; CIA, collageninduced arthritis; EAE, experimental autoimmune encephalomyelitis; GrB, granzyme B; GVHR, graft-

versus-host reaction; GVT, graft-versus-tumor; IBD, inflammatory bowel disease; iNOS, inducible nitric oxide synthase; MPTP, 1-methyl-4-phenyl-1.2.3.6tetrahydropyridine; α-MSH, melatonin-stimulating hormone; LPS, lipopolysaccharide; PACAP, pituitary adenylate cyclase activating polypeptide; PAC1, PACAP-preferring receptor; PD, Parkinson’s disease; PLC, phospholipase C; PLD, phospholipase D; RA, rheumatoid arthritis; SP, substance P; TH, tyrosine hydroxylase; TLR, Toll-like receptors; TNBS, trinitrobenzene sulfonic acid; Treg, regulatory T-cells; VIP, vasoactive intestinal peptide; VPAC1/2, VIP receptors.

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C H A P T E R

6 Immune-derived Opioids: Production and Function in Inflammatory Pain HALINA MACHELSKA AND CHRISTOPH STEIN

I. INTRODUCTION 159 II. PERIPHERAL OPIOID RECEPTORS 160 III. OPIOID PEPTIDES PRODUCED BY IMMUNE CELLS 161 IV. MIGRATION OF OPIOID-CONTAINING IMMUNE CELLS TO INFLAMED TISSUE 163 V. RELEASE OF OPIOID PEPTIDES FROM IMMUNE CELLS 164 VI. ANALGESIA PRODUCED BY IMMUNE-DERIVED OPIOID PEPTIDES 165 VII. CLINICAL IMPLICATIONS 166 VIII. PERSPECTIVES 166

peptides. These effects occur without central opioid side effects such as depression of breathing, clouding of consciousness, or addiction. Future research should elucidate the selective targeting of opioid peptidecontaining cells to sites of painful tissue injury and the augmentation of opioid peptide and receptor synthesis.

I. INTRODUCTION Acute transient pain serves as a physiological warning to guard the integrity of the organism. An immediate reflex, e.g., withdrawal of a body part from a heat source, prevents tissue damage. If tissue damage occurs, an inflammatory response develops triggered by various pro-inflammatory and pro-algesic mediators. These include protons, hydrogen ions, bradykinin, adenosine triphosphate, prostaglandins, proinflammatory cytokines, chemokines, sympathetic amines, nerve growth factor, and neuropeptides (e.g., substance P, calcitonin-gene–related peptide), which are produced at the site of injury, in plasma, in immune cells, and in the nervous system. In consequence, specialized sensory neurons called nociceptors are activated. Trigeminal and dorsal root ganglia contain nociceptor cell bodies, which give rise to myelinated Aδ and unmyelinated C fibers. Peripheral terminals of Aδ and C fibers transduce and propagate noxious stimuli from peripheral tissues (such as skin, muscles, joints, viscera) to the dorsal horn of the spinal cord

ABSTRACT Endogenous pain control is not only mediated within the central nervous system. In inflammation an interaction between immune cell-derived opioid peptides and peripheral opioid receptors produces potent, clinically relevant inhibition of pain. Opioid receptors are present on peripheral terminals of sensory neurons and are upregulated in inflammation. Their endogenous ligands, opioid peptides, are synthesized in circulating immune cells, which migrate to injured tissues directed by chemokines and adhesion molecules. Under stressful stimuli or in response to releasing agents (e.g., corticotropin-releasing factor, cytokines, catecholamines), immunocytes can secrete opioids. These peptides activate peripheral opioid receptors and produce analgesia by inhibiting the excitability of sensory nerves and/or the release of excitatory neuroPSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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from where these stimuli are transmitted to the brain. At the spinal and supraspinal sites, pro-algesic neurotransmitters, environmental, and cognitive factors contribute to the sensation of pain (Woolf and Salter, 2000). Concurrently, however, endogenous mechanisms which counteract pain develop. They have been well established in the brain and spinal cord where they constitute so-called descending pain inhibitory pathways, which contain mostly opioid peptides, noradrenaline and serotonin, and their receptors (Akil et al., 1998; Terman et al., 1984; Willer et al., 1981). In the peripheral inflamed tissue similar effects can be produced by anti-inflammatory cytokines (Cunha and Ferreira, 2003). However, the best characterized mechanisms of intrinsic pain inhibition (analgesia) directly at the site of injury are based on interactions between leukocyte-derived opioid peptides and peripheral nociceptor endings carrying opioid receptors (Machelska et al., 2002; Stein et al., 2003). This chapter will give an overview of current information on the anatomy and function of peripheral opioid receptors, production and release of opioid peptides from immune cells, mechanisms of opioid-containing leukocyte migration to inflamed tissue, and on analgesia produced by immune-derived opioids.

II. PERIPHERAL OPIOID RECEPTORS A. Localization Opioid peptides are the natural ligands at opioid receptors. Three cDNAs and their genes have been identified, encoding the μ-, δ-, and κ-opioid receptors (MOP, DOP, and KOP), respectively (Kieffer and Gaveriaux-Ruff, 2002). All three receptors can mediate pain inhibition, and they are found throughout the nervous system, including somatic and visceral sensory neurons, spinal cord projection and interneurons, midbrain and cortex. Recent interest has focused on the identification of opioid receptors on nociceptors. All three opioid receptor mRNAs and proteins are expressed in cell bodies of these neurons in dorsal root ganglia (Stein et al., 2001). Opioid receptors are intraaxonally transported into the neuronal processes and are detectable on peripheral sensory nerve terminals in animals and in humans. Colocalization studies confirmed the presence of opioid receptors on neurons expressing substance P and/or calcitonin gene-related peptide, on Aδ- and C-fibers. The latter carry the capsaicin receptor TRPV1 (transient receptor potential vanniloid subtype-1) and are considered the dominant fibers in clinical pain. The binding characteristics

(affinity) of peripheral and central opioid receptors are similar, but the molecular mass of peripheral and central μ-receptors appear to be different (references in Stein et al., 2003). If these findings are confirmed, a search for selective ligands at such distinct receptors may be warranted. It has been suggested that opioid receptors are also located on sympathetic postganglionic neuron terminals. However, there are reports arguing against this notion, and studies attempting the direct demonstration of opioid receptor mRNA in sympathetic ganglia have produced negative results. In addition, thorough morphological investigations have clearly demonstrated the presence of δ-receptors on unmyelinated primary afferent neurons and the absence of such receptors on postganglionic sympathetic neurons in skin, lip, and cornea. Moreover, chemical sympathectomy with 6-hydroxydopamine does not change the expression of opioid receptors in the dorsal root ganglion or the peripheral analgesic effects of μ-, δ-, and κ-receptor agonists in a model of inflammatory pain. Together, these findings have corroborated the notion that peripheral opioid receptors mediating analgesia are exclusively localized on primary sensory neurons (references in Stein et al., 2001). Opioid binding sites and the expression of opioid receptor transcripts have also been demonstrated in immune cells. Opioid-mediated modulation of the proliferation of these cells and of their functions (e.g., chemotaxis, superoxide and cytokine production, mast cell degranulation) has been reported. These immunomodulatory actions can be stimulatory as well as inhibitory and have been ascribed to the activation of opioid receptors (Sacerdote et al., 2003; Sharp, 2003). However, the significance of such effects with regard to pain transmission has not been investigated as yet.

B. Opioid Receptor Signaling in Sensory Neurons All three types of opioid receptors mediate the inhibition of high-voltage activated calcium channels in cultured primary afferent neurons (Figure 1). These effects are transduced by G-proteins (Gi and/or Go) (references in Stein et al., 2001, 2003). Although it is well known that opioids induce membrane hyperpolarization due to increased potassium currents in central neurons, this could not be detected in dorsal root ganglion neurons so far (Akins et al., 1993). Thus, the modulation of calcium channels appears to be the primary mechanism for the inhibitory effects of opioids on peripheral sensory neurons. In addition, opioids—via inhibition of adenylyl cyclase—suppress

6. Immune-derived Opioids: Production and Function in Inflammatory Pain Opioid receptor +

Na channel

Adenylyl cyclase

ATP

TABLE 1 NH2

++

Ca channel

Gi/o COOH

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Structure of endogenous opioid peptides

β-Endorphin

Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-LysSer-Gln-Thr-Pro-Leu-Val-Thr-Leu-PheLys-Asn-Ala-Ile-Val-Lys-Asn-Ala-HisLys-Lys-Gly-Gln-OH Methionine-enkephalin Tyr-Gly-Gly-Phe-Met-OH Leucine-enkephalin Tyr-Gly-Gly-Phe-Leu-OH Dynorphin A Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-ArgPro-Lys-Leu-Lys-Tyr-Asp-Asn-Gln-OH Endomorphin-1 Tyr-Pro-Trp-Phe-OH Endomorphin-2 Tyr-Pro-Phe-Phe-OH

cAMP

FIGURE 1 Opioid receptor structure and signaling in primary afferent neurons. Opioid receptors consist of seven transmembrane domains (green). Upon activation by exogenous or endogenous opioids, they couple to inhibitory G-proteins (Gi/o). This leads to direct or indirect (via decrease of cyclic adenosine monophosphate [cAMP]) closing of calcium (Ca++) and/or sodium (Na+) channels, and to subsequent analgesia. Cyclic AMP is produced from adenosine triphosphate (ATP) by the enzyme adenylyl cyclase.

tetrodotoxin-resistant sodium- and nonselective cation currents stimulated by the inflammatory agent prostaglandin E2 (Gold et al., 1994; Ingram and Williams, 1994). Interestingly, tetrodotoxin-resistant sodium channels are selectively expressed in nociceptors; they are important for impulse initiation and action potential conductance; they mediate spontaneous activity in sensitized nociceptors; and they accumulate at the site of injury in damaged nerves, leading to ectopic impulse generation (Laird et al., 2002; Porreca et al., 1999). The latter observations may explain the notable efficacy of peripheral opioids in inflammatory and neuropathic pain (Stein et al., 2001, 2003). Consistent with their effects on ion channels, opioids attenuate the excitability of peripheral nociceptor terminals, the propagation of action potentials, the release of excitatory pro-inflammatory neuropeptides (substance P, calcitonin gene-related peptide) from peripheral sensory nerve endings, and vasodilatation evoked by stimulation of C-fibers (references in Stein et al., 2001). All of these mechanisms result in analgesia and/or antiinflammatory actions.

C. Peripheral Opioid Receptors and Inflammation Peripheral opioid analgesic effects are augmented under conditions of tissue injury such as inflammation, neuropathy, or bone damage (Stein et al., 2001, 2003). One underlying mechanism is an increased

number (“upregulation”) of peripheral opioid receptors. In neuronal cell cultures, μ-receptor transcription is upregulated by the cytokine interleukin (IL)-4 through binding of STAT6 transcription factors to the μ-receptor gene promoter (Kraus et al., 2001). In dorsal root ganglia, the synthesis and expression of opioid receptors can be increased by peripheral tissue inflammation (Ji et al., 1995; Zöllner et al., 2003). Subsequently, the axonal transport of opioid receptors is greatly enhanced, leading to their upregulation and to enhanced agonist efficacy at peripheral nerve terminals (Jeanjean et al., 1995; Mousa et al., 2001). In addition, the specific milieu (low pH, prostanoid release) of inflamed tissue may increase opioid agonist efficacy by enhanced G-protein coupling and by increased neuronal cyclic adenosine monophosphate levels (Ingram and Williams, 1994; Selley et al., 1993; Zöllner et al., 2003). Inflammation also leads to an increase in the number of sensory nerve terminals (“sprouting”) and disrupts the perineurial barrier, thus facilitating the access of opioid agonists to their receptors (Stein et al., 2001, 2003).

III. OPIOID PEPTIDES PRODUCED BY IMMUNE CELLS Three families of these peptides are well characterized in the central nervous and neuroendocrine systems (Akil et al., 1998). Each family derives from a distinct gene and from one of the three precursor proteins proopiomelanocortin (POMC), proenkephalin (PENK), and prodynorphin, respectively. Appropriate processing yields their respective representative opioid peptides, the endorphins, enkephalins, and dynorphins (Table 1). These peptides exhibit different affinity and selectivity for the three opioid receptors μ (endorphins, enkephalins), δ (enkephalins, endorphins), and κ (dynorphin). Two additional endogenous opioid peptides have been isolated from bovine brain:

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endomorphin-1 and endomorphin-2. Both peptides are considered highly selective μ-receptor ligands. Their precursors are not known yet. All of these opioid peptides have been detected in immune cells, but the POMC and PENK families have been studied most extensively.

A. POMC-derived Opioid Peptides POMC-related opioid peptides have been found in immune cells of many vertebrates and non-vertebrates (references in Machelska and Stein, 2002). To determine whether these immune-competent cells actually synthesize POMC rather than simply absorb related peptides from plasma, mRNA-encoding POMC was sought for and demonstrated in many of these studies. The pituitary POMC gene is organized into three exons separated by intervening sequences which are removed during processing following transcription to produce the full-length, 1200 nt transcript. Initially, truncated POMC transcripts were found in leukocytes (references in Machelska and Stein, 2002). Lyons and Blalock re-examined the question of POMC mRNA expression using novel polymerase chain reaction procedures (Lyons and Blalock, 1997). With this exacting and sensitive methodology, they found expression of fulllength transcripts encoding all three POMC exons in rat mononuclear leukocytes. This POMC transcript is spliced in the same way as the pituitary transcript and contains the sequence for the signal peptide, which is necessary for the correct routing into the regulated secretory pathway. The POMC protein is also proteolytically processed in a way consistent with the pituitary gland (Lyons and Blalock, 1997; Mousa et al., 2004). These results unequivocally demonstrate that immune cells can produce full-length POMC transcripts. Apparently this production is stimulated by various immune and inflammatory mediators (Smith, 2003).

B. PENK-derived Opioid Peptides PENK-derived opioid peptides have also been detected in human and rodent immune cells. Both the mRNA and Methionine (Met)-enkephalin protein were detected. Preproenkephalin mRNA was found in Tand B-cells, macrophages, and mast cells. In subpopulations of immune cells, this mRNA was shown to be highly homologous to brain PENK mRNA, abundant and apparently translated, since immunoreactive enkephalin is present and/or released. The appropriate enzymes necessary for post-translational processing of PENK have also been identified in immune cells (references in Machelska and Stein, 2002).

C. Immune-derived Opioid Peptides in Inflammation Immune-derived opioid peptides apparently play a substantial role in the modulation of inflammatory pain (Machelska and Stein, 2002; Stein et al., 2003). Persistent inflammation is a pathophysiological in vivo stimulus for the immune system and represents a condition that is closer to the clinical setting than in vitro studies. POMC mRNA and β-endorphin, as well as Met-enkephalin and dynorphin, were found in cells derived from lymph nodes, and in leukocytes in the blood and within inflamed tissue (Cabot et al., 1997, 2001; Machelska et al., 2003; Mousa et al., 2004). In the pituitary POMC processing begins as the nascent polypeptide enters the endoplasmic reticulum directed by the signal peptide, and POMC cleavage begins in the trans-Golgi network (references in Mousa et al., 2004). The POMC prohormone is directed to the regulated secretory pathway at the trans-Golgi network by binding to a sorting receptor, membranebound carboxypeptidase E. The prohormone convertases PC1 (also called PC1/3) and PC2 cleave POMC within the trans-Golgi network. PC1 mediates the initial cleavage into adrenocorticotropic and βlipotropic hormones. The inactive pro-PC2 is bound to 7B2 (a chaperone-like binding protein) and is transported from the endoplasmic reticulum to later compartments of the secretory pathway, where it matures to active PC2; thereafter, PC2 converts β-lipotropic hormone into β-melanocyte-stimulating hormone and β-endorphin (references in Mousa et al., 2004). Recently, we detected β-endorphin and POMC alone and colocalized with PC1, PC2, carboxypeptidase E, and 7B2 in leukocytes in the blood and within inflamed tissue in a rat model of unilateral hind paw inflammation with complete Freund’s adjuvant (Mousa et al., 2004). This demonstrates that immune cells express the entire machinery required for POMC processing into functionally active β-endorphin. In the same model of inflammatory pain, mRNAs encoding POMC and PENK and the corresponding opioid peptides βendorphin and Met-enkephalin are abundant in cells of inflamed but not in non-inflamed tissue (Przewlocki et al., 1992). Histomorphological procedures and flow cytometry have identified the opioid-containing cells as T- and B-lymphocytes, granulocytes, and monocytes/macrophages (Przewlocki et al., 1992; Rittner et al., 2001). Also, it was shown that β-endorphin is present in activated/memory T-cells within inflamed tissue (Cabot et al., 1997; Mousa et al., 2001). Thus, opioid peptides are processed and present both in circulating and inflammatory cells infiltrating injured tissue.

6. Immune-derived Opioids: Production and Function in Inflammatory Pain

IV. MIGRATION OF OPIOID-CONTAINING IMMUNE CELLS TO INFLAMED TISSUE The recruitment of leukocytes from the circulation into areas of inflammation involves a well-orchestrated set of events. Leukocytes undergo multiple attachments to and detachments from the vessel’s endothelial cells prior to transendothelial migration. This includes slowing and rolling along the endothelial cell wall that is mediated predominantly by the interaction of selectins expressed on leukocytes (L-selectin) and on endothelial cells (P- and E-selectin) with their ligands on endothelium or immune cells, respectively. The rolling immunocytes can then be activated by chemoattractants such as chemokines released from

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inflammatory cells and presented on endothelium. This leads to the upregulation and increased avidity of integrins. These mediate the firm adhesion of leukocytes to endothelial cells via ligands of the immunoglobulin superfamily, e.g., intercellular adhesion molecule-1 (ICAM-1). Finally, the immune cells transmigrate through the endothelial wall mediated by immunoglobulin superfamily members (e.g., platelet-endothelial adhesion molecule-1; PECAM-1) and are directed to the sites of inflammation to initiate a host defense (reviewed by Petruzzelli et al., 1999; von Andrian and Mackay, 2000). Recent findings indicate that these events can also be involved in the endogenous control of inflammatory pain. In the model of unilateral hind paw

FIGURE 2 Migration of opioid-producing cells and opioid secretion within inflamed tissue. Adhesion molecules are upregulated on vascular endothelium of blood vessels (red) and are co-expressed by circulating immune cells producing opioid peptides. These cells also co-express receptors for chemokines which are released from leukocytes and presented on endothelium. Certain adhesion molecules mediate rolling of opioid-containing leukocytes along the endothelium. The rolling leukocytes can then be activated by chemokines which upregulate another class of adhesion molecules mediating adhesion, and finally cells transmigrate through the endothelium. Once extravasated, these cells can be stimulated by stress (e.g., swim stress, surgery) or releasing agents such as corticotropin-releasing factor (CRF), interleukin-1β (IL-1), and noradrenaline (NA) to secrete opioid peptides. CRF, IL-1, and NA (derived from postganglionic sympathetic neurons; PGSN) elicit opioid release by activating CRF receptors (CRFR), IL-1 receptors (IL-1R), and adrenergic receptors (AR) on immune cells, respectively. Opioids bind to peripheral opioid receptors (produced in dorsal root ganglia and transported to peripheral endings of primary afferent neurons; PAN) and lead to analgesia (see Figure 1).

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inflammation, we have shown that integrin α4 and the chemokines CXCL1 (i.e., keratinocyte-derived chemokine) and CXCL2 (i.e., macrophage inflammatory protein-2) are expressed by leukocytes, while adhesion molecules such as P- and E-selectins, ICAM1, and PECAM-1 are upregulated on endothelium in inflamed paw tissue (Brack et al., 2004c; Machelska et al., 2002, 2004; Mousa et al., 2000) (Figure 2). Expression of CXCL1 and CXCL2 mRNAs and protein contents significantly increased in inflamed tissue during the course of inflammation (Brack et al., 2004b, 2004c). Importantly, L-selectin, integrins β2, and the CXC chemokine receptor 2 (CXCR2) are co-expressed by opioidcontaining leukocytes that have migrated to inflamed subcutaneous paw tissue (Brack et al., 2004c; Machelska et al., 2004; Mousa et al., 2000). Furthermore, pretreatment of rats with a selectin blocker (fucoidin), selective antibodies against ICAM-1, integrins α4 and β2, or chemokines CXCL1 and CXCL2 substantially decreases the number of opioid-containing immunocytes infiltrating the inflamed tissue (Brack et al., 2004c; Machelska et al., 1998, 2002, 2004) and in consequence abolishes endogenous peripheral opioid analgesia (see section VI). This suggests that circulating opioidproducing immune cells home to inflamed tissue where they secrete the opioids to inhibit pain. Afterwards they travel to the regional lymph nodes, depleted of the opioid peptides (Cabot et al., 1997; 2001). Thus, local signals apparently not only stimulate the synthesis of opioid peptides in resident inflammatory cells, but also attract opioid-containing cells from the circulation to the site of injury to reduce pain. This is controlled by specific chemotactic and adhesive mechanisms.

V. RELEASE OF OPIOID PEPTIDES FROM IMMUNE CELLS Regulated secretion of peptides requires secretory granules deriving from the Golgi network for transport to the cell membrane. As discussed in Section III.C. immune cells in the blood and in inflamed tissue co-express the entire machinery required for POMC processing into functionally active β-endorphin (Mousa et al., 2004). Furthermore, our ultrastructural observations show that β-endorphin-immunoreactive macrophages, monocytes, granulocytes, and lymphocytes contain rough endoplasmic reticulum and Golgi apparatus, similar to pituitary cells. Immunostaining of βendorphin was detectable in secretory granules, which were grouped in small or large membranous vesicular

structures. The smaller β-endorphin-immunoreactive secretory granules were localized within cytoplasm, and the larger ones were arranged at the cell periphery ready for the exocytosis, similar to the pituitary (Mousa et al., 2004). In the pituitary β-endorphin and other POMC-derived peptides are released by CRF and IL1β (IL-1) (references in Schäfer, 2003). Similar mechanisms can trigger opioid release within peripheral inflamed tissue (Figure 2). CRF is present in immune cells, fibroblasts, and vascular endothelium, and peripheral CRF expression is enhanced in inflamed synovial and subcutaneous tissue of animals and humans (Schäfer et al., 1996). CRF- and IL-1–receptors and their upregulation were demonstrated in inflamed lymph nodes and paw tissue. Their pharmacological characteristics were similar to the high-affinity CRFand IL-1–binding sites in the pituitary (Mousa et al., 1996). Furthermore, the co-expression of CRF-1 and CRF-2 receptors with β-endorphin in monocytes/macrophages, granulocytes, and lymphocytes was recently shown in the blood and in inflamed paw tissue (Mousa et al., 2003). In contrast, CRF-receptors were not detected on peripheral endings of sensory nerves (Mousa et al., 1996, 2003). The activation of CRF- and IL-1–receptors on cells from lymph nodes results in the secretion of opioid peptides. In those studies βendorphin, Met-enkephalin, and dynorphin were dose-dependently released by CRF, while IL-1 released β-endorphin and dynorphin but not Met-enkephalin. These effects were specific to CRF- and IL-1–receptors (Cabot et al., 1997, 2001; Schäfer et al., 1994). Moreover, this release of opioid peptides was calcium-dependent and mimicked by elevated extracellular concentrations of potassium (Cabot et al., 1997, 2001). This is consistent with a regulated pathway of release from secretory vesicles, as in neurons and endocrine cells. Adrenergic-receptor agonists have also been shown to secrete β-endorphin from human peripheral blood mononuclear cells (Kavelaars et al., 1990). Similar mechanisms are involved in β-endorphin release from leukocytes in the rat model of inflammatory pain (Binder et al., 2004) (Figure 2). Double labeling demonstrated adrenergic α1-, β2- and, to a lesser degree, α2-receptors expressed on β-endorphin–containing inflammatory cells in paw tissue. β-endorphin– containing cells and adrenergic α1- and β2-receptor– expressing cells were localized in close proximity to sympathetic nerve fibers, and chemical ablation of these fibers abolished intrinsic opioid analgesia. Finally, noradrenaline induced adrenergic receptorspecific release of β-endorphin from immune cells in vitro (Binder et al., 2004). Taken together, CRF, IL-1, and sympathetic neuron-derived noradrenaline can


act on their respective receptors on immune cells, resulting in release of opioid peptides (Figure 2).

VI. ANALGESIA PRODUCED BY IMMUNE-DERIVED OPIOID PEPTIDES A. Analgesic Effects of CRF, Cytokines, and Noradrenaline CRF-, IL-1–, and noradrenaline-induced release of opioids from immune cells also occurs in vivo (Figure 2). CRF and IL-1 injected into inflamed paws produce dose-dependent analgesia reversible by their respective antagonists (Schäfer et al., 1994). CRF can produce analgesia both in early and late stages of inflammation (2 hours to 6 days) and, in accord with anatomical findings (see section V), CRF-analgesia involves both CRF-1 and CRF-2 receptors (Brack et al., 2004c; Machelska et al., 2003; Mousa et al., 2003). Intravenous administration of these agents in locally effective doses does not change pain thresholds, demonstrating a peripheral site of action (Brack et al., 2004c; Machelska et al., 2003; Mousa et al., 2003; Schäfer et al., 1994). Leukocytes apparently are the target for CRF and IL-1 because immunosuppression with cyclosporine A, depletion of granulocytes, blockade of chemokines (CXCL1 and CXCL2), as well as anti-selectin and antiICAM-1 treatments result in a significant reduction of opioid-containing cells and of CRF- or IL-1–induced analgesia (Brack et al., 2004c; Machelska et al., 1998, 2002; Schäfer et al., 1994). Also, cyclosporine A-induced attenuation of CRF-analgesia could be restored by injection of activated lymphocytes (Hermanussen et al., 2004). CRF- and IL-1–induced analgesia is blocked by an antibody against β-endorphin, suggesting that this opioid plays a major role. In addition, Met-enkephalin appears to be involved in CRF-, and dynorphin in IL-1–induced analgesia (Schäfer et al., 1994). These results are in line with other studies on local analgesic effects of CRF (Hargreaves et al., 1989; McLoon et al., 2002) and of the cytokines IL-6 and tumor necrosis factor-α (Czlonkowski et al., 1993) but are in contrast to hyperalgesia induced by IL-1α, IL-1β, IL-6, and tumor necrosis factor-α (Cunha and Ferreira, 2003). Importantly, however, the latter hyperalgesic effects of exogenous cytokines were observed after injections into noninflamed tissue (Cunha and Ferreira, 2003; Safieh-Garabedian et al., 1995; Woolf et al., 1997). Apparently, hyperalgesia in highly inflamed tissue (4 days after complete Freund’s adjuvant) has already reached a ceiling effect (Machelska et al., 2003) and therefore cannot be further increased. Instead, in such tissue opioid-containing immune cells became

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the predominant target for cytokines to produce analgesia. Contribution of endogenous pro-inflammatory cytokines to the generation of pain was mostly observed either in early stages (3–6 hours) when cytokine blockade preceded induction of complete Freund’s adjuvant inflammation (Woolf et al., 1997) or in short-lasting (30 minutes–3 hours) inflammation, e.g., induced with carrageenan, lipopolysaccharide, or acetic acid (Cunha and Ferreira, 2003). Thus, the presence or absence of inflammation and the duration and/or model of inflammatory pain are factors to be taken into consideration. Noradrenaline administered directly into inflamed tissue has been shown to produce analgesia, reversible by α1-, α2-, and β2-adrenergic receptor antagonists. Further, this effect was dose-dependently blocked by μ- and δ-opioid receptor antagonists and antibody against β-endorphin (Binder et al., 2004). These data suggest that this catecholamine produces analgesia via opioid peptides activating peripheral opioid receptors (Figure 2). In noninflamed tissue, noradrenaline did not influence pain behavior in this model, consistent with the lack of opioid-containing cells and with the scarcity of adrenergic receptors (Binder et al., 2004). However, the role of peripheral adrenergic receptors in nociception appears controversial. In noninflamed tissue noradrenaline has been shown to produce hyperalgesia via an α2-adrenergic receptor mechanism. Others found that α2-agonists could also produce peripheral analgesia. It has even been postulated that hyperalgesia is mediated via α2B and analgesia is mediated via α2C receptors (references in Binder et al., 2004). Thus, different receptor subtypes, receptor localization, microenvironment, and the presence or absence of inflammation are important parameters to be considered.

B. Endogenous Opioid Analgesia Stress is a natural stimulus triggering inhibition of pain (Terman et al., 1984; Willer et al., 1981). In rats with unilateral hind paw inflammation, stress induced by cold water (4°C) swim (for 1 minute) elicits potent analgesia in inflamed but not in noninflamed paws (Machelska et al., 2003; Stein et al., 1990a). In early inflammation (6 hours) this swim stress-induced analgesia was only partially attenuated by peripherally selective doses of different opioid peptide antibodies and receptor antagonists but was fully reversed by centrally acting doses of an opioid receptor antagonist (Machelska et al., 2003). At later stages of inflammation (4–6 days) swim stress-induced analgesia was completely abolished by peripherally selective doses

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of antibody against β-endorphin and by μ- and δantagonists (Stein et al., 1990a, 1990b). Together, these data indicate that at early stages of Freund’s adjuvantinduced inflammation, all three families of opioid peptides and receptors are involved, while at later stages β-endorphin acting at μ- and δ-receptors dominates. Whereas at early stages both peripheral and central opioid receptors are involved, at later stages endogenous analgesia is mediated exclusively by peripheral opioid receptors. Thus, peripheral opioid mechanisms of pain control become more prevalent with the duration and severity of inflammation. Endogenous triggers of swim stress-induced analgesia are locally produced CRF and sympathetic nerve-derived catecholamines because this effect is abolished by local neutralization of CRF (Machelska et al., 2003; Schäfer et al., 1996), and by sympathetic blockade (Binder et al., 2004). Various types of immune cells are the source of opioids as demonstrated by the abolishment of stressinduced analgesia by immunosuppression with cyclosporine A or whole body irradiation, and by depletion of monocytes/macrophages (Brack et al., 2004a; Przewlocki et al., 1992; Stein et al., 1990b). Moreover, this effect is also extinguished by inhibiting the extravasation of β-endorphin–containing immune cells following blockade of L- and P-selectins, α4 and β2 integrins, or of ICAM-1 (Machelska et al., 1998, 2002, 2004). These adhesion molecules apparently regulate the migration of opioid-containing immune cells and the subsequent generation of intrinsic pain control in injured tissue (Figure 2). A future challenge is to identify factors that will increase homing of opioidcontaining cells to injured tissue and will enhance analgesia. To this end, we have shown that hematopoetic growth factors strongly mobilized granulocytes in the blood but resulted only in a minor increase in the number of opioid-containing leukocytes in inflamed paws, and in no change of CRF- and swim stressinduced analgesia (Brack et al., 2004b). Another approach was to increase the migration of opioidcontaining cells to inflamed tissue with local injections of chemokine CXCL2. However, this did not result in stronger CRF- or swim stress-induced analgesia either, most probably as a result of the small number of peripheral opioid receptors at this very early (2 hours) stage of inflammation (Brack et al., 2004d). Indeed, our previous studies had shown that intrinsic analgesia increases with the duration of inflammation (2 hours–4 days), in parallel with the number of opioid-containing immunocytes, with the number of peripheral opioid receptors and with the efficacy of opioid receptor-G–protein coupling in sensory neurons (Mousa et al., 2001; Rittner et al., 2001; Zöllner et al., 2003).

VII. CLINICAL IMPLICATIONS Peripheral endogenous opioid analgesia has found many clinical applications. Opioid receptors have been demonstrated on peripheral terminals of sensory nerves in human synovia (Stein et al., 1996), and these receptors mediate analgesia in patients with various types of pain (e.g., in chronic rheumatoid arthritis and osteoarthritis; bone pain; after dental, laparoscopic, urinary bladder, and knee surgery) (references in Stein et al., 2003). Opioid peptides are found in human synovial lining cells, mast cells, lymphocytes, and macrophages. The prevailing peptides are βendorphin and Met-enkephalin, while only minor amounts of dynorphin are detectable (Stein et al., 1993; 1996). The interaction of synovial opioids with peripheral opioid receptors was examined in patients undergoing knee surgery. Blocking intra-articular opioid receptors by the local administration of naloxone resulted in significantly increased postoperative pain (Stein et al., 1993). Taken together, these findings suggest that in a stressful (e.g., postoperative) situation, opioids are tonically released from inflamed tissue and activate peripheral opioid receptors to attenuate clinical pain. Importantly, these endogenous opioids do not interfere with exogenous morphine, i.e., intra-articular morphine is an equally potent analgesic in patients with and without opioid-producing inflammatory synovial cells (Stein et al., 1996). This suggests that, in contrast to the rapid development of tolerance in the central nervous system, the immune cell-derived opioids do not readily produce cross-tolerance to morphine at peripheral opioid receptors.

VIII. PERSPECTIVES Effective control of inflammatory pain can result from interactions between leukocyte-derived opioid peptides and their receptors on peripheral sensory neurons. These findings provide new insights into intrinsic mechanisms of pain control and open strategies to develop new drugs and alternative approaches to treatment of pain. Immunocompromised patients (e.g., in AIDS, cancer, diabetes) frequently suffer from painful neuropathies that can be associated with intraand perineural inflammation, with reduced intraepidermal nerve fiber density and with low CD4+ lymphocyte counts (Polydefkis et al., 2003). Thus, it may be interesting to investigate the opioid production/release and the migration of opioid-containing leukocytes in these patients. The important role of certain adhesion molecules and chemokines in the trafficking of opioid-containing cells to injured tissues


indicates that anti-adhesion or anti-chemokine strategies for the treatment of inflammatory diseases may in fact carry a significant risk to exacerbate pain. It would be highly desirable to identify stimulating factors and strategies that selectively attract opioid-producing cells and increase peripheral opioid receptor numbers in damaged tissue. Augmenting the synthesis and/or secretion of opioid peptides and opioid receptor numbers within injured tissue may be accomplished by gene therapy: delivery of PENK-, POMC-, and μopioid receptor-cDNAs has been shown to decrease chronic pain and inflammation (Braz et al., 2001; Lu et al., 2002; Xu et al., 2003). Importantly, opioid analgesia resulting from neuroimmune interactions occurs in peripheral tissues and therefore is devoid of central opioid side effects (such as respiratory depression, nausea, dysphoria, addiction, and high rate of tolerance) and of typical side effects produced by non-steroidal anti-inflammatory drugs (such as gastric erosions, ulcers, bleeding, diarrhea, and renal toxicity). Many efforts have been undertaken to develop peripherally acting analgesics by aiming at individual excitatory receptors or channels on sensory neurons (Simonin and Kieffer, 2002). The major advantage of targeting opioid receptors is their mechanism of action: The inhibition of calcium (and possibly sodium) channels simply renders the nociceptor less excitable to the plethora of stimulating molecules expressed in damaged tissue. Thus, peripherally acting opioids can prevent and reverse the action of multiple excitatory agents simultaneously, in contrast to blocking only one single noxious stimulus. Uncovering mechanisms that can enhance the availability of endogenous opioids within injured tissue and the signal transduction of peripheral opioid receptors will open exciting possibilities for pain research and therapy.

Acknowledgment We wish to thank Christine Voigts for the preparation of the illustrations.

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Woolf, C. J., and Salter, M. W. (2000). Neuronal plasticity: increasing the gain in pain. Science, 288, 1765–1769. Xu, Y., Gu, Y., Xu, G. Y., Wu, P., Li, G. W., and Huang, L. Y. (2003). Adeno-associated viral transfer of opioid receptor gene to primary sensory neurons: a strategy to increase opioid antinociception. Proc. Natl. Acad. Sci. USA, 100, 6204–6209. Zöllner, C., Shaqura, M. A., Bopaiah, C. P., Mousa, S. A., Stein, C., and Schäfer, M. (2003). Painful inflammation-induced increase in mu-opioid receptor binding and G-protein coupling in primary afferent neurons. Mol. Pharmacol., 64, 202–210.


C H A P T E R

7 Crosstalk between Insulin-like Growth Factors and Pro-inflammatory Cytokines ROBERT H. McCUSKER, KLEMEN STRLE, SUZANNE R. BROUSSARD, ROBERT DANTZER, ROSE–MARIE BLUTHÉ, AND KEITH W. KELLEY

I. INTRODUCTION 171 II. THE INSULIN-LIKE GROWTH FACTOR (IGF) SYSTEM 172 III. IGF TYPE I RECEPTOR SIGNALING 175 IV. PRO-INFLAMMATORY CYTOKINE SIGNALING 177 V. CYTOKINE-INDUCED IGF RESISTANCE; IN VITRO—MECHANISMS OF ACTION 179 VI. CYTOKINE—IGF INTERACTIONS; IN VIVO— BEHAVIOR AND LEARNING 184 VII. CONCLUSIONS 187

ultimately relate to health and well-being. The chapter is broken into two general sections. The first describes how IGFs and pro-inflammatory cytokines interact at the cellular level, with an emphasis on proinflammatory cytokine-induced hormone resistance. The second section relates these cytokine and hormone interactions to the field of psychoneuroimmunology by presenting evidence of their interactive effects on behavior and cognition. The endocrine and immune systems are biosensors that constantly monitor the body and its environment to fine-tune cellular growth, metabolism, and behavior. These systems coordinate the body’s adaptive mechanisms. This adaptation is necessary for growth and survival, permitting the body to resist the stressors imposed by insults such as environment extremes, exposure to pathogens, or limited nutritional resources. Adaptation requires not only positive actions, but more often than realized, adaptation requires downregulatory, negative actions such as cytokine-induced resistance to the actions of hormones and growth factors. This cytokine-induced resistance is critical to curb the activity of growth factors and to maintain homeostasis. Homeostasis is a carefully controlled balance between responses to the deleterious effects of environmental and pathological stressors (both external and internal) and the drive for normal growth, development, and eventual maintenance of the status quo (Figure 1). The intricate balance maintained by homeostasis is partially coordinated by the secretion and action of hormones (Frystyk, 2004; Valverde et al.,

I. INTRODUCTION The path of least resistance is the path of the loser. H.G. Wells 1866–1946

The immune and endocrine systems interact to control growth, development, health, and mental wellbeing. An important aspect of this interaction is the regulation of immune events by the endocrine system, as we highlighted for growth hormone, insulin-like growth factor-I (IGF-I) and prolactin (Arkins et al., 2001). The two major pro-inflammatory cytokines, TNFα and IL-1β, have now been shown to interact with the IGFs to regulate each others’ actions, such as occurs when cytokines induce hormone resistance (Kelley, 2004). The purpose of this chapter is to highlight this exciting and relatively new area of research by describing the mechanisms underlying how the immune system affects various cellular functions that PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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2005) and cytokines (Hehlgans and Pfeffer, 2005; Valverde et al., 2005). For instance, infectious agents are perceived by the immune system, which in turn can initiate stress signals in the form of pro-inflammatory cytokines that act not only on leukocytes but also on other tissues, including the brain. The reliance on hormone and cytokine release for homeostasis is necessary to coordinate the multitude of processes that simultaneously occur in complex organisms. Such a reliance on biosensor systems is uniquely different from growth regulation of simpler organisms that respond to environmental stimuli with only a limited ability to communicate between cells. For decades, the endocrine and immune systems were largely studied independently of one another. However, it is now clear that the two systems interact at several levels to create an intricate network that controls everything from animal behavior to body growth and cancer progression. Pro-inflammatory cytokines act in the brain to cause sickness behavior (Kelley et al., 2003) and contribute to depressive-like symptoms (Schiepers et al., 2005). They act on skeletal muscle to cause wasting (Spate and Schulze, 2004) and on cancer cells to suppress proliferation or induce cell death (Mocellin et al., 2005; Wajant et al., 2005). Although there are differences in the ultimate consequence on cells, all of these effects share one common mechanism: pro-inflammatory cytokine-induced IGF resistance. Hormone resistance may be more common

than first believed as it is now becoming clear that pro-inflammatory cytokines can induce resistance to several important hormones besides IGF, including insulin (Hotamisligil, 2003), glucocorticoids (Avitsur et al., 2005; Silverman et al., 2005), and growth hormone (Lang et al., 2005). Why is there a need for the immune system to induce hormone resistance? The answer is by no means simple. One rudimental possibility is that hormone resistance is needed because the human body has limited resources. A system of checks and balances has evolved to control the use of these resources between tissues, within tissues, and indeed within different compartments of individual cells. Only in developed countries of the current era have humans found themselves in a “protected” environment, with almost unlimited essentials for life, i.e., food, shelter, and medicine. However, throughout most of history, humans have had limited resources. The body adapted to these limited resources by making careful decisions about how to use those resources, with the major choices being aimed at three main processes: (a) growth and nutrient storage, (b) reproduction, and (c) survival processes, such as combating disease. In response to many types of stressors, one of the global consequences of pro-inflammatory cytokines is to depress growth and nutrient storage, making more resources available for more fundamental survival processes. This goal is partially achieved by blocking the anabolic effects of IGF via hormone resistance. With this in mind, this chapter focuses on interactions between two major pro-inflammatory cytokines, TNFα and IL-1β, and an important growth factor, IGF. The main theme is aimed at describing how these pro-inflammatory cytokines indirectly alter normal cellular processes by interfering with the actions of IGF.

II. THE INSULIN-LIKE GROWTH FACTOR (IGF) SYSTEM

FIGURE 1 Adaptation via the interaction of two biosensor systems. The immune and IGF systems act synergistically on some functions yet are in opposition on others. One of the keys to regulating their actions is a clear understanding of how the two systems interact. It is now apparent that intracellular receptor signaling pathways of pro-inflammatory cytokines and IGFs communicate with each other to regulate cellular function.

The IGFs are unique among growth factors. Virtually all cells of the body are responsive to IGF-I and IGF-II, either acting through their own receptor or the insulin receptor. This fact underlies the global importance of these peptides in controlling cellular function. Indeed, IGF-I and IGF-II are key endocrine proteins driving cellular growth, development, and survival (McCusker, 1998). In particular, IGF-I is critical for normal postnatal growth and development. Together with its primary regulator, GH, IGF-I accounts for 83% of postnatal body growth (Lupu et al., 2001). IGF-I and IGF-II drive the growth of essentially every tissue

7. Crosstalk between Insulin-like Growth Factors and Pro-inflammatory Cytokines

within the body, including proper brain size and development (D’Ercole et al., 2002). Although GH regulates serum IGF-I and thereby body growth, it appears that the brain is privileged in this respect. GH deficiency does not reduce brain IGF-I, brain size, or cognitive function, thus creating a protective mechanism for the brain against GH deficiency. Indeed, growth hormone-deficient mice have higher concentrations of IGF-I in the brain than do non-deficient mice (Sun et al., 2005). IGF-I is also necessary for muscle growth and development. Early work clearly established that elevated GH enhances serum IGF-I concentrations, mRNA expression within muscle and overall muscle growth (Frost and Lang, 2003). Similarly, an enhanced GH/IGF axis increases the number of muscle precursor cells (McCusker and Campion, 1986). Until recently, it was believed that, over a large physiological range, the IGFs did not have major adverse consequences. However, from research that began with Caenorhabditis elegans and recently extended to mammals, it has become increasingly clear that postnatal circulating IGF-I is inversely related to life span (Kenyon, 2005) and elevated embryonic IGF-II leads to fetal overgrowth and early postnatal death (Lau et al., 1994). Also, since the IGFs increase proliferation of many cells, serum IGF is positively correlated to progression of several different types of tumors (Foulstone et al., 2005; Hofmann and Garcia-Echeverria, 2005; Khandwala et al., 2000; LeRoith and Helman, 2004). In terms of life span, the divergent outcomes of IGF signaling appear to converge mechanistically at the level of FOXO, a member of the forkhead family of transcription factors that inhibits cell proliferation and is associated with increased resistance to cellular stressors and prolonged life span. Inactivation of forkhead proteins by IGF promotes cell survival. Conversely, IGF activation of PI3-K and subsequently the downstream kinase, Akt, leads to phosphorylation of FOXO, which sequesters it to the cytoplasm and inhibits its transcriptional activity. Despite IGF inactivation of FOXO for cell survival, IGF inactivation of this factor appears to be associated with decreased life span (Kenyon, 2005). This disparate relationship underlies our lack of a comprehensive view of IGF physiology. However, it emphasizes a real and pressing goal of independently controlling the various intracellular signaling pathways of IGF in a specific fashion. The paradox that IGFs, which are necessary for growth and development, are inversely correlated with life span of the whole organism is intriguing and suggests that mechanisms should be present to precisely control IGF activity. Indeed, IGF activity is intri-

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cately regulated by several mechanisms, including binding to three distinct receptors (IGF type 1 receptor, or IGF-1R; the insulin receptor, or InsR; and the IGF type 2 receptor, IGF-2R, which is an inhibitory decoy) and several high-affinity binding proteins (IGFBPs, which control IGF access to cell surface IGF receptors). Pro-inflammatory cytokines from the immune system have now been defined as an IGF-regulating mechanism. An activated immune system, via pro-inflammatory cytokine production, causes IGF resistance by decreasing some aspects of intracellular receptor signaling, as will be described below. These findings indicate that the outcomes of IGF receptor signaling are defined by a complex network of interactions, and also point to the conclusion that there are major gaps in understanding just how IGF and each of its divergent signaling pathways are regulated. To completely grasp the intricate and complex regulation that has evolved to control IGF activity, we need to define the various members of the system (Table 1). IGF-I and IGF-II are both small peptides, 67 to 70 amino acids, respectively, that are present in varying levels in all body fluids. Encoded by separate genes, the two peptides share complete physiological redundancy. The IGFs act through the same receptors and have identical effects on cells, differing only in relative sensitivities. IGF-I binds to the IGF-1R with approximately twice the avidity of IGF-II (McCusker, 1998) and thus has similar activity at half the dose. Insulin binds the IGF-IR with a 200-fold lower affinity. Care must be taken when interpreting in vitro studies using high levels of any of these three ligands (IGF-I, IGF-II, or insulin). Due to receptor cross-reactivity, cellular changes resulting from IGF-1R, IGF-2R, and InsR cross-reactivity may not occur in vivo with physiological levels of ligand. This is especially important for insulin, which is frequently added at > 1 μg/ml to cell culture supplements. For instance, the popular B27 medium supplement used for neuronal serum-free cultures (Gibco) contains 4 μg/ml insulin, which likely activates both the InsR and the IGF-1R. Although most of the work, both in vivo and in vitro, has been done with IGF-I, cytokine-induced IGF resistance would occur for all three ligands—IGF-I, IGF-II, and insulin. Four major mechanisms exist to regulate IGF activity. First and foremost is the distribution of receptors; the presence or absence of receptors determines the ability of a cell to respond. A clear example is presented during development of skeletal muscle. Myoblasts, which are skeletal muscle precursor cells (Wagers and Conboy, 2005), express abundant IGF1Rs but fewer InsRs (McCusker and Novakofski, 2003,

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I. Neural and Endocrine Effects on Immunity TABLE I Size (kDa)

Ligands IGF-I & IGF-II Insulin Receptors IGF-1R

7.6 5.8 303.7

InsR (a/b isoforms)

306.7

IGF-specific regulators IGFBPs (1–6) IGF-2R

∼30.0 540.0

Members of the IGF System Primary role

Metabolism, proliferation, survival, differentiation Nutrient metabolism for storage Ligand activated tyrosine kinase Binds IGF-I > IGF-II >> Insulin at 1 : 2 : 200 ∼relative affinities Ligand-activated tyrosine kinase Binds Insulin ≥ IGF-II > IGF-I at 1 : 2 : 30 ∼relative affinities IGF inhibition/potentiation IGF inhibition

Location: source

All body fluids: all tissues Circulation / pancreas

Transmembrane: one or both receptors are present on most if not all cells of the body

Body fluids, cell surfaces: all tissues

IGF-1R and IGF-2R = IGF type 1 and 2 receptors. IGFBP = IGF binding protein. InsR = insulin receptor. Sizes based on amino acid composition (receptors = size of multimer) but vary with glycosylation. (Entingh et al., 2003; McCusker, 1998).

2004). In contrast, once differentiated, mature myofibers contain abundant InsRs but fewer IGF-1Rs (Brunetti et al., 1989). There is a general correlation of greater IGF-1R expression in rapidly growing cells (and thus transformed cells) and a greater relative InsR expression in post-mitotic cells (e.g., myofibers, hepatocytes, and adipocytes). This general trend in relative receptor expression led to the characterization of IGFs as growth factors, since they act on most mitoticcompetent cells at relatively low concentrations. In contrast, insulin was characterized as a metabolic hormone, since it is active at low concentrations in stimulating nutrient storage (e.g., sugars as glycogen, lipids as fat, and amino acids as protein). Relative receptor expression (IGF-1R/InsR) on a particular cell type is largely predetermined, and relatively little is known about the regulatory mechanisms determining which receptor is expressed on a particular cell. Other than knockout or forced expression of the IGFIR or InsR to study the in vitro and in vivo actions of each respective ligand (Baserga, 2005; Kitamura et al., 2003), there has been little effort devoted toward regulating relative receptor expression as a means of controlling cellular responsiveness among the three ligands. Second is the rate of IGF secretion. Clearly, postnatal secretion of IGF-I is more dynamically controlled than is the secretion of IGF-II. IGF-I secretion is affected by multiple hormones, including growth hormone, insulin, thyroid hormones, and glucocorticoids (McCusker, 1998). Similarly, insulin secretion has been extensively studied. From a physiological standpoint, this is a relatively mature subject. Thousands of research papers and numerous reviews have summarized the regulation of IGF secretion in mammals.

There remains great potential for the therapeutic use of IGF for human maladies, including treatments for wound healing (Grazul-Bilska et al., 2003), pediatric (Rosenfeld, 2005) and adult growth hormone deficiency (Savage et al., 2004), diabetes (Ranke, 2005), neurological disorders (Leinninger and Feldman, 2005), and immunoenhancement (Burgess et al., 1999; Welniak et al., 2002). A recent report established that rats partially deficient in GH for their entire life do not display increased longevity (Sonntag et al., 2005). However, treatment of GH-deficient rats with GH prior to adulthood increases longevity and improves cognitive function later during senescence. In humans, there is positive correlation between IGF levels and progression of cancer (Foulstone et al., 2005; Hofmann and Garcia-Echeverria, 2005) and diminution of life span (Kenyon, 2005). These findings have led to a huge controversy about the role of GH and IGF in human medicine (Consensus, 2001). For example, adults with low IGF-I have reduced risk for a variety of cancers. However, subjects with low IGF-I concentrations also have an increased risk for diabetes and osteoporosis, as well as both heart and neurodegenerative diseases (Yang et al., 2005). Importantly, there is as yet no evidence that reduced IGF-I blood concentration in humans is associated with longer life (Janssen and Lamberts, 2004). The current view is that not enough IGF or too much IGF is detrimental to health, but there is a certain amount of IGF that is required and necessary for optimal health. This conclusion is similar to the classic U-shaped curve in exercise physiology, which reflects the concept that too little or too much exercise can impair health, with moderate exercise being recommended for most of us.


Third are the IGFBPs. Six high affinity IGFBPs and an additional IGF receptor control IGF activity (see Table 1). The IGF-2R, residing on the cell surface, has no known signaling ability. Instead, it serves to aide internalization and target IGF-II for degradation. By this mechanism, the IGF-2R directly decreases the amount of active IGF-II and thus decreases IGF activity. The IGF-2R is also found in the circulation (McCusker et al., 1989), where it acts as an additional soluble IGFBP with unproven although presumed IGF-delaying activity similar to that of the smaller IGFBPs. In extracellular fluids, the six IGFBPs delay IGF activity, as demonstrated both in vivo and in vitro. By association with the IGFs, the IGFBPs prevent immediate IGF access to the IGF-1R (Arkins et al., 1995; Li et al., 1996; Liu et al., 1998; McCusker et al., 1990; McCusker et al., 1991). The IGFBPs and IGF-2R increase the half-life of circulating IGF and delay IGF activity until the IGF is released, wherein it can then associate with the IGF-1R. Fourth, and most critical to this review, is the regulation of IGF-induced intracellular signaling. The three mechanisms, briefly described above, control overall IGF activity with no specificity regarding the various effector functions of IGF such as cell survival, differentiation, proliferation, or metabolism. However, considering the requirement of IGF activity for these various cellular activities, the ability to specifically and independently control these various actions has the greatest potential to result in therapeutic application. Since all IGF-mediated actions are via the single cell surface receptor, IGF-1R, functional divergence depends on branching of intracellular signaling pathways that are initiated following binding of either IGFI or IGF-II to the IGF-IR.

III. IGF TYPE I RECEPTOR SIGNALING The heterotetrameric IGF-1R is a ligand-activated transmembrane tyrosine kinase composed of two α (105kDa each) and two ß (95kDa each) subunits. Ligand binding to the α-subunits induces conformational changes in the ß subunits and trans-autophosphorylation of critical tyrosine residues in the ß subunit kinase domain/C-terminus of the receptor. These residues allow for docking of IRS and Shc proteins, which are then tyrosine phosphorylated by the IGF-1R. Phosphorylated docking proteins recruit additional signal transducing factors, including the p85 regulatory subunit of PI3-K and Grb2/SOS (Dupont and LeRoith, 2001; Leroith and Nissley, 2005) activating two major signaling cascades, the PI3-K and the MAPK pathways, respectively. Various signaling components

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of these pathways regulate cell metabolism, hypertrophy, proliferation, survival, and differentiation (Figure 2). MAPK Pathway: IGF-1R activation and the subsequent tyrosine phosphorylation of Shc and IRS-1 allow Grb2 to bind to these adaptor proteins via its SH2 domain. Grb2 then recruits SOS, a guanine nucleotide release protein that catalyzes the conversion of inactive Ras-GDP into its active form, Ras-GTP. Ras-GTP promotes membrane localization and activation of Raf-1, which subsequently activates downstream ERK-1/2; components of an MAPK pathway. ERKs, rather than c-jun N-terminal kinase (JNK) or p38 stress-activated protein kinase (SAPK), are the major IGF-activated MAPKs that confer cell-cycle progression/proliferation of a diverse variety of cell types, including myoblasts and many cancer cells. PI3-K Pathway: Unlike the MAPK pathway, the PI3-K pathway does not require guanine nucleotide exchange and is activated by direct binding of IRS-1/2 to the phosphorylated IGF-1R. IGF-1R–induced tyrosine phosphorylation of IRS (pIRS) recruits the p85 regulatory subunit of PI3-K via two SH2 domains. Binding of p85 to pIRS activates PI3-K’s catalytic domain, p110. Activated PI3-K converts membrane 3,4-inositol-diphosphate to 3,4,5-inositol-triphosphate; which activates two distinct PDKs, followed by Akt. Akt is a major divergence point that ultimately regulates IGF-induced cell hypertrophy, proliferation, survival, and differentiation. Distinct Akt-dependent pathways are critical to specific IGF functional outcomes (Figure 2). Although neither the MAPK nor the PI3-K pathways shown in Figure 2 are completely defined, several important points about the pathways are obvious. (1) The two pathways are connected. This is specifically illustrated by showing that both pathways link the IGF-1R to cyclin-dependent kinase (CDK) activation. (2) Both pathways are branched; this is particularly true for the PI3-K pathway, which arborizes extensively after the activation of Akt. (3) Each branch has overlapping functions, but each is involved in specific effects of IGF. The overlapping properties are not necessarily obvious, but each of the branches after Akt has been found to have different functions in different systems. For instance, a cell must survive to differentiate. Hence, to a large degree, the pathways that one laboratory indicates to be involved in cell survival may be found by another laboratory as necessary for differentiation, since dead cells cannot differentiate. Similarly, transcription is necessary for proliferation. To put this another way and iterate this point, induction of a particular pathway does not promote only one particular function, but in many cases just one particu-

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FIGURE 2 A simplified IGF-1R signaling map. Upon IGF binding to the IGF-1R, or insulin to the InsR, a series of intracellular signaling events occurs that result in enhanced cell metabolism, hypertrophy, proliferation, differentiation, or survival. Two major pathways are activated by the IGF-1R: the MAPK pathway (left) and PI3-K pathway (right). The PI3-K pathway diverges after activation of Akt. Interference with any of the steps prior to Akt activation will affect all or part of IGF’s downstream actions. A more detailed signaling pathway and several excellent reviews can be found at STKE Connections Map (White, 2003). IRS—insulin receptor substrates, PI3-K— phosphoinositide 3-kinase, PDK—3-phosphoinositide-dependent protein kinase, Akt—(PKB, protein kinase B), TSC—tuberous sclerosis complex/tuberin, MDM—murine double minute, mTOR—mammalian target of rapamycin, GSK—glycogen synthase kinase; SOS—son of sevenless.

lar pathway may be limiting for that function to occur. (4) It is currently unclear whether a single receptor molecule activates both the MAPK and PI3-K pathways simultaneously; the possibility that specific IGF1R heterodimers promote only the MAPK or the PI3-K pathway is the reason for distinct receptor proteins linked to each pathway in Figure 2. The effect initiated

by IGF signaling may depend on the type of protein scaffolding that occurs at the intracellular face of individual receptors. For example, IGF-I activates the mitogenic ERK1/2 pathway very well in many cell types but poorly in primary hippocampal neurons despite all the hardware for IGF-1R signaling being present (Zheng and Quirion, 2004). (5) Finally, there


are inhibitors of specific components in the pathway. For example, the tumor suppressors PTEN (phosphatase and tension homolog deleted on chromosome 10) and tuberous sclerosis (TSC) lie upstream and downstream of Akt, both of which act to constrain the activity of Akt (Hay, 2005). Undoubtedly, more activators and inhibitors in the IGF signaling pathway will be discovered, thereby offering new targets for pharmacological interventions. It is important to note that all of the in vitro physiological effects of IGF can be considered positive, i.e., increased cell survival, increased cell proliferation, increased cell differentiation, increased transcription, increased metabolic activity, and increased cell hypertrophy. Although most approaches to modify intracellular IGF actions have focused on early upstream events induced by IGF-1R activation, such as IRS recruitment or PI3-K activation, controlling specific steps along either the MAPK or the PI3-K pathways could yield novel approaches to control a specific effect of IGF. For example, it should be possible to develop a way to depress IGF-induced proliferation of cancer cells without diminishing IGF’s protective actions within the brain or on skeletal muscle.

IV. PRO-INFLAMMATORY CYTOKINE SIGNALING The key aspect of pro-inflammatory cytokine action, relative to this chapter, is duality of action. Unlike the effects of the IGFs that can all be considered positive at the cellular level (i.e., increased in proliferation, differentiation, survival, or hypertrophy), pro-inflammatory cytokines affect cells in both positive and negative ways. Another important point is that various cytokines share many physiological functions and common intermediate receptor signaling molecules (Hanada and Yoshimura, 2002; Liu, 2005), although there are specific and important differences in signaling intermediates between each of the cytokines. With this in mind, TNFα acting through TNF-R1 is used as a prototypical pro-inflammatory cytokine model in this chapter. The multiple redundancies in receptor signaling between cytokine receptors, despite differences in the physiological outcomes of cytokines, is not unlike the remarkable similarity in signaling pathways shared by the IGF-1R and InsR, despite important differences in the perceived major physiological roles of insulin and the IGFs (nutrient storage vs. cell proliferation). These enigmas are most certainly due to lack of clear and complete definition of receptor distribution between cell types within tissues, as mentioned earlier for the IGF-1R and InsR, and additional unknown sig-

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naling differences that occur between and within the context of mammalian cells. TNF-R1 is a homotrimer without intrinsic enzymatic ability, i.e., absence of kinase activity. Ligand binding changes the conformation TNF-R1, leading to exchange of proteins that bind the intracellular face of the trimer. Receptor signaling is usually suppressed by the Silencer of Death Domain (SODD), which prevents association of early upstream signaling intermediates with the TNF-R1. Upon TNFα binding, SODD dissociates from the receptor allowing other proteins to bind to the receptor complex. Since the receptor lacks enzymatic activity, exactly how signaling is initiated upon ligand binding is currently unclear. TNF-R1 has two major domains for signal initiation: the death domain (DD) and the neutral sphingomyelinase domain (NSD). DD was so named because early work indicated that proteins bound to TNF-R1 by this region, such as TRADD and FADD, could initiate apoptosis via caspase activation (Figure 3). Similarly, factor-associated with neutral sphingomyelinase (FAN) binding to the NSD results in neutral sphingomyelinase (N-SMase) activation and ceramide generation. This pathway also can cause apoptosis of certain cells. Albeit TNFα was first characterized by its ability to cause tumor cell death, TNF-R1 signaling can also promote cell survival via NFκß-dependent transcription of anti-apoptotic factors (Sheikh and Huang, 2003). Although the last decade has witnessed remarkable advances in the field, it still remains unclear how cells choose between life and death following TNFα stimulation (Mocellin et al., 2005). TNFα, via the DD and several MAPK pathways, can activate ERK1/2 and p38MAPK, which enhance cell proliferation (Baud and Karin, 2001). However, TNFR1 activation has a notable cytostatic effect (Shen et al., 2002), albeit by largely uncharacterized mechanisms. Activation of TNF-R1 also suppresses differentiation of several cell types, particularly skeletal muscle cells. However, there is no direct signaling pathway associated with either cytostasis or inhibition of differentiation (Figure 3). Although the intricate details associated with IGF-1R signaling relative to a particular function are still being defined, there is consensus that a positive response will occur. However, TNFα action is difficult to predict based upon the seemingly opposing possible effects mediated by TNF-R1; numerous reviews have come to the same conclusion regarding the ambivalent prediction of TNFα activity. The manuscripts cited in the reviews contain excellent experiments attempting to elucidate the specific mechanism associated with a particular effect of TNFα. Most experiments, however, are not designed to consider the possibility that many

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FIGURE 3 A simplified TNFα signaling diagram. Proinflammatory cytokine signaling is a highly arborized process, and the net effect depends on the relative strength of each pathway [adapted from (Mocellin et al., 2005)]. TRADD—TNF-R associated death domain; FADD—Fas-associated death domain; PPA2—protein phosphatase 2A; SMase—sphingomyelinase; MADD—MAPK-activating death domain; RIP—receptorinteracting protein; TRAF—TNFR-associated protein; TNF-R1—TNF receptor type 1/ p55; MAPK—mitogen-activated protein kinase; JNK—c-Jun N-terminal kinase; SODD— silencer of death domain; NSD—N-SMase activation domain.

of the effects induced by TNFα are not direct. To clearly understand TNFα action requires the consideration of its interaction with growth factors and hormones, in particular the IGFs. Most in vitro experiments are conducted using cells grown in the presence of fetal bovine serum (FBS) or one of several medium supplements such as ITS, N2, or B27. FBS contains substantial amounts of both IGF-I and IGF-II, and the three aforementioned supplements contain insulin at

levels that activate the IGF-1R. As discussed below, many of the biological actions associated with TNFα may not be direct but instead may be the result of TNFα causing resistance to IGFs (and/or insulin). Similarly, since all in vivo fluids contain IGF, many of the results that have been reported with animal studies may well be the result of growth factor resistance in addition to direct effects of the pro-inflammatory cytokine.


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FIGURE 4 Signaling pathways for one cytokine and one growth factor. Proinflammatory cytokine and IGF signaling have both distinct and similar effects on cells. Similar pathways may be a means to amplify an action, whereas distinct pathways may either complement each other or be antagonistic.

V. CYTOKINE-INDUCED IGF RESISTANCE; IN VITRO—MECHANISMS OF ACTION In live animals, cells are not under the influence of a single growth factor or cytokine. This is also true in vitro, as many cells secrete several factors in culture, including pro-inflammatory cytokines and either IGF-I or IGF-II. As already mentioned above, few cells in vitro are grown in serum-free conditions, and even then they are generally supplemented with a hormone mixture which usually contains a member of the IGF ligand family. It has been difficult to unravel the particular pathways activated by a single factor due to pathway arborization (see Figures 2 and 3), and few investigators have attempted modeling the interaction of multiple factors within a cell. Diagrams rapidly become complex when simplified pathways for even a single cytokine and growth factor are considered jointly (Figure 4). The intracellular signaling pathways of receptors for pro-inflammatory cytokines and IGF coexist in many cells. Therefore, the undefined negative effects of TNFα (cytostasis, decreased differentiation, altered metabolism, and apoptosis) could be explained by attributing these actions not to a direct effect of the

cytokine but to cytokine-induced IGF resistance. As shown in Figure 4, at least two pro-inflammatory cytokine signaling pathways, FAN-ceramide and MAPKJNK, can depress the IGF-induced PI3-K pathway by interfering with IRS tyrosine phosphorylation or Akt activity. In contrast to the IRS/PI3-K/Akt pathway, it is important to note that TNFα-ERK1/2 and IGFERK1/2 are parallel pathways and thus are unlikely to have negative interactions. Evidence supporting proinflammatory cytokine-induced IGF resistance is presented below, using several distinct in vitro models.

A. Cell Proliferation: A Case for Cancer Therapy Cancer cells provide an excellent model to unravel the role of growth factors and pro-inflammatory cytokines during cell proliferation. Due to their transformation, many cancerous cells are immortal. Most of these cells grow rapidly, thus providing a robust proliferative signal and do not generally differentiate unless they are experimentally induced to do so. Many of these cells are responsive to both growth factors and pro-inflammatory cytokines despite their transformation. In fact, inhibition of IGF-1R activity and

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B. Proliferation; cytokine depressed IGF-induction of cell proliferation, [3H]-thymidine into DNA C Pro yt -i ok nf in lam e Si ma gn to al ry lin g

A.

C. Early signaling; cytokine depressed IGF activation of Akt

D. Cell cycle; cytokine suppressed IGF activation of Cdk2

FIGURE 5 Cytokines depress the proliferation of breast cancer cells by blocking the ability of IGF to enhance CDK activity. The effects of cytokines, IL-1β in this case, entails blockage of early IGF-1R events such as Akt phosphorylation, which blocks IGF-I–induced Cdk-2 enzymatic activity. (Shen et al., 2004a; Shen et al., 2002). Reprinted with permission.

administration of cytokines are two approaches that have been validated in animal models and have entered clinical trials to control some forms of cancer (Dranoff, 2004; Khandwala et al., 2000). IGF-1R activation drives cell cycle progression of several types of cancer cells. Recent studies indicate that components of cytokine receptor signaling pathways interact with an IGF-1R signaling pathway to reduce IGF activity and thereby reduce cell proliferation. 1. IGFs and Cancer The IGF system, particularly the IGF-1R, is closely associated with progression and metastasis of numerous human cancers, including those of the breast, prostate, lung, colon, and bladder (LeRoith and Helman, 2004). Many components of the IGF-activated PI3-K

pathway are constitutively active in cancer cells (Figure 5A). For example, the catalytic p110 domain is persistently activated in many transformed cells (Martin, 2003). Similar observations have been noted with Akt, although this may be due to PI3-K activation. These in vitro studies were confirmed with animal models showing that increased IGF-I promotes tumor number and growth (Baserga et al., 2003; LeRoith and Helman, 2004; Wu et al., 2003). The same appears true in humans. Patients with circulating IGF-I in the upper 25% of a normal range have an increased incidence of breast, bladder, colon, lung, and prostate cancer (LeRoith and Helman, 2004). Conversely, reduced IGF-I levels correlate with increased tumor latency and decreased growth and metastasis (Baserga et al., 2003; LeRoith and Helman, 2004). It should be noted that IGF activity does not cause transformation but instead enhances


the rate of transformation and/or the growth of transformed cells such that tumors appear earlier. 2. Cytokine-induced IGF Resistance during Cell Proliferation Numerous in vitro studies have confirmed that proinflammatory cytokines inhibit the growth of several types of cancer cells, including melanoma (LazarMolnar et al., 2000), leukemia (Mudipalli et al., 2001), rhabdomyosarcoma (Storz et al., 2000), breast (Rozen et al., 1998), salivary gland (Katz et al., 1999), sarcoma (Shalita-Chesner et al., 2001), and ovarian adenocarcinoma (Simonitsch and Krupitza, 1998). Unfortunately, parenteral administration of pro-inflammatory cytokines leads to development of the systemic inflammatory response syndrome (SIRS), which is often lethal. However, clinical trials attest to the fact that administration of cytokines directly into localized areas has substantial therapeutic benefit (Dranoff, 2004). What is intriguing is that inhibition of IGF activity induces much the same effects as the site-directed administration of cytokines. Until recently, no link has been drawn between these two approaches to treat cancer. One of the contributing factors is that the effect of cytokine treatment was believed to be direct, but most of the studies were done in the presence of serendipitous IGF. For example, most in vitro experiments with primary or transformed cancer cells use culture conditions that maintain cells in medium supplemented with FBS. Consequently, the inhibitory actions of cytokines observed in cancer models could be due to their blocking the IGF activity in the FBS (Figure 5A). Also, since many cancer cells synthesize IGF, the cytotoxic actions of cytokines could blunt the proliferative actions of endogenous IGF. In fact, several studies have reported that in cells grown in medium without serum, the cytostatic actions of pro-inflammatory cytokines were prominent only when the cells were treated with a growth factor (Shen et al., 2002; Shen et al., 2004a; Shen et al., 2004b). Indeed, TNFα, IL-1β, or IL-6 had no effect on cell proliferation when added to cells grown in serum-free conditions (Shen et al., 2002). However, cytokines inhibited proliferation of IGF-I treated cells (Figure 5B). This was one of the first indications that cytokines may suppress cancer cell growth by inhibiting IGF action. As discussed above, IGF binding results in IGF-1R autophosphorylation, recruitment of IRS docking proteins, and activation of downstream molecules. Cytokines alone do not affect MCF-7 cell proliferation, but they do depress the mitogenic effect of IGF (Figure 5B). Cytokines do not change the autophosphorylation state of the IGF-1R, but TNFα and IL-1β both inhibit IGF-induced activation of IRS-1 (Shen et al., 2002). The

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actions of cytokines on upstream IGF-1R signaling are reflected in decreased activation of downstream effector kinases. Thus, cytokines act by blocking IGF-1R induction of the PI3-K pathway including Akt activation (Figure 5C). After the major Akt branching point, cytokine action is reflected as a block in IGF-induced activation of the cell cycle machinery, Cdk2 activity, as assessed by reducing phosphorylation of its substrate, the retinoblastoma (Rb) tumor suppressor (Figure 5D). This occurs by IL-1β depression of PI3-K activity and subsequent lack of stimulus for the formation of Cdk2/ cyclin A complexes (Shen et al., 2002; Shen et al., 2004a). Consequently, one way that the pro-inflammatory cytokines act on breast cancer cells is by inhibiting cytoplasmic signaling components in the IGF cascade, which leads to cytostasis. This cytostatic effect is possible because the PI3-K pathway is limiting for the proliferation of MCF-7 breast cancer cells. However, this limitation is not true for all types of cells. For example, skeletal muscle myoblast proliferation is dependent upon IGF-induced MAPK activity. Apparently, the IGF-dependent MAPK pathway is limiting for myoblast proliferation. In these cells, proinflammatory cytokines, which activate various MAPK pathways, do not induce myoblast cytostasis but actually increase cell cycle progression (Stewart et al., 2004).

B. Differentiation and Hypertrophy: Myogenesis and Cachexia Skeletal muscle development provides an excellent model for the investigation of cellular differentiation and hypertrophy, especially in regard to interactions between cytokines and IGF. There are several wellcharacterized cell lines that faithfully mimic how these processes occur in primary cells. Of particular interest are murine C2C12 cells, which differentiate by cell cycle withdrawal and the upregulation of myogenic regulatory proteins, such as the transcription factor myogenin (Miller, 1990) and myosin, a functional skeletal muscle protein. C2C12 and other myogenic cells are sensitive to both IGF and pro-inflammatory cytokines. Using these cells, the interaction between cytokines and growth factors during differentiation and hypertrophy can be studied in a well-characterized and controlled in vitro paradigm. 1. IGFs and Muscle The anabolic effects of IGF drive differentiation and hypertrophy of skeletal muscle (Rommel et al., 2001) and overexpression of IGF within muscle enhance muscle formation and hypertrophy (Coleman et al., 1995; Musaro and Rosenthal, 1999). Several years ago

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it was shown that the major stimulus for myogenesis was indeed the IGFs (Florini et al., 1991). Using a variety of human, mouse, rat, or avian primary cultures or cell lines, the control of myogenesis (commitment to differentiation followed by fusion of mononucleated myoblasts into myofibers) and muscle hypertrophy (primarily via the synthesis and subsequent accumulation of protein) has been defined. Supporting very early work, it has been repeatedly confirmed that IGF increases myoblast differentiation into myotubes, an effect that requires expression of myogenic regulatory proteins such as myogenin. Subsequently, IGF increases hypertrophy and maturation by increasing synthesis of muscle-specific proteins, including myosin (Florini et al., 1991; Miller, 1990). Both differentiation and hypertrophy (Figure 6A) require IGF activation of the PI3-K pathway (Coolican et al., 1997; Rommel et al., 2001; Tureckova et al., 2001). In contrast, myoblast proliferation requires IGFinduced MAPK activity (Coolican et al., 1997). 2. Cytokines and Muscle Similar to cancer cells, myoblasts and myofibers respond not only to growth factors such as IGF, but they are responsive to a wide variety of proinflammatory cytokines, including TNFα and IL-1β. Pro-inflammatory cytokines markedly diminish both differentiation and hypertrophy of myoblasts (Langen et al., 2001; Li et al., 1998; Stewart et al., 2004; Szalay et al., 1997). Myoblasts differentiate better in low serum compared to serum-free conditions due to poorly defined factors in horse serum that are necessary for myotube formation. Therefore, most of the studies with pro-inflammatory cytokines were performed by switching cells from 10% FBS to 1–2% horse serum to induce differentiation. Until recently, it was believed that the inhibitory effects of cytokines would be attributed to specific signaling pathways that directly inhibit differentiation or hypertrophy. However, as shown in Figure 3, there are as yet no defined signaling pathways induced by cytokines that directly block these events. It is known that TNFα directly stimulates myoblast proliferation via its ability to activate ERK1/2 pathways (Stewart et al., 2004). This later action and mechanism are analogous to that of IGF, but only recently has the mechanism by which cytokines block differentiation and hypertrophy been defined. 3. Cytokine-induced IGF Resistance during Differentiation and Hypertrophy Similar to IGF-induced proliferation of certain cells, skeletal muscle differentiation and hypertrophy

(protein synthesis) are IGF-driven events, requiring activation of the PI3-K pathway. Differentiation and hypertrophy require a common branch of the IGFinduced Akt pathway involving p70S6K activation (Figure 6A). With IGF-1R activation being necessary for myogenesis and the fact that pro-inflammatory cytokines can often act in a fashion opposing IGF, there was probable cause to assume an interconnection between the two. We found that under serum-free conditions and in the absence of added IGF, neither TNFα or IL-1β affect myoblast differentiation or hypertrophy (Broussard et al., 2003; Broussard et al., 2004; Strle et al., 2004). For example, basal myogenin expression is not affected by IL-1β in C2C12 myoblasts, but IGF-I significantly increases myogenin expression (Figure 6B). An interaction was detected by showing that physiological levels of IL-1β, which do not affect basal myogenin expression, dose-dependently diminish the ability of IGF-I to increase myogenin. Similarly, IGF-I increases protein synthesis of C2C12 cells (Figure 6C), whereas TNFα neither stimulates nor depresses protein synthesis when added alone. However, TNFα potently depresses IGF-induced protein synthesis. Thus, both differentiation and hypertrophy are depressed by proinflammatory cytokines, and both effects are a result of diminished IGF activity. The effect of cytokines occurs very early in the IGF signaling cascade, as both IL-1β (Broussard et al., 2004) and TNFα (Figure 6D) depress IRS-1 and IRS-2 tyrosine phosphorylation induced by IGF-I. The inhibition caused by cytokines on both differentiation and protein synthesis requires ceramide synthesis (Strle et al., 2004). A ceramide analog completely mimics the inhibition caused by both TNFα and IL-1β (Figure 6E), as does exogenous sphingomyelinase (which generates ceramide). Blocking ceramide synthesis in C2C12 cells abrogates the ability of both TNFα and IL-1β to inhibit IGF activity. Consequently, one way that the pro-inflammatory cytokines act on muscle is by inhibiting cytoplasmic signaling components in the IGF cascade, with a resultant decrease in hypertrophy and a decrease in the differentiation of precursor cells. As previously mentioned, however, this limitation is not true for myoblast proliferation (Stewart et al., 2004). Thus, cellular differentiation, not proliferation, is inhibited because IGF-induction of the PI3K pathway is limiting for only one of the two processes. This clearly illustrates that cytokines can be used to block only a portion of the IGF signaling cascade. As mentioned earlier, extracellular tools that depress IGF activity, such as the soluble IGFBPs, lack specificity. They block all IGF effects. Similarly, recent pharmaceuticals that block IGF-1R kinase activity such as


A.

C Pro yt -i ok nf in lam e S i ma gn to al r y lin g

B. Differentiation; IL-1β depresses IGF-induction of myogenesis, as assessed by myogenin expression

C. Hypertrophy; TNFα depresses IGF-induction of protein synthesis in cultured C2C12 muscle cells

D. Early Signaling; TNFα depresses IGF-induction of IRS phosphorylation/activation.

E. Early Signaling; A ceramide analog (Cer) mimics TNFα blockage of IGF→IRS-1

FIGURE 6 Cytokines depress hypertrophy and differentiation of muscle by blocking IGFinduced protein synthesis and myogenin expression. The effects of either TNFα or IL-1β entails blockage of early IGF-1R events such as IRS-1 phosphorylation, which diminishes downstream signaling effects. Ceramide, a signaling intermediate, mimics the cytokine inhibition, and preventing ceramide synthesis can block cytokine activity (Broussard et al., 2003; Broussard et al., 2004; Strle et al., 2004). Reprinted with permission.

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tyrphostins or cyclolignans block all IGF-1R activity (Girnita et al., 2004; LeRoith and Helman, 2004; Levitzki and Gazit, 1995); because they block the first step in IGF signaling. In contrast, cytokines clearly block some but not all of the effects of IGF. A careful dissection of cytokine actions and mechanisms will continue to provide important new insights into the regulation of specific actions of IGF and thus allow tailoring of IGF resistance toward a specific goal.

C. Cell Survival: Lessons from Neurons It has long been established that IGF protects neurons against a variety of excitotoxic/ischemic insults (Cheng and Mattson, 1992; Mattson and Cheng, 1993), reviewed in (Carro et al., 2003; Dore et al., 2000). Considerable evidence supporting the survival properties of IGF justifies the potential use of IGF-I as a treatment for a variety of neurological diseases (Leinninger and Feldman, 2005; Wilczak and de Keyser, 2005). Indeed, overexpression of IGF-I in the cerebellum prevents neuronal death in Weaver mice, and in doing so, prevents ataxia (Zhong et al., 2005), probably by activation of the PI3-K pathway. Unfortunately, genetic models to study IGF depletion in vivo are not available because mice with a null mutation in the IGF-1R die at birth (Liu et al., 1993), and mice with a mutation to IGF-I die perinatally (Powell-Braxton et al., 1993). Hence, most of the mechanistic studies regarding IGF activity on neurons necessitate the use of in vitro models. A general theme has emerged from IGF studies, which is that IGF saves neurons via the PI3-K pathway. A few selective manuscripts indicate that IGF saves cerebellar granule neurons from depolarization (Subramaniam et al., 2005; Wiedmann et al., 2005) and hippocampal neurons from either hypoglycemic damage (Cheng and Mattson, 1992) or growth factor deprivation (Zheng et al., 2002). IGF also saves cortical neurons from corticosterone-induced death (Nitta et al., 2004). All of these actions are apparently mediated via the PI3-K pathway. IGF also is neuroprotective in models of ischemic damage and several other types of death, including dopamine-induced neurotoxicity [a model of Parkinson’s disease (Offen et al., 2001)], N-methylD-aspartate toxicity of cortical neurons (Takadera et al., 1999), and development of sensitivity to kainate neurotoxicity (Leski et al., 2000). IGF-I is a strong activator of the PI3-K pathway in hippocampal neurons, and inhibition of IGF activity in vivo leads to neuronal degeneration (Zheng and Quirion, 2004; Zhong et al., 2002). These data underlie the critical need of the PI3-K signaling cascade in neurons. IGF-I and the phosphatase inhibitor, sodium orthovanadate, appear to exert their neuroprotective

activities in vivo against ischemic damage by enhancing the PI3-K pathway (Fukunaga and Kawano, 2003). This makes the PI3-K downstream protein, Akt, a potential molecular target for ischemic therapy. The PI3-K pathway is also involved in IGF actions during neuronal differentiation. As examples, IGF-I induces the expression of glutamate receptors on neurons (Zona et al., 1995) and potentiates expression of neuronal L calcium channels (Bence-Hanulec et al., 2000; Blair et al., 1999). IGF is necessary for the induction of glutamate sensitivity of developing neurons (Calissano et al., 1993; Gonzalez de la Vega et al., 2001) and enhances field excitatory postsynaptic potentials (Ramsey et al., 2005). With this in mind, IGF-I infusion into old rats increases complexity of synapses within the hippocampus (Shi et al., 2005) and diminishes the age-associated loss of neurogenesis within the hippocampus (Lichtenwalner et al., 2001). All of these findings imply a cognitive benefit associated with IGF signaling in neurons, primarily through activation of the PI3-K pathway. They also suggest that diminished signaling through this pathway will markedly affect neuron activity, including those associated with behavior and learning. Considering this and the ability of cytokines such as TNFα and IL-1β to block IGF-induction of the PI3-K pathway, it was logical to assume that cytokine effects on behavior and cognition may involve antagonizing the positive effects of IGF.

VI. CYTOKINE—IGF INTERACTIONS; IN VIVO—BEHAVIOR AND LEARNING A. IGF Reduces Sickness Behavior Critical to the development of therapeutic approaches to increase quality of life is defining the role of proinflammatory cytokines in the induction of sickness behavior [reviewed in (Kelley et al., 2003; Parnet et al., 2002)]. Several key experiments highlighted in these reviews indicate that induction of cytokine expression within the brain causes a myriad of symptoms collectively defined as sickness behavior. The symptoms include decreased activity, decreased social investigation, anhedonia, general malaise, fatigue, depressed appetite, and changes in sleep pattern. It is now well accepted that pro-inflammatory cytokines act within the central nervous system to modulate behavior, albeit with ill-defined targets. However, with the antagonist relationship between cytokines and IGF described above, a natural extension of these experiments was to determine if IGF would counteract the sickness-inducing properties of LPS. In these experiments, LPS or bovine serum albumin was injected i.c.v. into mice, which was immediately followed by


A. Behavior; IGF-l reduces TNFαdepressed social exploration of a novel juvenile.

TNFa TNFa

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B. Immobility; IGF-l prevents TNFαinduced immobility.

TNFa TNFa

FIGURE 7 TNFα-induced sickness behavior is antagonized by IGF-I. TNFα, when administered i.c.v., depresses social exploration of a novel juvenile and induces immobility, two well-characterized indices of sickness behavior. Central administration of IGF-I antagonizes the effect of TNFα (Bluthé et al., 2006). Reprinted with permission.

i.c.v. administration of IGF-I (100 ng or 1000 ng). There was no effect of either dose of IGF-I following administration of albumin. As expected, LPS reduced exploration of a novel object, suppressed rearing behavior and increased immobility. In all these cases, the higher dose of IGF-I, but not the lower one, significantly inhibited development of all of these sickness behaviors (Dantzer et al., 1999). Although unproven, the beneficial effect of IGF-I on sickness behavior may be associated with increased glucose utilization throughout the brain (Cheng et al., 2000). This increase in glucose utilization is likely to be a consequence of IGF increasing tyrosine phosphorylation of the critical IRS docking proteins and PI3-K in both neurons and glia following activation of the IGF-1R. The beneficial effects of IGF-I on LPS-induced sickness behavior were interpreted to suggest that IGF-I either reduces the production of cytokines in the brain that are synthesized in response to LPS or that IGF-I impairs the action of these LPS-induced central cytokines (Dantzer et al., 1999). In order to test the possibility that IGF-I interferes with the action of proinflammatory cytokines, mice were injected i.c.v. with either recombinant TNFα or IL-1β. The doses of TNFα (50 ng) and IL-1β (2 ng) were carefully selected in order to induce equivalent degrees of sickness behavior. In the absence of cytokine, IGF-I (100 ng) did not affect behavior, as assessed by investigation of a conspecific juvenile (Figure 7A) or duration of immobility (Figure 7B). As expected, TNFα impaired both these behavioral readouts. However, when mice were pre-

treated i.c.v. with IGF-I, changes in both social investigation and immobility in response to TNFα were ameliorated. Interestingly, this dose of IGF-I did not impair development of sickness behavior induced by i.c.v. injection of a similar sickness-inducing dose of IL-1β. However, a higher dose of IGF-I (1000 ng) significantly attenuated IL-1β-induced sickness behavior. These data extend earlier results in which IGF-I inhibited LPS-induced reduction in several sickness indices, including investigation, rearing, and freezing (Dantzer et al., 1999). These findings offer strong evidence in a whole animal model of the antagonism that exists between pro-inflammatory cytokines and IGF-I by showing that enhanced IGF-1R activation is able to overcome the probable TNFα-induced decrease in IGF signaling in vivo within the brain. Interestingly, IGF-I is more potent in ameliorating TNFα- than IL-1β-induced sickness behavior. Since TNFα-induced IL-1β synthesis is responsible for inducing most symptoms of sickness behavior (Bluthé et al., 1991), at least part of the IGF effect may not be caused by a direct effect on neurons but may involve an impairment in the ability of TNFα to induce central synthesis of IL-1β.

B. Spatial Learning Deficits Are Prevented by IGF Low levels of IGF-I and elevated pro-inflammatory cytokines have been linked to progressive inflammatory neuropathologic disorders, including Alzheimer’s

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disease (Dik et al., 2003; Wilson et al., 2002a). New results indicate that IGF-I protects against the development of amyloid plaques (Carro & Torres-Aleman, 2004; Carro et al., 2002; Dik et al., 2003; Wilson et al., 2002b). Consistent with data in other physiological systems, this neuroprotective effect of IGF-I is antagonized by TNFα (Carro et al., 2002). In this study, TNFα blocked the ability of IGF-I to promote transport of amyloid across the blood-brain barrier. Although the potential role of IGF-I in improving cognitive functions is in its infancy, a review of the literature in 2000 (van Dam et al., 2000) noted the very strong positive relationship between plasma IGF-I and a variety of cognitive functions, particularly in the elderly. Since that time, it has been reported that humans with serum IGF-I < 70 ng/ml have lower information processing speed (Dik et al., 2003). This correlative study is supported by recent cause-and-effect studies with growth hormone–deficient adult humans who suffer from a variety of psychological disturbances (Stouthart et al., 2003). Growth hormone replacement therapy, which causes an increase in circulating IGF-I, reduces anxiety and improves quality of life in these patients. Elevated serum IGF-I is positively related to memory and negatively related to depression, tension, and anxiety. Serum IGF-I is well known to be elevated by moderate exercise, an effect that has now been extended to exercise-induced increases in brain IGF-I and protection of the brain against a variety of insults (Carro et al., 2001). Although natural methods that are associated with healthy lifestyles can increase brain IGF-I, the roles of IGF in regulating both sickness behavior and cognitive events have not been fully defined. More importantly, the intracellular protective mechanisms that are mediated by the IGF-1R must be refined. Antagonizing IGF-II action in the brains of young animals impairs both learning and memory (Lupien et al., 2003) suggesting that other treatments that alter IGF activity, for instance, cytokines, would also result in impaired learning and memory. An in vivo model, kainate (KA, a neurotoxic glutamate receptor agonist)induced deficits in spatial memory, show that the deficits are blocked by an inhibitor of TNFα synthesis (Bluthé et al., 2005). KA-induced spatial memory deficits are also inhibited by i.c.v. administration of IGF-I. Coupled with other data showing that KA induces synthesis of TNFα in the brain (de Bock et al., 1996; Dinkel et al., 2003; Minami et al., 1991) by microglia (Noda et al., 2000), this new work begins to define KA as an inflammatory stimulus. Consistent with this effect, IGF-I promotes neuronal survival following exposure of neurons to glutamate or cytokines (Kenchappa et al., 2004; Vincent et al., 2004; Yadav et

al., 2005). However, unlike cytokine-induced sickness behavior, KA does not diminish general activity of mice when assessed 2 weeks after KA injection (Bluthé et al., 2005). Instead, at a dose of KA that is not neurotoxic (15 mg), KA causes deficits in spatial memory. The cognitive effect of KA, without gross behavioral effects, is due primarily to the localized effect of KA, which is largely limited to the hippocampus (Jarrard, 2002). The hippocampus is directly involved in spatial memory but not necessarily involved in all aspects of sickness behavior. KA induces pro-inflammatory cytokine production within the hippocampus (de Bock et al., 1996; Lehtimaki et al., 2003; Minami et al., 1991; Vezzani et al., 2002) and at higher doses results in neuronal death (Jarrard, 2002). However, under controlled conditions, KA causes spatial memory deficits without concurrent neuronal loss, as assessed by Fluorojade B staining (Bluthé et al., 2005). The cognitive effects of KA are blocked by an inhibitor of TNFα synthesis, pentoxifylline, (Figure 8A) as well as by chronic (5 days), but not acute (1 day) i.c.v. administration of IGF-I (Figure 8B). These findings further support an antagonistic relationship between pro-inflammatory cytokines and IGF within the brain. Although the mechanisms responsible for the CNS effects are ill defined, they are consistent with the idea that pro-inflammatory cytokines reduce IGF activity, causing IGF resistance. This relationship is strengthened by the ability of IGF administration to ameliorate both inflammation and cytokine-induced sickness behavior and cytokine-dependent spatial memory deficits. It is possible that sickness behaviors induced by TNFα can be overcome by maintaining higher levels of IGF in the brain, such as might be induced by exercise. Experimental data, coupled with clinical correlative studies, provide strong evidence for a link between IGF activity, behavior, and cognition in otherwise healthy subjects. The experimental animal trials clearly indicate that sickness behavior and at least some cognitive deficits are mediated by proinflammatory cytokines within the brain. Many of the clinical and experimental changes can be abrogated by treatments aimed at enhancing IGF activity, including GH injection, exercise, or direct i.c.v. IGF injection. Critical to preventing sickness behavior and cognitive deficits is a clear understanding of the mechanisms by which pro-inflammatory cytokines elicit their negative actions within the brain. Based upon these data, investigations on how pro-inflammatory cytokines cause adverse effects in the CNS should include studies directed at understanding the ability of cytokines to interact with the IGFs and possibly other growth factors, such as brain-derived neurotrophic factor.


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A. KA inhibits spatial memory. PTX, B. KA administration depresses which blocks TNFa synthesis, prevents hippocampal-dependent spatial memory. KA-induced spatial memory loss. IGF-l inhibits this TNFα-dependent action.

FIGURE 8 The spatial memory deficit caused by KA requires TNFα and is antagonized by IGF-I. Blocking TNFα secretion with PTX (A) or i.c.v. treatment with IGF-I both prevent the cognitive deficits associated with KA (Bluthé et al., 2005). Reprinted with permission.

VII. CONCLUSIONS Hormone and immune interactions have been classically defined by experiments using synthetic glucocorticoids and the stress- or inflammation-induced release of adrenal glucocorticoids. These hormones have long been known to have a variety of effects on nearly all cells of the immune system. The overall theme of this chapter is that the GH/IGF axis, which controls over 80% of postnatal growth, represents another important example of how the endocrine system interacts with the immune system. A number of examples have been used not only to demonstrate the existence of the interaction between pro-inflammatory cytokines and IGF, but also to provide insights into the mechanism by which this occurs. These data make it clear that advances at any one level of understanding in either the immune or endocrine systems will not be fully understood until there is a more complete understanding of the crosstalk that occurs between these two systems.

Acknowledgment This research was supported by grants from National Institutes of Health to Keith W. Kelley (AI50442 and MH51569) and Robert Dantzer (MH071349).

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genetically induced mammary tumors. Cancer Res., 63, 4384–4388. Yadav, A., Kalita, A., and Banerjee, K. (2005). JAK/STAT3 pathway is involved in survival of neurons in response to insulin like growth factor and negatively regulated by suppression of cytokine signaling-3. J. Biol. Chem., 36, 31830–31840. Yang, J., Anzo, M., and Cohen, P. (2005). Control of aging and longevity by IGF-I signaling. Exp. Gerontol., 40, 867–872. Zheng, W. H., Kar, S., and Quirion, R. (2002). Insulin-like growth factor-1–induced phosphorylation of transcription factor FKHRL1 is mediated by phosphatidylinositol 3-kinase/Akt kinase and role of this pathway in insulin-like growth factor-1– induced survival of cultured hippocampal neurons. Mol. Pharmacol., 62, 225–233. Zheng, W. H., and Quirion, R. (2004). Comparative signaling pathways of insulin-like growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival. J. Neurochem., 89, 844–852. Zhong, J., Deng, J., Ghetti, B., and Lee, W. H. (2002). Inhibition of insulin-like growth factor I activity contributes to the premature apoptosis of cerebellar granule neuron in Weaver mutant mice: in vitro analysis. J. Neurosci. Res., 70, 36–45. Zhong, J., Deng, J., Phan, J., Dlouhy, S., Wu, H., Yao, W., Ye, P., D’Ercole, A. J., and Lee, W. H. (2005). Insulin-like growth factor-I protects granule neurons from apoptosis and improves ataxia in Weaver mice. J. Neurosci. Res., 80, 481–490. Zona, C., Ciotti, M. T., and Calissano, P. (1995). Human recombinant IGF-I induces the functional expression of AMPA/kainate receptors in cerebellar granule cells. Neurosci. Lett., 186, 75–78.


C H A P T E R

8 The Neuroendocrine System and Rheumatoid Arthritis: Focus on the Hypothalamic-Pituitary-Adrenal Axis HEATHER E. GORBY AND ESTHER M. STERNBERG

I. INTRODUCTION 193 II. GENERAL CONCEPTS OF NEURAL-IMMUNE INTERACTIONS 194 III. ANIMAL MODELS OF RHEUMATOID ARTHRITIS 196 IV. HPA AXIS FUNCTION FINDINGS IN RHEUMATOID ARTHRITIS PATIENTS 197 V. OTHER NEUROENDOCRINE FACTORS IMPLICATED IN RA PATHOGENESIS 199 VI. SUMMARY 201

lage and bone. Persistent synovial inflammation, chronic pain, joint damage, and progressive disability occur over the course of RA. RA etiology remains unclear. RA is frequently accompanied by a variety of other known autoimmune or autoimmune-associated disorders such as systemic lupus erythematosus and Sjogren’s syndrome that are characterized by autoantibody production, suggesting a generalized autoimmune susceptibility in affected individuals. Indeed, over 20 different regions on 15 different chromosomes, many of which are immunerelated genes, have been linked to inflammatory arthritis in animal studies (Wilder et al., 1999), indicating a multi-genic, polygenic component to this syndrome. Furthermore, the cytokines TNF-α and interleukin-1 (IL-1) are elevated in patients with RA, suggesting that they have a pathological role in the disease (Chikanza et al., 1995; Saxne et al., 1988). Animal studies and human clinical trials using pharmacological agents that block TNF show clinical improvement in RA and decreased rate of progression of erosions (Barrera et al., 1996; Maini et al., 2004; Schiff, 2004; Thorbecke et al., 1992; Williams et al., 1992). There is also abundant evidence that the nervous system and endocrine systems are involved in the pathogenesis of RA (Harle et al., 2005). The interaction of the neural, endocrine, and immune systems in RA will be the focus of this chapter.

I. INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory disease effecting an estimated 0.5–1.1% of the North American population (Alamanos and Drosos, 2005). The age of onset is usually between 20 and 40 years of age with a female:male ratio of 3 : 1. There is a 12–20% concordance rate in monozygotic twins, and a 4–5% concordance rate in dizygotic twins, suggesting that there may be a genetic component in RA predisposition (MacGregor et al., 2000; Silman et al., 1993). The first stage involves the classical signs of inflammation in the synovial lining, including redness, swelling, warmth, pain, and stiffness. Late-stage disease is characterized by a release of cytokines and enzymes in the extracellular matrix that leads to degradation of cartiPSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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II. GENERAL CONCEPTS OF NEURAL-IMMUNE INTERACTIONS A. Interactions between the Immune and Neuroendocrine Systems: Bi-directional Communication Pathways 1. Peripheral Immune System Signaling to the Central Nervous System Peripheral inflammation results in the production of a variety of inflammatory mediators including cytokines, which can signal the brain via several routes. Peripherally released cytokines modulate brain function and can produce fever and sickness behavior (loss of appetite, decreased locomotion, loss of interest in sex and social interactions, and depressed mood). Cytokines produced in the periphery can cross the blood-brain barrier in small amounts at leaky points (the circumventricular organs) or via specific active transport mechanisms (Banks, 2005). Cytokines can also signal the CNS by stimulation of the vagus nerve or through activation of second messengers such as nitric oxide or prostaglandins. In addition, cytokines can also be produced in the brain by glia, neurons, or macrophages and can influence neuronal cell death or survival. 2. CNS Stress Response Effects on the Immune Response: HPA Axis, Sympathetic, Parasympathetic, and Peripheral Nervous System Regulation of Immunity The nervous system regulates the immune system systemically through hormonal routes, regionally in immune organs, and locally at sites of inflammation (Webster et al., 2002b). Endogenous glucocorticoids (GCs) are essential physiological regulators of the immune response and inflammation. Exogenous GCs have been used for several decades as potent suppressors of overactive immune responses. Extensive research has demonstrated the powerful pharmacological and physiological effects of GCs on multiple aspects of immune cell function [reviewed in (Webster et al., 2002b)]. Pharmacological doses of GCs suppress the immune response, including innate immune, cellular, and humoral acquired immune responses. More recently it has been suggested that physiological levels of GCs are immunomodulatory and result in a shift of cytokine production from primarily pro-inflammatory to a primarily anti-inflammatory pattern. Such patterns of cytokine production can be categorized as TH1 or TH2, and roughly correspond to cellular or humoral patterns of immune responses, respectively. A TH1 pattern of cytokines is characterized by production of

largely pro-inflammatory IL-12, with a primarily cellular immune response. A TH2 pattern of immunity is characterized by production of IL-4 and IL-10, and is associated with a primarily humoral or antibody response. At physiological concentrations, GCs inhibit TH1 and enhance TH2 cytokine production (Elenkov and Chrousos, 1999). Thus, immune suppression by GCs provides an important mechanism to prevent the immune response from continuing unchecked. Activation of the stress response also results in increased neural output to peripheral tissues. The sympathetic, parasympathetic, and peripheral nervous systems play important roles in regulating the immune response. The sympathetic and parasympathetic nervous systems modulate inflammatory responses through regional innervation of immune organs including the spleen, thymus, and lymph nodes (Bellinger et al., 1991). In animal studies, sympathetic denervation can have different effects on regional inflammation depending on the site and mode of denervation (Fleshner and Laudenslager, 2004; Madden et al., 1994a; Madden et al., 1994b; Sanders and Kohm, 2002). Natural sympathetic denervation of the spleen that occurs with aging may play a role in some of the immunosuppression seen in aging (Madden et al., 1998). Sympathetic postganglionic neurons are also key in regulating the fluid extravasation component of the early inflammatory response (Green et al., 1998). The parasympathetic nervous system modulates immune responses at a regional level though the efferent fibers of the vagus nerve. The afferent fibers of the vagus nerve also signal the presence of peripheral inflammation to the brain. Thus, during inflammation in the peritoneum, cytokines such as IL-1 bind to IL-1 receptors in paraganglia cells located in the peritoneum, rapidly inducing early gene expression and increasing activity in vagal brain stem centers (Watkins and Maier, 1999). The main parasympathetic neurotransmitter is acetylcholine (ACh), which binds to two general cholinergic receptor sub-types: nicotinic and muscarinic cholinergic receptors. Enhanced parasympathetic tone results in increased ACh, which suppresses production of pro-inflammatory cytokines (Tracey, 2002). Activation of the parasympathetic cholinergic response and its subsequent suppression of inflammation through the vagus nerve has been termed the “cholinergic inflammatory reflex” (Tracey, 2002). Interruption of this important route by cutting the vagus nerve results in exacerbation of inflammation and shock (Tracey, 2002). At sites of peripheral inflammation, peripheral nerves regulate local inflammatory responses through release of neuropeptides that are largely pro-

8. The Neuroendocrine System and Rheumatoid Arthritis: Focus on the Hypothalamic-Pituitary-Adrenal Axis

inflammatory. The peripheral nervous system affects inflammation through neuropeptides, such as substance P (SP) or vasoactive intestinal polypeptide (VIP), a 29-amino acid peptide released from peripheral sensory neurons. These may be released directly from nerve endings and synapses at sites of inflammation, or they can be synthesized and released by immune cells depending on the type of inflammation involved. Peripheral neuropeptides may be pro- or anti-inflammatory. Thus, while SP is primarily pro-inflammatory, VIP is predominantly anti-inflammatory, inhibiting the production of proinflammatory cytokines (Pozo et al., 2000). As would be expected, VIP administration in animal models leads to clinical improvement and suppression of pro-inflammatory cytokine production from T-cells (Delgado et al., 2001; Foey et al., 2003). Studies have also shown that both peripheral sympathetic noradrenergic (NA) nerves and SP-containing nerves play a role in joint inflammation (Keeble and Brain, 2004). 3. Defining Stress and the Stress Response A wide range of physical, psychological, or immune/ inflammatory stimuli, including cytokines, can activate a series of neuroendocrine and neuronal responses, which produce a characteristic series of physiological responses that together comprise the physiological stress response [for review, see (Goldstein and McEwen, 2002)]. These include activation of the hypothalamic-pituitary-adrenal (HPA) axis and of the sympathetic nervous system. Activation of hypothalamic CRH stimulates the stress response, including secretion of ACTH from the pituitary and glucocorticoid secretion from the adrenal gland. Activation of central corticotrophin-releasing hormone (CRH) and the locus ceruleus produce behavioral effects of increased arousal and alertness. Sympathetic nervous system activation leads to increased heart rate, increased muscle blood flow, and sweating. Together these early behavioral and physiological responses comprise the stress or “fight or flight” response. The bi-directional communication between the immune system and the central nervous system (CNS), in which cytokines signal the brain and the brain responds by regulating the immune system in part through the anti-inflammatory effects of GCs released from the adrenal glands, constitutes the main hormonal negative feedback loop for CNS regulation of immunity (Figure 1) (Webster et al., 2002b). In addition to their role in regulating the immune system, GCs also downregulate the HPA axis itself, and are essential for the maintenance of several homeostatic mecha-

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nisms in the body, including the CNS, cardiovascular system, and metabolic homeostasis. A blunted HPA axis is associated with a wide range of autoimmune/inflammatory diseases across species (chickens, mice, rats, humans); and interruptions of the HPA axis in otherwise inflammatory-resistant hosts, through surgical interventions (adrenalectomy, hypophysectomy) or through pharmacological intervention with glucocorticoid antagonists (e.g., RU 486), renders otherwise inflammatory-resistant hosts highly susceptible to inflammation and increased mortality from septic shock [for a more detailed review, see (Webster and Sternberg, 2004)]. Stressful stimuli also activate brain-stem adrenergic pathways and the sympathetic nervous system outflow from the brainstem to the periphery. Several neuronal pathways project from the hypothalamus to noradrenergic areas in the brain stem, such as the locus ceruleus and A2 and C2 regions (Sawchenko et al., 1996), and thus activation of hypothalamic CRH can also stimulate these noradrenergic regions and sympathetic outflow to the periphery. In turn adrenergic outflow from the brain stem can stimulate the hypothalamus to secrete CRH (Figure 1). 4. Relationship between the Nature of the Stressor and the HPA Axis Response: Psychological versus Physical Stress, Acute versus Chronic Stress The components of the stress response that are activated vary with the type of stimulus to which the organism is exposed. Stressors can be categorized as being primarily physical in nature (illness, inflammation, exercise) or psychological (i.e., performance tasks). Different stressors yield different patterns of activation of brain stress response regions. The response to psychological stressors appears to be particularly dependent on the individual’s critical perception of the event (Ehlert and Straub, 1998). Additionally, even within the category of primarily physical stressors, the HPA axis response differs depending on the type of stressor. Thus, intermittent electrical foot shocks in rats activate different stress-responsive neurotransmitter regions and pathways within the brain as compared to a systemic cytokine (IL-1) challenge (Li et al., 1996). With regards to inflammatory stressors, the nature of the immune challenge also determines the degree of the HPA axis response (Nagano et al., 1999). The duration of the stressful stimulus is also an important qualitative and quantitative determinant of the HPA axis response. It is critical to consider the neuroendocrine consequences of any long-standing peripheral immune activation process when studying

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FIGURE 1 Schematic illustration of neural immune connections, including immune signaling of central nervous system via systemic routes and the vagus nerve (Vagus n.) and CNS regulation of immunity via the hypothalamicpituitary-adrenal (HPA) axis, sympathetic nervous system (SNS), parasympathetic nervous system, and peripheral nervous system (PNS). Asterisks within the brain represent cytokine expression within the CNS. Dotted lines represent negative regulatory pathways; solid lines represent positive regulatory pathways. Reprinted with permission from (Marques-Deak et al., 2005) (www.nature.com/mp). CRH, corticotrophin releasing hormone; AVP, arginine vasopressin; ACTH, adrenocorticotrophin hormone, SNS, sympathetic nervous system; PNS, peripheral nervous system; LC, locus ceruleus; A1, C1, A2, C2, brainstem adrenergic nucleii.

the HPA axis in chronic inflammatory conditions such as RA or erosive arthritis in animal models. Chronic stress stimulation results in readjustments of the neurotransmitter and neuroendocrine responses that are activated during acute stress. These physiological changes complicate data interpretation and make it difficult to establish causal relationships between HPA axis hyporeactivity and predisposition to inflammatory disease in the context of already established chronic inflammation. Thus, any observed differences between unaffected and diseased subjects may be a reflection of the illness.

Although not consistent across all rodent species, during chronic stress the control of ACTH secretion may shift from CRH to arginine vasopressin (AVP) in rats (Harbuz et al., 1997). This shift may be related to differential susceptibility of CRH and AVP secretion to negative feedback by corticosterone (Makino et al., 1995). CRH and AVP have synergistic actions as ACTH secretagogues; AVP significantly potentiates the stimulatory action of CRH on ACTH production. Chronic inflammatory conditions in humans are also associated with this shift in ACTH control from CRH to AVP. Thus, patients with multiple sclerosis and significant baseline hypercortisolemia have a blunted ACTH response to AVP with normal ACTH response to CRH. Of note, subjects in this study did not exhibit evidence of increased peripheral inflammation, such as elevated plasma sedimentation rates, or elevated IL-6, or IL-1β levels (Michelson et al., 1994). The neuroendocrine response pattern changes seen in chronic stress may represent central adaptation to a continuous exposure to the stimulus. It is also possible that peripheral immune signaling to and the activation of the CNS evolve as patterns of cytokine production change over the course of chronic inflammation. Certainly, patterns of cytokine production differ in peripheral blood mononuclear cells from patients with early versus chronic RA (Butrimiene et al., 2004; Kanik et al., 1998). In addition to a shift in the stress hormonal response pattern, there is a dampening of the stress response to acute stimulation in animals with chronic inflammation, which varies with the type of acute stressor studied (Aguilera et al., 1997; Shanks et al., 1998). Hypothalamic CRH mRNA expression changes in rats developing adjuvant arthritis (Aguilera et al., 1997). When the nature of the acute stressor is inflammatory, the blunted response is not observed (Harbuz et al., 1997). A pre-existing stressor may dampen the inflammatory response to an immune challenge. Thus, repeated psychological stress is associated with a decreased severity in collagen-induced arthritis in female Lewis (LEW/N) rats (Miller et al., 1995).

III. ANIMAL MODELS OF RHEUMATOID ARTHRITIS A. Animal Models of Neuroendocrine Responses and Resistance and Susceptibility to Chronic Erosive Arthritis Multiple animal models have been developed to study RA, which partially but incompletely mimic the human situation. RA is a multifactorial disease related


to an interaction of genetic susceptibility, infection, and environmental factors. Multiple antigenic and pro-inflammatory triggers have been used to induce RA in rats and mice, including adjuvant and streptococcal cell walls. The most widely used of these is collagen-induced arthritis (Holmdahl et al., 2002). Transgenic mouse models, such as TNFα transgenic mice, have also been developed (Campbell et al., 2001). Evidence from inbred rat strains indicate that a blunted HPA axis plays a role in autoimmune disorders including inflammatory arthritis. Lewis (LEW/N) rats with a hyporesponsive HPA axis are susceptible to autoimmune/inflammatory disease, while Fischer (F344/N) rats, which exhibit a hyperresponsive HPA axis, are relatively resistant to inflammation (Sternberg et al., 1989a; Sternberg et al., 1989b). Interruption of the HPA axis by a variety of methods including RU486, a GR antagonist, renders F344/N rats susceptible to autoimmune/inflammatory diseases (Sternberg et al., 1989a), and conversely, in LEW/N rats, reconstitution of the HPA axis by intracerebroventricular fetal hypothalamic tissue transplantation reverses their susceptibility to autoimmune disease (Misiewicz et al., 1997). Removal of endogenous GCs, through chemical or surgical adrenalectomy, results in increased severity of inflammation and decreased survival (Harbuz et al., 1993) in other animal models of RA, such as adjuvant arthritis, as well as in other autoimmune disease models (Mason et al., 1990). These animal models of RA have been useful in testing neuroendocrine agents as therapies for RA. Treatment with a non-peptide CRH antagonist diminishes synovitis and clinical joint scores in inflammatory arthritis in LEW/N rats, but has no effect on F344/N rats (Webster et al., 2002a). These results suggest that multiple endocrine pathways play a role in rheumatoid arthritis and that peripheral blockade of the pro-inflammatory effects of CRH may play a primary role in the beneficial effects of this agent. Estrogen also plays a role in RA development, and manipulations of the HPA axis have been used in animal models of RA. Ovariectomy in Sprague-Dawley rats increases the susceptibility to collagen-induced arthritis, as well as the severity of erosions and bone loss (Yamasaki et al., 2001), while estrogen replacement reversed this effect. Tamoxifen, an estrogen receptor antagonist, is anti-inflammatory in the carrageenan inflammation in LEW/N rats (Misiewicz et al., 1996), and inhibits receptor activation of NF-κB in human RA (Mitani et al., 2005), suggesting that further study of this category of agents may yield additional anti-inflammatory treatments for RA.

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IV. HPA AXIS FUNCTION FINDINGS IN RHEUMATOID ARTHRITIS PATIENTS As in animal models, accumulating evidence from human studies shows that neuroendocrine abnormalities exist in RA patients, suggesting a link between the neuroendocrine system and the development of human autoimmune disease.

A. HPA Axis Studies in Human Rheumatoid Arthritis Various methods have been used to assess HPA axis function in patients with RA, including plasma ACTH and cortisol levels measured at regular intervals over the circadian rhythm, and after CRH stimulation. 1. HPA Axis Activation Patterns in RA The anti-inflammatory effects of the glucocorticoids (GCs) have been known since the 1940s, and the Nobel Prize was awarded for their use in the treatment of RA in 1950 (Hench et al., 1949). However, it was not until the late 1980s that the important physiological role of glucocorticoids in regulating immunity and in pathogenesis of a susceptibility to inflammatory arthritis was recognized. Overall, RA patients exhibit a blunted HPA axis response (Chikanza et al., 1992; Gudbjornsson et al., 1996; Hall et al., 1994b; Kanik et al., 2000; Neeck et al., 1990; Straub et al., 2002b) and a shift in the circadian rhythm of cortisol production, with cortisol maximum and minimum shifted to earlier times in the day (Neeck et al., 1990). Some RA patients are reported to have increased ACTH, but normal cortisol levels compared to controls (Gudbjornsson et al., 1996; Hall et al., 1994b; Kanik et al., 2000), indicating a relative adrenal insensitivity to ACTH stimulation. RA patients also show disproportionately lower levels of cortisol in response to the pro-inflammatory cytokines IL-6 and TNF-α (Straub et al., 2002b). This suggests an under-responsive HPA axis to increased inflammatory drive. Generally, inappropriately low cortisol secretion is a typical feature of RA patients. It is unknown whether the HPA axis abnormalities in RA patients are merely a consequence of the disease or a predisposing factor. Manipulations of the HPA axis can also effect susceptibility to RA. RA activity is exacerbated by the inhibition of glucocorticoid synthesis with the 11-β hydroxylase inhibitor metyrapone (Panayi, 1992; Saldanha et al., 1986). Patients with Cushing’s disease (characterized by hyperproduction of cortisol) who undergo adrenalectomy, and therefore removal of endogenous GCs, show enhanced susceptibility to RA

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(Uthman and Senecal, 1995; Yakushiji et al., 1995). GCs have long been used as a treatment option in patients with RA. Prednisolone reduces the rate of RA disease progression, and joint destruction resumes when patients discontinue the use of the drug (Hickling et al., 1998; Kirwan, 1995). The development of more specific GC therapeutics, such as time-release formulas and drugs with lower toxicity, may help curb harmful side effects (Bijlsma et al., 2005). For example, selective GC receptor agonists preferentially induce transrepression of GR, which leads to repressed transcription of pro-inflammatory factors (Schacke et al., 2004). This suggests that GCs and their derivatives will continue to be a useful therapy in RA and that new preparations may increase their efficacy, while lowering side effects. 2. Genetic Polymorphisms in the HPA Axis Genes in RA Genetic polymorphisms have been reported in the CRH gene and the glucocorticoid receptor gene in human RA. Recent evidence indicates that CRH gene polymorphisms are present in RA patients and could contribute to RA susceptibility (Baerwald et al., 2000; Gonzalez-Gay et al., 2003). Although no functional abnormality of CRH was reported in these studies, CRH polymorphisms could be a useful genetic marker for RA susceptibility. Impaired glucocorticoid control of inflammation may also result from resistance at the level of the glucocorticoid receptor (GR)—a member of a superfamily of nuclear hormone receptors (including the progesterone, estrogen, androgen, mineralocorticoid, and thyroid hormone receptors) that reside in the cytoplasm or nucleus. When glucocorticoid hormone binds to the ligand-binding domain of the GR, the receptorhormone complex dimerizes and moves into the nucleus and, facilitated by many co-factors and accessory proteins, binds to DNA and activates or inhibits transcription and translation (Webster et al., 2002b). Abnormalities of any of these molecules may lead to impaired GR function and glucocorticoid resistance. There are two forms of the GR, α and β. GRβ is an inactive form of the GR that does not activate DNA (Bamberger et al., 1995). Enhanced expression of GRβ, polymorphisms or impaired function of the GRα, GRβ, or associated co-factors may all contribute to glucocorticoid resistance (DeRijk et al., 2002; Webster et al., 2002b). Glucocorticoid resistance has been increasingly reported in autoimmune/inflammatory allergic diseases [for review, see (Marques-Deak et al., 2005)]. GRβ over-expression may contribute to glucocorticoid insensitivity in RA (Chikanza, 2002). A GRβ polymor-

phism associated with enhanced stability of this receptor has also been reported in RA patients, suggesting a potential source of glucocorticoid resistance in this sub-population of RA patients (DeRijk et al., 2001). Finally, there is some evidence of alterations in GR number in RA patients (Schlaghecke et al., 1992). Some studies report a downregulation of GR number in the early course of the disease (Huisman et al., 2002; van Everdingen et al., 2002). However, studying RA patients previously treated with glucocorticoids may confound these findings, since glucocorticoids downregulate GR expression. Indeed, GC-treated RA patients show decreased GR density in lymphocytes, whereas untreated RA patients do not show changes in GR number when compared to healthy controls (Neeck et al., 2002). Given this finding, evidence of alterations in GC receptor number should be interpreted with caution in GC-treated RA patients.

B. Stress and Depression and RA 1. Stress and RA Anecdotal reports have long linked environmental stressors to precipitation or exacerbation of disease in RA. Minor daily stressors have been linked to increases in inflammation in RA (Affleck et al., 1987; Thomason et al., 1992), and a 5-year longitudinal study found that RA patients with higher daily stress levels had more bony erosions over time (Feigenbaum et al., 1979). Interestingly, major stressors do not appear to be linked to RA, and may even be associated with a decrease in disease activity (Potter and Zautra, 1997). Psychological stress is reported to exacerbate RA (Herrmann et al., 2000; Straub et al., 2005), and stress management may lead to reductions in pain in RA patients (Rhee et al., 2000). 2. Depression and RA More than 50% of patients with RA also experience symptoms of depression (Frank et al., 1988; MarquesDeak et al., 2005; Sternberg, 1993). The underlying mechanisms between these relationships are not well understood; however, recent studies showing dysregulations in the HPA axis in both RA and depressive disorders suggest that a common underlying neuroendocrine dysregulation may underlie both these disorders in patients who experience RA and depression, since both syndromes are associated with dysregulations of hypothalamic CRH responsiveness. RA patients who concurrently experience depressive symptoms and stress also had increased levels of the pro-inflammatory cytokine IL-6 in peripheral blood


mononuclear cells (Zautra et al., 2004), supporting the hypothesis that an underlying biological process may predispose to both syndromes in some individuals. Indeed, the induction of depressive symptomatology in patients treated with IL-6 for other conditions (cancer, bone marrow suppression) and prevention of these symptoms by pre-treatment with anti-depressants (Musselman et al., 2001) raise the possibility that depression in RA may be, in part, secondary to elevated peripheral cytokine production. Other factors that may contribute to the association include pain and psychosocial factors (Marques-Deak et al., 2005). Chronic pain is often associated with both stress and depression (Blackburn-Munro and Blackburn-Munro, 2001), suggesting that the development of psychiatric symptoms in patients with RA may be partially due to chronic pain (Blalock and DeVellis, 1992). Psychosocial factors such as interpersonal stress may also contribute to neuroimmune dysregulation and worsening of disease severity in RA patients (Zautra et al., 1997).

V. OTHER NEUROENDOCRINE FACTORS IMPLICATED IN RA PATHOGENESIS A. Sex Hormones Females of all species show a higher incidence and/or severity of autoimmune/inflammatory disease compared to males, suggesting a role for sex hormones in the pathogenesis of the disease (Exploring the biological contributions to human health: Does sex matter? 2001). Conditions in which hormone levels are in flux are also associated with remissions or exacerbations of RA (Masi et al., 1995). Thus, pregnancy is associated with remission, while the postpartum period is associated with exacerbations of RA (Silman et al., 1992). During times of estrogen decline or insufficiency (i.e., postpartum and in menopausal women), the incidence of RA is increased. The severity of RA is also increased post-menopause, but this may be age-related rather than related to changes in sex hormone levels alone (Kuiper et al., 2001). The relationship between exogenous hormones, such as oral contraceptives, and rheumatoid arthritis disease progression and pathogenesis is unclear. Early evidence suggested that oral contraceptives were not protective against RA occurrence (Moskowitz et al., 1990), and a more recent study also failed to show that oral contraceptives influence RA expression (Drossaers-Bakker et al., 2002). Clinical trials of hormone replacement therapy showed no improvement in RA (Doran et al., 2004; Hall et al., 1994a; van den Brink et al., 1993). However, a population-based

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study found that oral contraceptives were associated with a decreased incidence of rheumatoid arthritis (Doran et al., 2004). Interestingly, this study also found that women who took oral contraceptives before 1970, when higher doses of estrogens and progestins were used, had a further lowering of risk to RA development. Importantly, synovial tissue from RA patients has been shown to express estrogen receptors (Ushiyama et al., 1995), suggesting that synovial tissue in RA could be responsive to fluctuating hormone levels. It is also possible that changing ratios of other hormones or their receptors in immune cells throughout development may contribute to changes in inflammatory disease expression. Animal studies show changes in ratios of estrogen and glucocorticoid receptors in Bcells throughout early development, with higher ratios of estrogen receptors compared to glucocorticoid receptors during puberty (Igarashi et al., 2001). Since estrogens are generally regarded as immunostimulatory [e.g., estrogen upregulates bcl-2, which interferes with induction of B cell tolerance (Bynoe et al., 2000)], this suggests the possibility that enhanced tissue sensitivity to the immunostimulatory effects of estrogen that occurs during puberty may account for some of the higher incidence for disease in post-pubertal females. In contrast, androgens are generally immunosuppressive (Cutolo et al., 2002). Low levels of androgens have been reported in both male and female RA patients (Cutolo et al., 2002; Masi et al., 2002; Tengstrand et al., 2002). These include reduced levels of dehydroepiandrostenedione (DHEA)/DHEA sulphate, and testosterone/dihydrotestosterone. Low levels of DHEA in RA patients correlates with low waking cortisol values and high levels of the proinflammatory cytokine IL-6 (Cutolo et al., 1999). There is also evidence that low levels of DHEA and a dissociation of cortisol and DHEA occur before the onset of RA in young premenopausal women (Masi et al., 1999). Administration of androgens to these patients improves RA symptoms (Booji et al., 1996). Recent studies have reported normal estrogen levels, and low serum androgen levels in RA patients have been reported (Cutolo et al., 2002; Spector et al., 1988), suggesting that an imbalance in these hormones may contribute to disease in some patients.

B. Sympathetic Nervous System Activation in RA Chemical sympathectomy or surgically cutting the sympathetic nerve in vivo has shown that the SNS plays an important role in regulating regional immu-

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nity in both animal models of inflammatory arthritis and in human RA. Sympathectomy diminishes inflammatory responses in an animal model of arthritis (Felten et al., 1992), and regional chemical sympathetic blockade improves symptoms in human RA (Levine et al., 1986). The primary neurotransmitter released from sympathetic nerves is norepinephrine, which acts through adrenergic receptors (Felten and Felten, 1988). The β2adrenergic receptor (β2AR), a classic 7 membrane Gprotein coupled receptor, is the main adrenergic receptor expressed on lymphocytes (Bishopric et al., 1980). Binding of β2AR by norepinephrine stimulates cAMP and activates protein kinase A, which in turn suppresses cytokine production. Removal of this inhibitory control results in enhanced immune responses. In contrast, stimulation of αAR enhances immune responses through activation of protein kinase C (PKC) (Bergmann and Sautner, 2002). RA patients exhibit several abnormalities of the SNS. These include a decreased β2AR density in blood mononuclear cells, which results in an increase of α1AR-mediated effects (Baerwald et al., 1992; Rouppe van der Voort et al., 2000; Wahle et al., 1999), and alterations in neuropeptide Y (NPY), a neuropeptide that is synthesized and/or released with norepinephrine from sympathetic neurons. Thus, NPY levels in serum correlate with symptom severity in RA patients, with higher levels associated with shorter durations of pain (Holmlund et al., 1991). Synovium from RA patients also shows a loss of NPY-containing fibers (Mapp et al., 1990; Miller et al., 2000). Finally, norepinephrine administration acts synergistically with cortisol to inhibit pro-inflammatory cytokine secretion from RA synoviocytes in vitro (Straub et al., 2002a).

C. Parasympathetic Nervous System in RA Although the parasympathetic nervous system has rarely been studied in RA patients, there is some evidence that parasympathetic regulation in RA may be impaired (Louthrenoo et al., 1999; Maule et al., 1997).

D. Peripheral Nervous System in RA Neuropeptides of the peripheral nervous system may be involved in RA pathogenesis. These include NPY, vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP). SP is released from peripheral sensory nerves and has a pro-inflammatory effect on the immune system. Increased levels of SP have been shown in synovial fluid from RA patients (Agro and Stanisz, 1992). The

primary receptor for SP, the neurokinin 1 (NK1) receptor, is present in both normal synovium and in the synovial membrane of RA patients (Lambert et al., 1998). Evidence from animal models suggest that SP plays a role in joint inflammation (Keeble and Brain, 2004), indicating that SP antagonists may be useful therapeutic agents for RA. Specific NK1 receptor antagonists have been shown to reduce inflammation and pain in rodents, but have not been tested in humans (Keeble and Brain, 2004). The same sensory neurons that release SP also release CGRP, a 37-amino acid peptide. CGRP opposes the pro-inflammatory effects of SP. In rats, immunization with CGRP reduces the severity of adjuvantinduced lesions (Louis et al., 1990). Animal studies also report increased amounts of SP and CGRP in synovial fluid (Bileviciute et al., 1993). CGRP mRNA is contained in synovial cells from RA patients, and exogenous CGRP administration has an inhibitory effect on pro-inflammatory cytokines and matrix metalloproteinase (MMP) production in vitro (Takeba et al., 1999). Similar to CGRP, VIP mRNA is found in synovial cells from RA patients, and exogenous VIP has an inhibitory effect on pro-inflammatory cytokines, chemokines, and matrix metalloproteinase (MMP) production in vitro (Juarranz et al., 2004; Takeba et al., 1999). This suggests that VIP-related agents could play a therapeutic role in RA. Peripheral corticotropin releasing hormone (CRH) is present in higher concentrations in inflamed joints in Lewis compared to Fischer rats. While central CRH suppresses immune responses through activation of the HPA axis and release of glucocorticoids, peripheral CRH released at nerve terminals is pro-inflammatory and its immunoneutralization is associated with decreased cellular infiltration and volume of exudates in chronic inflammation (Karalis et al., 1991). Furthermore, treatment of rats with adjuvant arthritis with the non-peptide CRH antagonist antalarmin reduced arthritis scores by 50% (Webster et al., 2002a). The findings that neuropeptides influence RA pathology suggest that further investigations in their role in RA and inflammation could provide new avenues for treatment of the disease.

E. Prolactin Prolactin is a pro-inflammatory hormone released from the pituitary gland. Prolactin receptors are widely distributed and are found in the CNS, adrenals, and on lymphocytes. Studies in animal models of inflammatory arthritis and lupus have shown that prolactin antagonists inhibit disease activity (Berczi et al., 1984;


Walker et al., 1998). Elevated prolactin levels have been reported in human RA patients (Chikanza et al., 1992; Ram et al., 2004), and increases in the prolactin diurnal rhythm coincide with RA disease activity. Although the therapeutic effects of prolactin have not been tested in human RA, studies in human SLE indicate that prolactin is elevated in SLE and prolactin inhibitors ameliorate disease course (Walker et al., 1998).

VI. SUMMARY Research in recent decades shows that the HPA axis, the SNS, and the parasympathetic and peripheral nervous systems all play an important role in regulating the peripheral immune/inflammatory response. Advances in our understanding of the molecular, biochemical, and neuroanatomical pathways by which the immune system and the CNS communicate also provide evidence that dysregulations of these connections are associated with and contribute to RA pathogenesis and disease expression. There is no simple cause-and-effect relationship between RA and the responsiveness of these neural and neuroendocrine systems, but dysregulation in the bi-directional communication between the periphery and these axes are certainly present in RA and are likely to contribute to susceptibility, course, and expression of disease. Findings in both animal and human studies show a downregulated HPA axis in RA. While there is no definitive evidence in humans that a hypoactive HPA axis causes or directly contributes to the development of RA, animal studies in which the HPA axis is interrupted do suggest a causal relationship. Recent studies showing glucocorticoid receptor polymorphisms and GC resistance in RA suggest that differences in receptor sensitivity may also be an important factor contributing to disease susceptibility and individual variability in expression of RA. Peripheral neuropeptides and sympathetic and parasympathetic nervous system factors may also contribute to RA pathology, and further exploration of the role of these peptides and neurotransmitters in human RA may lead to additional therapeutic options. Finally, sex hormones and their receptors also play an important role in disease predisposition and severity. These are reviewed elsewhere in this text and in more depth.

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C H A P T E R

9 Sex Steroids and Immunity MAURIZIO CUTOLO AND ALESSANDRO CALVIA

I. INTRODUCTION: SEX HORMONES AND AUTOIMMUNE DISEASES 207 II. PERIPHERAL SEX HORMONE METABOLISM IN AUTOIMMUNE DISEASES 208 III. POSSIBLE MECHANISMS OF IMMUNOMODULATION BY SEX HORMONES 209 IV. PREGNANCY AND AUTOIMMUNITY 211

Sex hormones are implicated in the immune response, with estrogens as enhancers (at least of the humoral immunity), and androgens and progesterone (and glucocorticoids) as natural immune-suppressors on Th0 maturation and Th1/Th2 activities (4,5) (Figure 1). Estrogens in physiological concentrations enhance the Th1 cytokine production, and at pharmacological concentrations exert opposite effects, at least in models (Figure 2). In addition, estrogens enhance antibody production by activated B-cells (Figure 3). Low concentrations of gonadal and adrenal androgen [testosterone (T)/dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA) and its sulphate (DHEAS), respectively] levels, as well as reduced androgens/ estrogens ratio, have been detected in serum and body fluids (i.e., blood, synovial fluid [SF], smears, salivary) of male and female RA patients, as well as in SLE, supporting the possible pathogenic role for the decreased levels of the immune-suppressive androgens (6). However, in respect to serum levels of estrogens, interestingly, they are not significantly changed, which is in strict contrast to androgen levels in RA patients (4). Several physiological, pathological, and therapeutic conditions may change the serum estrogen milieu and/or peripheral conversion rate, including the menstrual cycle; pregnancy; postpartum period; menopause; elderly; chronic stress; inflammatory cytokines; use of corticosteroids, oral contraceptives, and steroid hormonal replacements; inducing altered androgen/ estrogen ratios and related effects (5–7). As a matter of fact, sex hormones can also exert local actions (paracrine) in the tissues in which they are formed or enter the circulation, and both T and 17-β estradiol (E2) seem to exert dose- and time-dependent

I. INTRODUCTION: SEX HORMONES AND AUTOIMMUNE DISEASES Epidemiological evidence indicates that during the fertile age, women are more often affected by rheumatic diseases than men, particularly autoimmune diseases (1). As a matter of fact, rheumatic disorders with autoimmune involvement such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) result from the combination of several predisposing factors, which include the relationships between epitopes of the trigger agent (i.e., virus) and histocompatibility epitopes (i.e., HLA), the status of the stress response system including the hypothalamicpituitary-adrenocortical axis (HPA) and the sympathetic nervous system (SNS), and mainly the effects of the gonadal hormones (hypothalamic-pituitarygonadal, or HPG, axis) (2). The pre- or post-menopausal serum sex hormonal status is a further factor influencing the rate of rheumatic diseases. It is therefore important, whenever possible, to evaluate epidemiologic data broken down into age (for example, 10-year age band) and sexspecific group before making inferences (3). Obviously, sex hormones seem to play an important role as modulators of both disease onset and perpetuation (4). PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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208


Estrogens (physiological - low)

Th1 Testosterone

(physiological - low)

Th2

(progesterone)

Th0

Estrogens

IL-2 RA IFN-γγ = MS

Testosterone (progesterone)

Th2

Estrogens

IL-4 IL-10 SLE IL-5 = β TGF-β

(pharmacological - high)

Estrogens Th1

IL-1 TNF-α α IL-6

Testosterone (progesterone)

M0

Estrogens

IL-4 Ab IL-10 = production IL-5 TGF-β β

(pharmacological - high)

FIGURE 1 Sex hormones are implicated in the immune response, with estrogens as enhancers (at least of the humoral immunity), and androgens and progesterone (and glucocorticoids) as natural immune-suppressors on Th0 maturation and Th1/Th2 activities.

(physiological - low)

B cell

IL-4 IL-10 = Ab IL-5 production β TGF-β

IL-1 TNF-α α IL-6

Estrogens (pharmacological - high)

FIGURE 2 Estrogens in physiological concentrations enhance the Th1 cytokine production, and at pharmacological concentrations exert opposite effects, at least in models.

effects on cell growth and apoptosis (2,4). These effects, as well as important influences on gene promoter of Th1/Th2 cytokines and the recently discovered increased SF estrogen concentrations, might suggest interesting new roles for estrogens, at least in RA (8–11).

II. PERIPHERAL SEX HORMONE METABOLISM IN AUTOIMMUNE DISEASES Several findings suggest an accelerated metabolic conversion of upstream androgen precursors to E2 in

FIGURE 3 Estrogens enhance antibody production by activated B cells.

RA and SLE patients. E2, as the aromatic product of the gonadal steroid metabolic pathway and the result of peripheral conversion from the adrenal androgen DHEA, recognizes as upstream precursors different hormones such as DHEA, testosterone, and progesterone. As a matter of fact, a very large number of studies and reviews in the last 20 years have shown reduced serum concentrations of DHEAS, testosterone, and progesterone in both male and female RA/SLE patients (12,13). These data strongly support an accelerated peripheral metabolic conversion of upstream androgen precursors to E2 (see Figure 1). High estrogen concentrations have been found particularly in synovial fluids of RA patients of both sexes. How can one explain the recent detection of lower androgen and higher estrogen levels in both female and male RA synovial fluids? The appropriate explanation might originate from recent studies showing that the inflammatory cytokines (i.e., TNFα, IL-6, IL-1), particularly increased in RA synovitis, are able to markedly stimulate the aromatase activity in peripheral tissues (14,15). As a matter of fact, the aromatase enzyme complex is involved in the peripheral conversion of androgens (testosterone and androstenedione) to estrogens (estrone and estradiol, respectively). In tissues rich in macrophages, a significant correlation was found between the aromatase activity and the IL-6 production, and aromatase has been found also in synoviocytes (16). Therefore, the increased aromatase activity induced by locally produced inflammatory cytokines (i.e. TNFα, IL-1, IL-6) might explain the altered balance resulting in lower androgens and higher estrogens in the synovial RA fluids, as well as their effects on syno-

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vial cells, first described in our research (17) (see Figure 1). The role of local sex hormone concentrations at the level of inflammatory foci is of great value in order to explain the modulatory effects exerted by these hormones on the immune-inflammatory reaction. Men with rheumatoid arthritis (RA) have a higher than normal frequency of low testosterone levels. Interestingly, in a recent study, DHEAS and estrone concentrations have been found lower and estradiol was found higher in male RA patients compared with healthy controls (18). In this study, estrone did not correlate with any disease variable, whereas estradiol correlated strongly and positively with all measured indices of inflammation. Men with RA had aberrations in all sex hormones analyzed, although only estradiol consistently correlated with inflammation (18). The low levels of estrone and DHEAS may depend on a shift in the adrenal steroidogenesis towards the glucocorticoid pathway, whereas increased conversion of estrone to estradiol seemed to be the cause for the high estradiol levels (effect of the 17beta-hydroxysteroid dehydrogenase). In SLE patients, the aromatase activity evaluated in skin and subcutaneous tissue showed a tendency toward an increase when compared to control subjects (19). Among SLE patients, the aromatase activity varied inversely with disease activity, and the patients had decreased androgen and increased estrogen serum levels (19). Therefore, tissue aromatase activity showed significant direct correlation with estrogen levels in SLE patients. These data suggest that abnormal regulation of aromatase activity (i.e., increased activity) may partially explain the abnormalities of peripheral estrogen synthesis (i.e., increased availability of E2 and possible metabolites) in SLE, as well as the altered serum sexhormone levels and ratio (i.e., decreased androgens and DHEAS) (see Figure 1). Similarly, in a recent study by Straub, it is thought that the urinary excretion of hydroxyestrogens (namely, 16α-hydroxyestrone and 2-hydroxyestrogens) reflects the production in the tissues, since no respective hydroxylase activity is expected in the urine (20,21). On the other hand, as recently reviewed, peripheral estrogen hydroxylation was found increased in both men and women with SLE, and the estrogenic metabolites have been reported to increase B-cell differentiation and activate T-cells (22).

A. The Role of Estrogen Metabolites The elevated serum levels of 16α-hydroxyestrone, already described in SLE patients, indicate that dis-

eases in men differ from diseases in women to the extent that only 16 alpha-hydroxyestrone was elevated in men, whereas women had elevations of both 16 alpha-hydroxyestrone and estriol (23). These data suggest abnormal patterns of E2 metabolism, which may lead to increased estrogenic activity in SLE patients. A similar phenomenon is described in the synovial fluids of RA patients where 16α-hydroxyestrone/4hydroxyestradiol were found significantly higher when compared with control fluids (17,20) (see Figure 1). In these studies, molar ratio of free estrogens/free androgens also was found significantly higher in RA synovial fluids. Two important aspects must be considered. Total serum levels of E2 are not typically outside physiologic ranges in RA, as well as in SLE patients of both sexes, and the reported alterations in estrogen metabolism are again observed in both male and female patients, as recently reviewed (24–26). Therefore, it is intriguing that gender may not influence the entire phenomenon and that the gonadal production of the sex hormones is not responsible for the observed metabolic results, since most of the measured metabolites are converted in the periphery which is largely independent of gender. The phenomenon seems only dependent on the inflammatory state of the tissues, and the common mechanisms in both RA and SLE patients might indicate this is also a diseaseunspecific phenomenon. Furthermore, E2 is thought to play a dual proand anti-inflammatory role in chronic inflammatory diseases that were found related to low and high concentrations, respectively. In light of these data, it is possible that the phenomenon might just depend on different dose-related rate of peripheral E2 conversion to pro- or anti-inflammatory metabolites, such as 16αhydroxyestrone or naturally occurring antagonists (i.e., 2-hydroxyestrogens), respectively (27).

III. POSSIBLE MECHANISMS OF IMMUNOMODULATION BY SEX HORMONES A. Clinical Evidence Macrophages release cytokines, such as tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1), and IL-6, which, for example, modulate the symptoms at least of RA. Macrophage release of these cytokines can be modulated by estrogen in different ways. Fc gamma receptor type IIIA (CD16a) is a receptor expressed on macrophages that selectively binds IgG molecules, an important rheumatoid factor in RA. Binding of CD16

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by anti-CD16 monoclonal antibodies stimulates macrophage cytokine release. In a recent study, estrogen can modulate pro-inflammatory cytokine release from activated monocytes and/or macrophages, in particular through modulation of CD16 expression (28). On the other hand, recent studies have shown that 16α-hydroxyestrone was far more potent than E2 in exerting proliferative activities (27). More recently we tested in cultured human myeloid monocytic cells (THP-1), differentiated into activated macrophages (M), the effects of E2 and testosterone in order to evaluate their influence on cell proliferation and apoptosis. The effects were evaluated on the NFk-B activity, as a complex of molecules modulating cellular activation. Testosterone was found to exert pro-apoptotic effects and to reduce M proliferation, whereas E2 induced opposite effects by interfering with NFk-B activities. Therefore, these results might support the hypothesis of a sex hormone modulation of cell growth and apoptosis. The next obvious step will be to test the effects of 16α-hydroxyestrone and 2-hydroxyestrogens on the same cells. In another study, E2 was found to increase IgG and IgM production by peripheral blood mononuclear cells (PBMC) from SLE patients, which leads to elevated levels of polyclonal IgG, including IgG antidsDNA, by enhancing B-cell activity via interleukin-10 (IL-10) (19). These latter results should also be replicated in presence of 16α-hydroxyestrone as well as with the naturally occurring 2-hydroxylated antiestrogen. In fact, in a recent study it has been shown that disease activity in SLE patients was negatively correlated to urinary concentrations of 2-hydroxylated estrogens (21). Estrogens are confirmed as one of the risk factors in autoimmunity. Interesting changes of serum estrogens have been found during pregnancy in SLE patients and have been found to correlate with cytokine variations (29,30) . The major hormonal alteration observed during pregnancy in SLE patients was an unexpected lack of estrogen serum level increase and, to a lesser extent, progesterone serum level increase, during the second and—even more—the third trimester of gestation (29). This lack of increase probably was due to placental compromise. In addition, a lower than expected increase of IL-6 in the third trimester of gestation and persistently high levels of IL-10 during pregnancy seem to be the major alterations of the cytokine milieu in the peripheral circulation of SLE pregnant patients (30). Therefore, these steroid hormone and cytokine variations may result in a lower humoral immune response activation, probably related to a change in

the estrogen/androgen balance, which in turn could account for a more immunosuppressive effect exerted by cytokines on disease activity as observed during the third trimester in pregnant SLE patients (29). Since cyclophosphamide-induced ovarian failure has been reported to be protective against flares of SLE, a recent study evaluated whether patients with SLE experience a decrease in disease activity after natural menopause (31). Differences in disease activity scores (mean and maximum) and the number of visits to a rheumatologist’s office were only significant when the fourth year before menopause was compared with the fourth year after menopause. Disease activity was found mild during the pre-menopausal and postmenopausal periods in women with SLE. A modest decrease, especially in the maximum disease activity, was seen after natural menopause (31).

B. Role of Sex Hormones in Cell Proliferation and Apoptosis Monocytes/macrophages contribute to the autoimmune process, mainly acting as antigen-processing/ presenting cells and sources of inflammatory cytokines, in particular at the level of the synovial tissue in RA (32). Moreover, sex hormones can exert local actions (paracrine) in the tissues in which they are formed, including the synovial tissue (17,20). Activated THP-1 cells differentiate into macrophages for long-term cultures. On the contrary, synovial macrophages are characterized by a short life during in vitro culture. The present study shows opposite effects by sex hormones on cultures of activated monocytic/macrophage cells (THP-1 cells) concerning their modulatory effects on cell proliferation and/or apoptosis. The signaling pathways modulating the pro- and anti-inflammatory mechanisms seem to involve the steroidal hormone receptor activation and the NFk-B complex factors; in addition estrogens may differently regulate NF-kB activation depending on cell type tested (33,34). Therefore, E2 increased the expression of markers of cell growth and proliferation, whereas T induced an increase of the PARP-cleaved expression, indicating DNA damage and apoptosis. In addition, to support the proliferative role exerted by E2, the THP-1 cells pre-treated with the estrogens showed a decrease of staurosporine-induced apoptosis when compared with T-treated and untreated cells. Furthermore, the increased NFk-B p65 expression and the evident NFkappaB-binding to DNA in E2-treated cells, when compared with untreated or T-treated cells, as well as the increased levels of the IkB-•• phosphorylated


form, seem to support the major enhancing role exerted by estrogens on immune/inflammatory response by activating the NF-kB complex. On the contrary, the observed positive upregulation of the IkB-α exerted by T treatment presumably dampens the pro-inflammatory effects mediated by the NF-kB activation and therefore might represent a further mechanism by which androgens exert antiinflammatory effects. Recent studies support these results, showing that E2 inhibits apoptosis in different cell types (cardiac myocytes and others), whereas androgens have been found to induce apoptosis (35,36). The increased concentrations of estrogens (and low androgens) recently described (17) at the level of the synovial fluid of RA patients of both sexes seem to support their possible modulator roles on synovial tissue hyperplasia and chronic synovial cell activation, by considering the estrogenic effects on cell proliferation and apoptosis. These observations have been recently obtained also in human breast cancer cells (37). To explain increased estrogen concentrations in RA synovial fluids, the pro-inflammatory cytokines (TNFα, IL-1, IL-6) have been found to accelerate the metabolic conversion on estrogens from androgens by inducing the synovial tissue aromatases (38–40). As a consequence, locally increased estrogen levels might exert activating effects on synovial cell proliferation, including macrophages and fibroblasts (41). In conclusion, the concentrations for E2 and T tested here seem to modulate the activity of NFk-B molecules in monocytic/macrophage cells line (THP-1) with opposite effects, and interfere with cell growth and apoptosis (42). These observations might provide a further biological link between gender effects and the complex inflammatory process involved in rheumatoid synovitis. Further studies using peripheral metabolites of estrogens and synovial macrophages from RA patients might extend the value of these observations.

IV. PREGNANCY AND AUTOIMMUNITY A. The Immune System at the Placental Level Feto-maternal unit organization starts with the invasion of maternal decidua by trophoblast. The maternal immune system does allow the trophoblast invasion anchoring the placenta to the uterus. In the initial phases, the progesterone produced by the corpus luteum induces the differentiation of Th0

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to Th2 lymphocytes. Th2 cells inhibit Th1 by the production of IL-4, IL-10, and IL-13, as well as the antigenpresenting cells (APC) activity secreting IL-4, IL-6, and IL-10. At the feto-maternal interface, APCs are almost all represented by dendritic cells. Th2 lymphocytes producing IL-4, IL-6, and IL-10 also stimulate differentiation of B-cells to plasma cells, and in particular, they stimulate the synthesis of some trophoblast-protecting antibodies, such as asymmetric IgG and anti-R80K antibodies. IL-4 produced by Th2 lymphocytes inhibits NK cells expressing the “Killer Activator Inhibitor” (KAR). These cells are also inhibited by anti-R80K antibodies. On the contrary, IL-4 stimulates KAR-expressing NK cells. This receptor forms a complex with HLA-G molecules expressed by the trophoblast, and this interaction blocks the killer activity of NK lymphocytes on trophoblastic cells. NK cells start to produce LIF and M-CSF (monocyte-stimulating growth factor), which regulate the invasion of trophoblastic tissue. Th2 cytokines are produced not only by immune cells but also by the placenta. In particular, IL-4, IL-6, and IL-10 stimulate hCG secretion by the placenta, which in turn stimulates the ovarium to produce progesterone. The same cytokines produced by the placenta can directly stimulate the differentiation of Th0 to Th2 lymphocytes. Estrogens at high levels, such as those produced by the placenta during the second and third trimester of pregnancy, can stimulate IL-6 production by the placenta itself or by macrophages. This cytokine plays a very important role during differentiation of Th0 to Th2 lymphocytes. Therefore, at the feto-maternal interface, the Th1/ Th2 shift plays a crucial role: Th1 cytokine reduction results in cell immune response inhibition, and Th2 increments results in humoral immune response stimulation. If Th1/Th2 balance does not shift, abortion can occur. This shift is mainly modulated by progesterone and estrogens (43). Progesterone participates in the first phase of pregnancy, whereas estrogens seem to be more important during the second period of pregnancy, during which they are produced in large amounts by the placenta.

B. Cytokine and Endocrine Network in the Periphery during Pregnancy Hormonal modifications during pregnancy are regulated by the feto-placental unit and depend on interactions between mother and fetus (44). Progesterone has a placental origin, since it is secreted by the corpus luteum during the first 6–8

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weeks of gestation. During physiological pregnancy, progesterone levels are 4–6 times higher than those out of gestation. Progesterone is the fundamental hormone during the first part of pregnancy and the precursor of some fetal hormones, and it also has other functions, some of which are still partially unknown. Deoxicorticosterone, one of its metabolites, is found in concentration 1,000 times higher than that out of pregnancy, but the physiologic role of this hormone is still not known (44). Also, the estrogen concentration is significantly increased during pregnancy, reaching levels 3–8 times higher compared to normal levels (44). This increase results from a unique interchange between mother and fetus. The fetus uses the pregnenolone produced by placenta in order to produce adrenal dehydroepiandrosterone and dehydroepiandrosterone sulphate. These hormones are metabolized to androstenedione and testosterone at the level of the placenta; finally, they are rapidly converted to estrose and estradiol and let into maternal circulation (44). The metabolite of dehydroepiandrosterone, which is 16α idroxilate, is converted to estriol in the fetal liver throughout the same pathways. One of the main side effects of elevated levels of estrogens during pregnancy is the increased protein synthesis in maternal liver, thus increasing the concentrations of several proteins in maternal circulation (44). It has also been shown that the increase of several coagulation proteins is probably responsible for the ipercoagulability state observed during pregnancy. The feto-placental hormones—in particular, progesterone, estrogens, and placental lactogenic hormone— are responsible for an altered metabolism of carbohydrates observed during gestation. In fact, during the second part of pregnancy, the mother develops a slight peripheral resistance to insulin, inducing an increased production and release of insulin by the pancreas (44). Due to glucose tolerance– induced physiological stress, almost 5% of pregnant women develop gestational diabetes during the second part of pregnancy. Probably, the concomitant corticosteroid therapy might predispose to this condition (44). Pregnancy and postpartum represent a paradigmatic example of how the modification of steroid hormone concentration can influence the immune and inflammatory responses in women and can modify the concentrations of immunoregulatory molecules (i.e., cytokines) and thereafter the expression of autoimmune diseases.

The physiological increase of cortisol, progesterone, estradiol, and testosterone during the third trimester of pregnancy seems to be responsible systemically and at the feto-maternal interface of the Th2 cytokines’ polarization (45). In fact, elevated levels of Th2 cytokines, such as IL10, have been found in placenta and amniotic fluid during the third trimester of pregnancy (46). Therefore, the suppression of the immune response mediated by Th1 cytokines seems to be fundamental for fetal survival. It has been suggested that some autoimmune diseases, such as SLE, which are mediated mainly by Th2 cytokines and humoral immune response, tend to develop or relapse during pregnancy (47), whereas Th1-mediated diseases, such as rheumatoid arthritis, tend to improve. In both cases a flare or explosion of disease is shown during the postpartum, when the anti-inflammatory Th2 cytokines collapse. During normal pregnancy the IL-10 production by peripheral lymphocytes progressively increases (48,49). It also has been demonstrated that during pregnancy IL-6 serum levels gradually increase in maternal circulation and even more during labor (50). TNF-α serum levels do not vary during pregnancy, whereas the TNF-α-soluble receptors increase just to protect the fetus from TNF-α dangerous effects (51,52). It is known that glococorticoids inhibit IL-1, TNF-α, IFN-γ, and IL-2 production and that they stimulate IL-10, IL-4, and IL-13 synthesis, confirming a modulatory effect on the balance between the antiinflammatory/immunosuppressive responses during pregnancy (53). At physiological concentration, progesterone stimulates IL-4 (Th2 cytokine) synthesis, whereas estradiol stimulates TNFα production (Th1 cytokine). On the contrary, at pharmacologic levels, such as those observed during the second part of pregnancy, progesterone inhibits TNFα secretion and stimulates the IL-10 production in T lymphocyte clones, leading to an increased humoral immune response (54,55). Increased concentration of prolactin is associated with worsening of renal involvement in animal studies and has been found both in SLE males and females. A study carried out to detect serum autoantibodies in women during premenopausal periods has shown that in 20% of these, the prolactin levels were found increased, and high levels of anti-DNA antibodies were present at the same time. However, many women not diagnosed as SLE and followed at the Hyperprolactinemia Endocrinological Center showed increased levels of anti-nuclear antibodies (ANA) (56).


C. Cytokine Network in Non-pregnant SLE Patients Cytokine profile has been largely investigated in SLE. In T lymphocytes from SLE patients, an insufficient production of IL-2 in vitro has been observed, probably resulting from several factors, including downregulation of some Th2 cytokines. The recent finding of the IL-10 role in the pathogenesis of SLE supports this hypothesis. IL-10 is a Th2 cytokine that strongly stimulates B lymphocyte proliferation and differentiation, having a potential role as mediator of B-cell polyclonal activation in SLE. Recent studies have demonstrated that the spontaneous IL-10 production by B lymphocytes and peripheral blood mononuclear cells is significantly more elevated in SLE patients compared to controls (56,57). In addition, IL10 concentration in the serum was found higher in SLE patients and correlated with disease clinical and serological activity, as well as with anti-DNA antibodies (58–60). IL-10 increased production may be caused by a reduced Th1 in vitro response of T-cells in SLE. This phenomenon has been suggested in a study showing that the addition of IL-10–blocking antibodies significantly increased the proliferative response of peripheral blood mononuclear cells (61). IL-12 cytokine is a heterodimer produced by B lymphocytes, macrophages, and dendritic cells that promotes cell-mediated immune response and has some inhibitory activity on humoral immune response (62). It has been shown that IL-12 is insufficiently produced by peripheral blood mononuclear cells in SLE patients (62,63). The deficit of IL-12 production is probably characteristic of monocytes rather than B lymphocytes (64). On the contrary, IL-12 addition to SLE peripheral blood mononuclear cells significantly inhibits both spontaneous and IL-10–stimulated production of immunoglobulins and anti-DNA antibodies (65). Therefore, the altered regulation of IL-10/IL-12 balance plays a crucial role in the inadequate cell immune response observed in SLE patients.

D. Immunoendocrine Modifications in SLE Patients during Pregnancy In healthy subjects, the altered immunoregulation induced by pregnancy is manifested mainly at the fetomaternal interface, whereas systemic effects are negligible. The situation is different in patients affected by autoimmune rheumatic diseases (ARDs) during pregnancy. ARDs are already characterized by an altered immunoregulation, and in these patients the maternal immune system modifications can influence the course of the disease.

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One of the most important modifications is the Th1/ Th2 shift. It occurs both at the feto-placental barrier and in maternal circulation, and it is driven by the physiological increase of progesterone and estrogens that occurs progressively throughout pregnancy, with the maximum peak during the third trimester of gestation. In particular, progesterone plays an important role in the first part of pregnancy, and estrogens in the second part. In physiological conditions, it seems that estrogens stimulate both humoral and cell immune responses (Th1 and Th2 cytokines). Nevertheless, at concentrations higher than physiological, such as those reached during pregnancy, it seems that estrogens inhibit cellmediated immune response (Th1 cytokines), whereas they induce antibody production (Th2 cytokines). The polarization of Th2 response may explain why patients affected by rheumatoid arthritis generally improve during pregnancy, whereas those with SLE get worse. In SLE patients, pregnancy has always been considered a risk event both for the mother and fetus since the relapse of the disease is frequent during gestation. As a consequence, fetal loss, prematurity, and underweight babies are more common events in SLE women than in healthy subjects. Nevertheless, during the last years, the number of successful pregnancies has increased, although disease relapse still remains a frequent event during both gestation and postpartum. Recently, a reduced frequency of relapses has been reported during the third trimester of pregnancy compared to the second and postpartum. It has been shown that serum levels of some hormones are lower in SLE patients compared to healthy controls (29). In particular, the concentration of estradiol and progesterone in the serum is unexpectedly low in the second and most in the third trimester of pregnancy, periods during which these hormones are secreted mainly by the placenta. The reduced concentration of estradiol and progesterone may be due to the compromised placental activities, responsible for a lower activation of humoral response and leading to a lower disease activity during the third trimester of pregnancy in SLE patients (30). The effects of steroid hormones on disease activity during pregnancy seem to be mediated by Th2 cytokines. IL-10 strongly stimulates B lymphocytes and anti-DNA production in SLE. In healthy subjects IL-10 progressively increases during pregnancy, whereas in SLE patients IL-10 serum levels remain elevated before and after pregnancy in the case of both active and inactive disease. These effects support the hypothesis that

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SLE IL-10 is constitutively hyperproducted rather than modulated by gonadal hormones (steroids) (30). On the other hand, it is known that IL-6 is involved in Th0/Th2 shift (44). The production of IL-6 seems to be correlated with disease activity. In healthy subjects during pregnancy, IL-6 progressively increases in maternal circulation and even more during delivery. In SLE patients the peak of IL-6 during the third trimester seems lacking (30). The modulation of estrogens on IL-6 production seems dose-dependent; at physiological levels estrogens inhibit IL-6, but at pharmacological levels, such as those reached at the end of pregnancy in physiological conditions, estrogens inhibit IL-6 production (45). The low levels of IL-6 observed during the third trimester of pregnancy in SLE may depend on the low levels of estrogens observed during the same period of gestation and may explain the lower frequency of relapse reported in the same period (30).

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the expression of autoimmune diseases in pregnancy and the postpartum period? Mol Med Today, 3, 379–383. Russel, A. S., et al. (1997). Evidence for reduced Th1 function in normal pregnancy: a hypothesis for the remission of rheumatoid arthritis. J Rheumatol, 24, 1045–1050. Kupfermine, M. J., Peaceman, A. M., Aderka, D., Wallach, D., Peyser, M. R., Lessing, J. B., and Socol, M. L. (1995). Soluble tumor necrosis factor receptors in maternal plasma and second-trimester amniotic fluid. Am J Obstet Gynecol, 173, 900–905. Branch, D. W. (1992). Physiologic adaptation of pregnancy. Am J Reprod Immunol, 28, 120–122. Lin, H., Mosman, T. R., Guilbert, L., Tntipopipats, S., and Wegmann, T. G. (1993). Synthesis of T helper-2-cytokines at the maternal-fetal interface. J Immunol, 151, 4562–4573. Greig, P. C., Herbert, W. N., Robinette, B. L., and Teot, L. A. (1995). Amniotic fluid interleukin-10 concentrations increase through pregnancy and are elevated in patients with preterm labor associated with intrauterine infection. Am J Obstet Gynecol, 173, 1223–1227. Ramirez, F., Fowell, D. J., Puklavec, M., Simmonds, S., and Mason, D. (1996). Glucocorticoids promote a Th2 cytokine response by CD4+ T cells in vitro. J Immunol, 156, 2406– 2412. Piccinni, M. P., Giudizi, M. G., Biagiotti, R., Beloni, L., Giannarini, L., Sampognaro, S., Parronchi, P., Manetti, R., Annunziato, F., and Livi, C. (1995). Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones. J Immunol, 155, 128–133. Cutolo, M., Sulli, A., Seriolo, B., Accardo, S., and Masi, A. T. (1995). Estrogens, the immune response and autoimmunity. Clin Exp. Rheumatol., 13, 217–226. Lahita, R. G. (1999). The role of sex hormones in systemic lupus erythematosus. Curr Opin Rheum, 11, 352–356. Llorente, L., Richaud-Patin, Y., Wijdenes, J., Alcocer-Varela, J., Maillot, M. C., Durand-Gasselin, I., Fourrier, B. M., Galanaud, P., and Emilie, D. (1993). Spontaneous production of interleukin10 by B-lymphocytes and monocytes in systemic lupus erythematosus. Eur Cytokine Netw, 4, 421–427. Houssiau, F. A., Lefebvre, C., Vanden Berghe, M., et al. (1995). Serum interleukin 10 titers in systemic lupus erythematosus reflect disease activity. Lupus, 4, 393–395. Park, Y. B., Lee, S. K., Kim, D. S., Lee, J., Lee, C. H., and Song, C. H. (1998). Elevated interleukin-10 levels correlated with disease activity in systemic lupus erythematosus. Clin Exp Rheumatol, 16, 283–288. Grondal, G., Gunnarson, I., Ronnelid, J., et al. (2000). Cytokine production, serum levels and disease activity in systemic lupus erythematosus. Clin Exp Rheumatol, 18, 565–570. Lauwerys, B. R., Garot, N., Renauld, J. C., and Houssiau, F. A. (2000). Interleukin-10 blockade corrects impaired in vitro cellular immune responses of systemic lupus erythematosus patients. Arthritis Rheum, 43, 1976–1981. Horowitz, D. A., Gray, J. D., Behrendsen, S. C., Kubin, M., Rengaraju, M., Ohtsuka, K., and Trinchieri, G. (1998). Decreased production of interleukin-12 and other Th1-type cytokines in patients with recent-onset systemic lupus erythematosus. Arthritis Rheum, 41, 838–844. Liu, T. F., and Jones, B. M. (1998). Impaired production of IL-12 in systemic lupus erythematosus. I. Excessive production of IL10 suppresses production of IL-12 by monocytes. Cytokine, 10, 140–147.

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64. Liu, T. F., and Jones, B. M. (1997). Impairment of IL-12 production in SLE is due to defective monocytes, not B cells. Immuol Lett, 56, 314. 65. Houssiau, F. A., Mascart-Lemone, F., Stevens, M., Libin, M., Devogelaer, J. P., Goldman, M., and Renauld, J. C. (1997). IL-12 inhibits in vitro immunoglobulin production by human lupus

peripheral blood mononuclear cells (PBMC). Clin Exp Immunol, 108, 375–380. 66. Kovacs, E. J., Messingham, K. A., and Gregory, M. S. (2002). Estrogen regulation of immune responses after injury. Mol Cell Endocrinol, 193, 129–135.

C H A P T E R

10 Emerging Concepts for the Pathogenesis of Chronic Disabling Inflammatory Diseases: Neuroendocrine-immune Interactions and Evolutionary Biology RAINER H. STRAUB, ADRIANA DEL REY, AND HUGO O. BESEDOVSKY

I. INTRODUCTION 217 II. BALANCE BETWEEN IMMUNE TOLERANCE AND IMMUNE AGGRESSION 217 III. GENES RESPONSIBLE FOR ADVANTAGEOUS REACTIONS TO OVERCOME CHRONIC DISABLING INFLAMMATORY DISEASES WERE NOT EVOLUTIONARILY CONSERVED 219 IV. HOMEOSTATIC MECHANISMS ARE EVOLUTIONARILY CONSERVED FOR TRANSIENT INFLAMMATORY REACTIONS—THEIR MISUSE IN CHRONIC DISABLING INFLAMMATORY DISEASES 220 V. UNCOUPLING OF SUPERSYSTEMS DURING CDIDS 224 VI. THE HEN-AND-EGG PROBLEM OF CDIDs—WHO STARTS THE DISEASE? 226

well-programmed adaptive mechanisms to interfere with the chronicity of pathologies that are expressed after reproductive ages or have a polygenetic background.

II. BALANCE BETWEEN IMMUNE TOLERANCE AND IMMUNE AGGRESSION Immunological models such as “self-nonself” (Burnett, 1959), “infectious-nonself” (Janeway, Jr., 1989), the “danger model” (Matzinger, 2002), “missing self” (Ljunggren et al., 1990), and “altered self” (Medzhitov and Janeway, 2002) explain certain facets of CDIDs (including the relevance of auto-antigen or commensurate foreign antigen). It has been shown that tolerance against an antigen occurs when dendritic cells ingest and present this antigen in the context of low expression of MHC class II and co-stimulatory molecules and in a microenvironment in which the cytokine cocktail is of an anti-inflammatory nature (Lutz and Schuler, 2002). Different subsets of antigenpresenting cells (APCs)/macrophages guide the responses accordingly (Mantovani et al., 2004). In contrast, if this cocktail is pro-inflammatory, an aggressive response will follow (Lutz and Schuler, 2002). It became evident that the presence of self-reactive lymphocytes is rather normal in humans and mice

I. INTRODUCTION This chapter provides an integrated framework of the pathogenesis of chronic disabling inflammatory diseases (CDIDs) combining aspects of evolutionary biology, the main homeostatic supersystems, such as the immune, the nervous, and the endocrine systems, and others such as the reproductive system. The goal of this chapter is the integration of knowledge derived from different fields. The chronic inflammatory disease of rheumatoid arthritis (RA) is used as a prototypic disease to illustrate the proposal that there are not PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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many years before the first joint symptoms appeared (Figure 1) (Rantapää-Dahlqvist et al., 2003). Interestingly, such auto-antigens can be products of posttranslational modifications of harmless self-proteins that can allow immune recognition of neo-self epitopes (Anderton, 2004). The epidemiological study from Sweden mentioned above is outstandingly important because it demonstrates that auto-antigenicity is not immediately accompanied by symptomatic joint disease: “The immune response is restricted to hidden players.” When the disease becomes symptomatic, many other cell types such as neutrophils, fibroblasts,

se

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tig en –

rela

te d

phe no me na

(Agrawal et al., 1991; Tao et al., 1985). In the pre-symptomatic phase of an immune-mediated joint disease, a limited number of cell types are involved. If, as in the case of RA, the etiological concept of an antigen-driven disease (self or foreign) is accepted, T-cells, B-cells, and APCs will play the major role in the pre-symptomatic phase (as easily recognized in animal models, see below). Not many other cell types contribute to the initiation of the joint disease. In this context, it is worth mentioning that increased antibody titers against possible RA auto-antigens (cyclic-citrullinated peptide [CCP] and IgA rheumatoid factor) have been detected

a re inc

hiden players: T- & B-cells and APCs

FIGURE 1 Autoimmune phenomena are present during the pre-symptomatic phase in rheumatoid arthritis. In the pre-symptomatic phase of the disease, T-cells, B-cells, and antigen presenting cells (APCs) dominate the immune response. Since no symptoms appear, a homeostatic balance exists between aggression and tolerance towards a self or foreign antigen. In the symptomatic phase of the disease, aggression outweighs tolerance, and other cell types are involved in the inflammatory process. The inlay demonstrates an opened knee joint. Arrows indicate the proliferation zone of synovium (pannus formation) that destroys the cartilage. The interval between first detection of autoimmune phenomena (auto-antibodies) and first symptoms can last 10 years (Rantapää-Dahlqvist et al., 2003. Arthritis Rheum., 48, 2741–2749).

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10. Neuroendocrine-immune Interactions and Evolutionary Biology TABLE 1

Effect of Various Mediators of the Nervous and Endocrine System on Function of Dendritic Cells

Mediator

Effect on dendritic cells

Refs

norepinephrine substance P calcitonin gene-related peptide vasoactive inflammatory peptide (VIP) cortisol estrogens

Regulates antigen presentation by Langerhans cells Contributes to T-cell proliferation induced by dendritic cells Inhibits B7-2 expression Synergizes with TNF in inducing human dendritic cell maturation Inhibits the maturation of dendritic cells and the presenting capabilities Stimulate dendritic cell function and differentiation

testosterone

Downregulates dendritic cell density in the skin

Seiffert et al., 2002 Lambrecht et al., 1999 Torii et al., 1998 Delneste et al., 1999 de Jong et al., 1999 Komi et al., 2000; Komi et al., 2001 Galasso et al., 1996

leading to an unfavorable preponderance of aggressive factors must occur during aging. Age-related changes of all supersystems might play a critical role.

III. GENES RESPONSIBLE FOR ADVANTAGEOUS REACTIONS TO OVERCOME CHRONIC DISABLING INFLAMMATORY DISEASES WERE NOT EVOLUTIONARILY CONSERVED FIGURE 2 Increased incidence of rheumatoid arthritis during aging in women. It is obvious that incidence particularly increases in the peri- and post-menopausal time.

osteoclasts, endothelial cells, chondrocytes, mast cells, osteoblasts, nerve fibers, stem cells, and fat cells are involved in the local destructive process (Figure 1). The role of the initial secret players—the T-cell, B-cell, and APCs—decreases while other cell types become important and the characteristics of the disease gradually change. Thus, a pre-symptomatic and a symptomatic phase of arthritis can be defined (Figure 1). In most animal models of autoimmune diseases, co-injection of a strong immune stimulus such as Freund’s adjuvant is needed to guarantee a pro-inflammatory condition during ingestion and presentation of the antigen (pre-symptomatic phase). During situations that are not polarized regarding aggression versus tolerance, hormones and neurotransmitters may become decisive (Table 1). If an acute inflammatory disease is not rapidly resolved, aggression will predominate (see below) and the disease will become chronic. The response favored will mainly depend on the antigen (concentration, epitope, antigen receptor density), the dendritic cells involved, involvement of pattern recognition receptors, and the microenvironment (Mantovani et al., 2004; Murray, 1998). In the case of RA, it is interesting that the incidence of the disease increases during aging (Figure 2) (Masi, 1994). It seems rather obvious that homeostatic changes

A. Advantageous Reactions Were Not Conserved At the time of the homo habilis (2 Mio years ago), the reproductive period lasted about 25 years (Baur et al., 2001). However, we know now that many CDIDs appear after the age of 35 years (e.g., RA). This has not allowed retaining specific genes responsible for advantageous reactions to overcome late-manifesting CDIDs. Prolongation of life as consequence of hygienic and nutritive practices and modern medicine were not predicted by biological evolution. In contrast, under natural conditions the development of a chronic disease is a handicap for the individual (e.g., in competition for nutrients and vulnerability to predators), implying a strong negative selection pressure. In addition, systemic inflammatory episodes lead to the production of cytokines that mediate a strong inhibition of the hypothalamus-pituitary-gonadal axis at different levels, resulting in inhibition of reproduction (Adashi et al., 1989). It has been shown that such inhibition also occurs in humans (Tsigos et al., 1999): IL-6, injected into healthy male volunteers in doses that lead to circulating IL-6 levels of a magnitude similar to that observed during severe inflammatory stress, causes a significant decrease in testosterone levels, which return to baseline concentrations only 7 days later (Tsigos et al., 1999). Thus, a long-lasting increase of IL-6 levels, which is typical during RA or juvenile idiopathic arthritis, would hinder testosterone production. Even today under optimal therapy, CDIDs such as juvenile

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idiopathic arthritis significantly diminish the probability of successful conception (Ostensen et al., 2000). Most likely, such linkage between blunted reproductory functions and pro-inflammatory cytokines has not allowed the evolutionary acquisition of well-adjusted immuneneuro-endocrine responses to impede or modulate the chronic development of a disease. A typical example is the adult form of RA, a CDID with severe polyarticular inflammatory symptoms. We hypothesize that under natural conditions this disabling disease would be hardly expressed for a long time (as a chronic disease) because of the need to cope with the environmental threats and to compete for nutrients. However, an individual that would suffer from RA later in life can transmit before to the progeny the genetic background that predisposes to the disease. Conversely, advantageous mechanisms to prevent late-manifesting RA and other CDIDs might not have been evolutionarily conserved because possibly advantageous genes were not retained in the offspring. In addition, genes that may be beneficial in the early period of life (e.g., for reproduction) may be deleterious in an elderly person suffering from a CDID. In aging research, this is called “the pleiotropy theory of aging” (Kirkwood and Austad, 2000), but it can also be applied to CDIDs. To this, it should be added that some evolutionarily conserved programs may be dangerous for the individual but favorable for the species (Box 1).

Box 1 1. The antechinus Antechinus is a marsupial the size of a large mouse, with a pointed snout and a short-haired, medium-long tail. It has short broad feet of buff to yellow-brown color. Copulation occurs during a short season in winter. The males, driven to somewhat frenzied sexual activity due to raised testosterone levels, compete vigorously for females. Copulation can last up to 24 hours. Within 3 weeks, almost all the males in the population are dead. This male die-off is largely brought on by the high stress levels associated with the physiological changes brought on by the breeding period. The animals die due to severe infection, particularly, owing to parasites. Removing male antechinus from the natural habitat leads to a life expectancy similar to female animals. 2. The leaping salmon Salmon spend part of their life as freshwater fish (youth), part as saltwater fish (adulthood), and during reproduction again as freshwater animals. When they return to rivers from the ocean to give birth, salmon do not eat. Spawning salmon live solely on accumulated muscle reserves for

nearly a year. In rivers brimming with food, 20-pound salmon will drive themselves to the edge of starvation. Over the 3 to 5 months between arriving from the ocean, migrating upstream, and spawning in the late fall, salmon expose themselves to predators. Salmon in rivers will jump and roll and swirl on the surface of pools, for no apparent reason, even though this reveals their location to predators and makes them vulnerable to death or injury during that critical period before they mate. After mating, salmon die due to exhaustion and parasitic infections.

B. Prerequisites for CDIDS Are Conserved—The Accumulation Theory It is still necessary to explain why genetic (e.g., HLA-linked) predisposition for CDIDs that appear before or during the reproductive period has been retained. In our view, an explanation for this apparent paradox resides in the fact that CDIDs have a multifactorial genetically polymorph background. For example, HLA-B27 is important for ankylosing spondylitis (relative risk = 90). HLA-B27 could have been retained in the offspring because in most cases the disease does not develop due to the lack of additional factors (genetically inherited or environmental). In RA, many other genetic prerequisites, which depend on the population, have been described (Figure 3, grayshaded area). The genetic prerequisites for a CDID can be retained over generations via individuals that never express the disease because relevant co-factors may either not occur or are expressed following very irregular patterns and only after the reproductive phase. This scenario, which we have called the “accumulation theory” (Straub and Besedovsky, 2003), would allow the appearance of CDIDs in the young.

IV. HOMEOSTATIC MECHANISMS ARE EVOLUTIONARILY CONSERVED FOR TRANSIENT INFLAMMATORY REACTIONS—THEIR MISUSE IN CHRONIC DISABLING INFLAMMATORY DISEASES A. The Versatility of the Immune System—The Dilemma in CDIDs The immune system (including tissue repairing by mechanisms comparable to those used by the reproductory system), the nervous system, and the endocrine system are homeostatic supersystems that integrate many reactions occurring during transient inflammatory events (see Table 2 later in this chapter).

10. Neuroendocrine-immune Interactions and Evolutionary Biology

FIGURE 3 Integrated view of factors influencing chronic inflammatory rheumatic diseases. The basis of rheumatoid arthritis is a genetic background. Several polymorphisms have been suggested to play a role (gray area, left part, abbreviations, see below), and they are a prerequisite for the development of rheumatoid arthritis. During early life, an asymptomatic phase of the disease exists during which serologically detectable autoimmune phenomena are already present (yellow area, autoantibodies can be observed years before disease outbreak). Environmental triggers may contribute to initiate this early phase. Particularly, adaptive immune responses play a predominant role (dendritic cells, T-cells, B-cells). In addition, nervous and endocrine factors can intensely modulate leukocyte migration and distribution. In the symptomatic overt phase of the disease (red area), the role of the initial players—the T-cell, B-cell, and dendritic cell—simultaneously decreases while other cell types become involved in the inflamed area. Various mediators may stimulate the immune response in phase P1, but the same mediators can inhibit immune response in phase P2. These differential effects are due to the different components involved. Furthermore, in the asymptomatic phase of the disease (yellow area), neuroendocrine axes are probably intact, whereas in the symptomatic phase these axes are markedly altered possibly due to inherited deficits in responding to inflammation. Abbreviations: ACE, angiotensin converting enzyme; CRH, corticotropin-releasing hormone; FCγRIIIA, Fc-gamma receptor IIIA; GRβ, glucocorticoid receptor beta; MMP-3, matrix metalloproteinase 3; NK cells, natural killer cells; PADI, peptidylarginine deiminase 4 (initiates cyclic citrullination); SLC22A4, organic cation transporter; TNF, tumor necrosis factor; TNFRII, TNF receptor type II.

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During approximately 109 years, an extremely efficient pattern of receptors for recognition of foreign agents has been evolutionarily conserved in our genome (Marchalonis et al., 2002). Responses triggered by stimulation of these receptors are immediate, non-clonal, independent of gene re-arrangement, and the repertoire of pattern recognition receptors is small. In contrast to innate immunity, the younger adaptive immune system (arising later, particularly with vertebrates 450 Mio years ago) demonstrates a striking dissociation from these evolutionary older elements: Genes in Tand B-cells allow recognition of a huge number of 109 to 1011 antigenic determinants. In antigen-activated peripheral B-cells, every infection leads to a high rate of somatic point mutations (hypermutation, genes are not fixed in the genome), which improves affinity for the antigen. This versatility bears a highly significant risk for errors in the discrimination between self and foreign, which is most likely the dilemma in CDIDs (the starting point). The big advantage of the adaptive immune system is its memory for the antigen, the specificity, and the diversity (that allows the recognition of future emerging antigens) (Abbas et al., 1996; Marchalonis et al., 2002). This has been evolutionarily advantageous since about 94% of the human population profit from the adaptive immune system (during infections, etc.), whereas only 6% suffer from CDIDs. Which are the homeostatic mechanisms that have been evolutionarily conserved for transient inflammatory reactions but are mistaken in CDIDs?

B. The Immune System The evolutionarily conserved reaction against an unwanted antigen is the continuous attack to eliminate it, a process that is obviously beneficial during infection. In the case of a harmless self or foreign antigen, the continuous attack is no longer meaningful. In RA, a large list of self-antigens has been reported (Bläß et al., 1999), and some epitopes are identical for the human disease and in experimental models (Burkhardt et al., 2002). However, whether RA is an autoimmune disease with broken tolerance against self rather than a disease with an inflammatory reaction against a foreign antigen is still a matter of debate. Nevertheless, RA is a severe CDID with a continuous strong proinflammatory and destructive behavior (continuous aggression). In CDIDs, reactions of the immune system are accompanied by a permanent aggressive attack with the purpose of completely eliminating the probably harmless antigen. The evolutionarily conserved reaction used to overcome infectious agents is no longer meaningful in such a situation.

C. The Endocrine System In the early phase of an inflammatory episode, an immediate rise of cortisol, epinephrine, and norepinephrine in plasma is observed (Christeff et al., 1987; Jones et al., 1984; Suzuki et al., 1986). These endocrine reactions were most likely conserved to control inflammatory reactions and cytokine production (Besedovsky et al., 1986). In addition, it has been demonstrated that cortisol and norepinephrine are necessary to establish an adequate immune reaction (Del Rey et al., 2002; Kohm and Sanders, 2000; Madden et al., 1989) and an early mobilization and redistribution of immune cells (Dhabhar et al., 1995; Toft et al., 1994). However, a prolonged increase in the activity of the HPA axis and the sympathetic nervous system during immune processes predispose to severe infections. Furthermore, endotoxin–induced increase of cortisol levels is achieved at the expense of adrenal androgens (Straub et al., 2002d), which are anti-inflammatory (reviewed in Cutolo and Wilder, 2000). Thus, the fast cortisol rise and fall and the loss of adrenal androgens have been evolutionarily conserved to support a strong immune reaction during infection. In the case of a prolonged immune aggression such as in RA, these evolutionarily conserved mechanisms are used in a wrong way: A rapid loss of the anti-inflammatory mediators cortisol and androgens and their continuous inadequate production are unfavorable in CDIDs (Crofford et al., 1997; Cutolo et al., 1988; Masi et al., 1984; Straub et al., 2002c). Prolactin, another important endocrine mediator, should be taken into account. During an infectious disease, the levels of prolactin, a stimulator of the immune system (Matera et al., 1992; Spangelo et al., 1987), sharply increase (Feldman et al., 1989). The evolutionarily conserved increase in prolactin levels during an infectious episode particularly serves to shelter mother and baby during breast-feeding. In RA and other CDIDs, this endocrine response provides a continuous unfavorable pro-inflammatory condition (Jorgensen et al., 1995). Again, the evolutionarily conserved response to transient infectious diseases or other harmless inflammatory episodes has adverse consequences in CDIDs.

D. The Nervous System Following a physical injury of the skin, with or without infectious agents, a typical wound response is observed (Figure 4): (1) The sensory nervous system signals this event to the central nervous system. (2) In parallel, substance P is locally released from sensory

10. Neuroendocrine-immune Interactions and Evolutionary Biology

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FIGURE 4 Sequence of local events during wound healing. The normal wound-healing process lasts approximately 3 to 5 weeks. The behavior of sensory and sympathetic nerve fibers is indicated in red. Abbreviations: ECM, extracellular matrix.

nerve terminals and attracts monocytes and neutrophils (Roch-Arveiller et al., 1986; Ruff et al., 1985). Substance P activates these cells to produce pro-inflammatory cytokines such as IL-1β and TNF (Lotz et al., 1988). (3) In a later phase of the wound-healing process, substance P stimulates fibroblasts to generate matrix in order to close the wound (Nilsson et al., 1985). The reaction of the sensory nervous system is accompanied by peripheral and central sensitization that imprints pain pathways (Schaible and Grubb, 1993), an evolutionarily conserved learning phenomenon to heal the wound. (4) On the other hand, sympathetic nerves hinder the wound-healing process (Perez et al., 1987; Wucherpfennig et al., 1990). Thus, during 2 up to 21 days following a wound reaction, the preponderance of sensory over sympathetic nerve fibers was evolutionarily conserved for wound healing (Reynolds et al., 1995; Westerman et al., 1993). In the case of an improper immune reaction such as in RA, the same wound-healing processes (Miller et al., 2000; Pereira da Silva and Carmo-Fonseca, 1990) co-exist with an inadequate cortisol secretion (Straub et al., 2002a; Straub et al., 2002c) supporting the continuation of a local aggressive immune response. This evolutionarily conserved wound reaction is no longer advantageous in a CDID. Another phenomenon occurring during acute infectious disease is sickness behavior, in the form of malaise, fatigue, numbness, coldness, muscle and joint aches, reduced appetite, anxiety, and depressive mood triggered by pro-inflammatory cytokines in the central

nervous system (Bluthe et al., 1994; Reichenberg et al., 2001). The behavioral components of sickness represent, together with the fever response and the associated neuroendocrine changes, a highly organized strategy evolutionarily conserved and beneficial during acute infections. In a CDID such as RA, the same pro-inflammatory cytokines induce comparable symptoms such as depression, anxiety, hypersensitivity, and numbness (reviewed in Herrmann et al., 2000). If this evolutionary conserved behavior is prolonged, it becomes deleterious for the individual.

E. The Reproductive System Before the embryonic blastocyst can enter the mucosa, the mucosa must be prepared by estrogens and progesterone (Shifren et al., 1996). Progesterone and 17β-estradiol are strong stimulators of vascular endothelial growth factor (VEGF) (Shifren et al., 1996). Furthermore, adhesion molecules such as beta 1 integrins and alpha5 beta3 integrin are needed for attachment of the blastocyst (Yoshimura et al., 1998). The 17β-estradiol is a stimulator of these adhesion molecules (Shiokawa et al., 1996). The same adhesion molecules play an essential role in angiogenesis during a wound-healing process, and in leukocyte traffic and homing (Tonnesen et al., 2000). During the transient inflammatory wound-healing process, the repair system is stimulated so that multipotent stem cells can enter the tissue, which is prepared by estrogens and supported by vessel formation. During RA, the inflam-

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matory process leads to the local preponderance of estrogens versus androgens (Castagnetta et al., 2003). These estrogens upregulate adhesion molecules and most probably induce neovascularization via VEGF (Cid et al., 1994) and placental growth factor (PlGF) (Ziche et al., 1997). The same mechanisms are continuously used during an immune aggression against a harmless antigen, which adds to the continuation of inflammation (Bottomley et al., 2000; Koch et al., 1994). It is possible that these “erroneous” mechanisms particularly operate in women with a perfect estrogen/ progesterone-driven reproductive system during a CDID in later life (pleiotropy theory). Interestingly, the above-mentioned factors such as VEGF and PlGF are important repellents of sympathetic nerve fibers (Neufeld et al., 2002). One may speculate that these sympathetic nerve-repellent factors impede the sympathetic innervation of the placenta. Similar mechanisms are likely used during placental growth and wound repair, leading to sympathetic nerve fiber repulsion and preponderance of pro-inflammatory sensory nerve fibers (see above).

V. UNCOUPLING OF SUPERSYSTEMS DURING CDIDS Synchronization and desynchronization of hormonal/neurotransmitter systems have been evolutionarily conserved for normal life and transient inflammatory episodes in order to maintain bodily functions. For example, plasma levels of prolactin, growth hormone, and melatonin are increased and those of cortisol and norepinephrine are decreased during nighttime (evolutionarily conserved desynchronization). This particular desynchronization is thought to be important for circadian rhythms of immune system activation (Born et al., 1997). Compared with wakefulness, early nocturnal sleep induces a shift in the Th1/Th2 cytokine balance towards increased Th1 activity, as indicated by an increased ratio of IFN-γ/IL-4–producing T helper cells (Dimitrov et al., 2004). The nighttime hormonal profile probably supports the Th1 preponderance. Could it be an explanation that during sleep an animal is more susceptible to be attacked and wounded, and therefore, it would be beneficial to promote inflammatory reactions at this time? Another example is the evolutionarily conserved synchronization of cortisol and norepinephrine release during acute stress situations. Since cortisol supports several effects of norepinephrine and vice versa, blood pressure and energy supply are maintained during acute stress responses. These examples indicate that

synchronized and desynchronized programs were evolutionarily conserved to maintain homeostasis during normal life and non–life-threatening episodes (e.g., transient infectious disease). How does this concept apply to CDIDs? It has been recently demonstrated that in patients with Crohn’s disease and ulcerative colitis, two prototypic CDIDs, the synchronization of the sympathetic nervous system and the HPA axis is disturbed (Straub et al., 2002b). Under continuous inflammatory stress in these CDIDs, the sympathetic nervous tone increases, whereas the HPA axis tone remains relatively normal (Figure 5A). Despite direct activation of the adrenal glands by circulating cytokines such as IL-6 (EhrhartBornstein et al., 1998), cortisol secretion is inadequately low in relation to cytokine serum levels (Straub et al., 1998; Straub et al., 2002c). A very similar phenomenon with an increased tone of the sympathetic nervous system and a low normal activity of the HPA axis was observed in patients with RA and systemic lupus erythematosus (P. Härle and R.H. Straub, 2006). An “uncoupling” between the activities of the sympathetic nervous system and the HPA axis was also detected early during ontogeny in an animal model of systemic lupus erythematosus (A. del Rey et al., submitted). It seems that continuous high output steroid hormone secretion is not evolutionarily conserved, whereas continuous activation of the sympathetic nervous system is possible (= uncoupling of supersystems). We hypothesize that the increase of the sympathetic nervous tone may compensate for disturbed cortisol secretion and lower density of sympathetic nerve fibers in inflamed tissue (Figure 5B). In inflammatory diseases, coupling of the HPA axis and the sympathetic nervous system may be important to dampen peripheral inflammation due to cooperativity between cortisol and norepinephrine, which has recently been demonstrated in patients with RA (Straub et al., 2002a). Thus, uncoupling of these supersystems may be deleterious in CDIDs.

A. The Time-point When CDIDs Become Chronic Two, not mutually excluding explanations are provided which indicate a possible time-point when CDIDs become chronic: 1. It should be considered that the above-mentioned adaptation processes to transient inflammatory reactions are terminated within 3 to 5 weeks (Table 2). If the immune reaction against a harmless antigen (self or foreign) is not successful, the antigen is not eliminated after 3 to 5 weeks, but coordinated antiinflammatory responses of the other supersystems may not operate properly after this time. According to

FIGURE 5 Uncoupling of the HPA axis and the sympathetic nervous system. (A) In healthy subjects cortisol levels positively correlate with plasma levels of neuropeptide Y, indicating coupling of the activity of these two systems. In patients with ulcerative colitis, the correlation between these parameters is negative. (B) In the pre-symptomatic phase without overt inflammation, the sympathetic nervous tone is synchronized with the tone of the HPA axis. In the symptomatic phase of the disease, the activity of the two axes is uncoupled. We hypothesize that uncoupling is an important factor that drives to chronicity.

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I. Neural and Endocrine Effects on Immunity TABLE 2

Problem Infection in general Infection during pregnancy Wound Foreign body reaction Embryonic implantation Unwelcome small inanimate objects (e.g., pollen)

Evolutionarily Conserved Transient Inflammatory Reactions Reaction of the supersystems Continuous attempt to eliminate infectious agents Hormonal support of the immune system Closing of a wound including implantation of adult stem cells in a wounded tissue Continuous attempt to get rid of a foreign body Uterine implantation of the embryonic blastocyst Allergic-type immune response to get rid of the particular allergen

our proposal, this may reflect that anti-inflammatory adaptive neuroendocrine responses were evolutionarily conserved to cope only with transient inflammatory processes. Thus, uncoupling of supersystems after 3 to 5 weeks may be an important prerequisite for chronicity. 2. The responses of the supersystems change with time. In the early phase of a CDID (first weeks, presymptomatic phase), clonal expansion of highly affine aggressive immune cells is most important, and the adaptive immune system is the main player. After appearance of overt clinical symptoms, the local inflammatory process—for example, in a joint— involves many other cell types such as neutrophils, macrophages, natural killer cells, fibroblasts, endothelial cells, nerve fibers, and others (evolutionarily the old players). Since the supersystems affect these two phases of the disease in a different way, their influence on the course of a CDID must change when the local inflammatory process acquires a dominant role. This happens within the first weeks after appearance of clinical symptoms (symptomatic phase of arthritis) (see Figure 3). It is interesting that important hormonal and neuronal factors change during the course of CDIDs so that in the chronic phase (after 5 weeks) the systemic situation and the local micromilieu in the synovium demonstrate predominantly pro-inflammatory patterns (Figure 6).

VI. THE HEN-AND-EGG PROBLEM OF CDIDs—WHO STARTS THE DISEASE? A. The Erroneous Reaction against a SelfAntigen or a Harmless Foreign Antigen Starts the Disease At the beginning of CDIDs (in a pre-symptomatic phase), immune tolerance against self-antigens is

Typical duration of the reaction (days) ≤21 ≤21 ≤21 ≤21 ≤21 (until deep implantation) very fast, short-lasting

broken, or an inadequate immune response to harmless foreign antigens is triggered. T-cells, B-cells, and APCs are hidden players of the disease process in the pre-symptomatic phase of the disease (Figure 5B). Then, the disease, probably because of an additional trigger, strides into the symptomatic phase that involves many other cell types. Continuous aggression against an antigen leads to a dissociation between the immune system and the other supersystems (Figure 5B). Now, CDIDs become chronic due to the aberrant expression of programs that have been preserved to cope only with acute events during transient inflammatory reactions (uncoupling) (Figure 5B).

B. Age-related Changes of Supersystems Start the Disease We have mentioned that aging is an important prerequisite for the development of RA (Figure 2). During aging, adrenal cortisol production remains relatively stable, whereas the production of adrenal and gonadal androgens as well as of gonadal estrogens continuously decreases (more obvious in women than in men, particularly after menopause) (Straub et al., 2001). In addition, plasma levels of norepinephrine increase in relation to other steroid hormones including cortisol (Straub et al., 2001) (Figure 7). Aging is accompanied by a decrease of sympathetic innervation and a general increase of the sympathetic nervous tone (Bellinger et al., 2001; Straub et al., 2001) (Figure 7). Several other forms of uncoupling phenomena, which are beyond the scope of this chapter, are apparent during the aging process. During aging, uncoupling is a slowly progressive process (Figure 7), which may be paralleled by hidden immune phenomena as mentioned above (Figure 1). Autoimmunity seems to be a rather normal phenomenon in a healthy person, which is supported by a specific genetic background and largely controlled in the pre-symptomatic phase of CDIDs. It is conceivable that the uncoupling of homeostatic programs during the process of aging is enough to drive a process

FIGURE 6 Evolutionarily conserved changes of the supersystems related to acute immune challenges, whose maintenance in CDIDs induces an overall pro-inflammatory situation. Timepoint zero indicates the starting point of the overt inflammatory process. Day X indicates a later time-point in the symptomatic chronic inflammatory disease. Changes include inadequate low levels of systemic cortisol (A) (two different time courses of cortisol increase are demonstrated), decreased local androgens, progesterone, and 2-hydroxyestrogens (C), decreased local sympathetic neurotransmitters due to sympathetic nerve fiber repulsion (E), and elevated systemic levels of prolactin (B), locally increased 17β-estradiol and 16-hydroxylated estrogens (D), and high local levels of substance P due to sensory hyperinnervation (nerve fiber sprouting) (F). In relation to the initial levels, the systemic or local level of all mentioned factors changes into an aggressive direction (red zone) and never into an anti-inflammatory direction (blue zone). The larger symbol for the antigen indicates that sometimes access to a self or foreign antigen increases during the symptomatic phase of a CDID. Abbreviations: β-END, β endorphin (from sympathetic nerve terminals); A, adrenaline; NE, norepinephrine; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide.

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FIGURE 7 Uncoupling of the HPA axis and the sympathetic nervous system during the process of aging. This example shows that during early life (until 25 years of age) the activities of the HPA axis and the sympathetic nervous system are synchronized. Evolutionarily conserved synchronization of the response of these two systems is necessary in order to provide cooperative effects, which contribute to prevent overt immune reactions against self or foreign antigens. Uncoupling of the activity of the two systems during aging steadily drives the aggressive immune process. At a certain time-point, with or without an exogenous trigger, the symptomatic phase of the disease starts, and uncoupling of the two supersystems is boosted, leading to further increased uncoupling phenomena.

to a symptomatic disease and chronicity, with or without an additional environmental trigger (Figure 6). Probably both an inadequate immune response and age-related changes of the supersystems play a role in this process. For both theories, genetic prerequisites are important (Figure 3, gray-shaded area). The genetic background is not restricted to genes within the immune system. Background genes controlling the other supersystems are certainly equally important (Masi et al., 2003). The integrated framework provided in this chapter demonstrates that all supersystems concomitantly add to the nature of a CDID.

Acknowledgments This work was supported by several national grants from the Deutsche Forschungsgemeinschaft (DFG), the VolkswagenFoundation (VWS), and the Nationales Genom-Forschungsnetz (NGFN) (Straub: Str 511/5-1,2,3; Str 511/9-1,2,3; Str 511/10-1,2; Str

511/11-1,2; Wi 1502/2-1; SFB 585/B8, Str 511/14-1; Str 511/15-1; Str 511/16-1; Str 511/17-1; VWS I/68950; ReFormC program of the University Hospital Regensburg; Besedovsky and del Rey: RE 1451/2-2; SFB 297/C1; VWS I/67419; (NGFN) NV-S14T02).

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C H A P T E R

11 Neuroendocrine Regulation of Cancer Progression: I. Biological Mechanisms and Clinical Relevance ANIL K. SOOD, SUSAN K. LUTGENDORF, AND STEVEN W. COLE

I. INTRODUCTION 233 II. OVERVIEW OF THE STRESS RESPONSE SYSTEM 235 III. CANCER INITIATION 235 IV. CANCER METASTASIS 237 V. NEUROENDOCRINE EFFECTS ON CANCER-RELATED BIOLOGICAL PATHWAYS VI. SUMMARY 242

cellular, and molecular processes that might mediate those relationships.

A. Psychosocial Factors and Cancer Incidence 239

The question of whether psychosocial factors pose risks for development of cancer has been the focus of longstanding attention. (6,7) Galen, the ancient Greek physician, stated that women with a “melancholic” disposition were more likely to develop cancer than their counterparts with a more “sanguine” (positive) disposition. (8) Two recent meta-analyses and a review paper have found no overall link between stressful life events and development of cancer (9,10); and a third meta-analysis indicated that although some significant links were found between stressful life events, personality factors, and development of cancer, the moderate effect sizes suggested that the variance contributed by psychosocial factors to cancer initiation was relatively small. (11) However, when greater specificity is applied to these findings, relationships start to emerge. For example, presence of severe life events, or specific severe circumstances such as death of a child or spouse have been more consistently associated with increased cancer risk (6,9,12,13), with findings differing according to cancer site (14,15). The Lillberg (2003) study was notable for its large sample size of 10,808 women. (13) In a similar vein, Giraldi and colleagues found that the occurrence of severe stressful life events in the 6 months

I. INTRODUCTION Clinical and epidemiological studies over the last 25 years have identified psychosocial factors including stress, chronic depression, and lack of social support as risk factors for the incidence and progression of cancer. (1) Relationships between behavioral risk factors and cancer onset are inconsistent (see review in Chapter 41 of this volume), but behavioral factors are more consistently found to forecast differences in disease progression for already-established cancers. These clinical findings parallel a large animal research literature showing that experimentally imposed stress can alter cancer progression. (2–5) Recent molecular studies have begun to identify specific signaling pathways by which neuroendocrine correlates of behavior might impact tumor growth and metastasis. This chapter will briefly review the clinical relationship between behavioral factors and cancer pathogenesis, and then consider in more detail the neuroendocrine, PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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before a breast cancer diagnosis was associated with larger tumor volume at surgery (16). In a study unique for its repeated assessments of depression, Penninx and colleagues assessed depression prospectively (in 1982, 1985, and 1988) in 4,825 older adults in a population-based study (17). Those individuals reporting chronic depression (positive at each of the three time-points) had almost a two-fold (1.88) increase in cancer incidence compared to those with no depression. When depression at a single time point was used, it did not predict cancer incidence, suggesting a potential contribution of chronic, but not short-term depression to carcinogenesis (17). Particular combinations of psychosocial factors may be most conducive to the development of cancer. For example, Price and colleagues reported that the combination of a highly threatening life stressor and low social support was related to a nine-fold increase in breast cancer incidence, whereas, individually, neither life stressors nor low social support was related to cancer incidence (18). Furthermore, specific psychosocial risks may act synergistically in combination with biological vulnerabilities, genetic predispositions, or exposure to smoking or environmental toxins to increase the risk for cancer development (19). Examination of these potential risk factor interactions may increase understanding of the complex interactions between these phenomena.

B. Depression, Stress, and Cancer Progression In general, psychosocial factors have more strongly predicted variations in the progression of established cancers than differential risk of cancer onset. A classic prospective study of 2,020 healthy middle-aged men by Shekelle reported that psychological depression was prospectively associated with a two-fold increase in risk of death from cancer (20). This relationship was independent of adjustment for age, smoking, alcohol, family cancer history, and occupational status. In six major studies since 1990, depressive factors have been found to predict cancer progression or mortality (see review in Spiegel, 2003) (1). For example, Everson and colleagues found a dose-response relationship between hopelessness and cancer mortality in a large-scale, prospective population-based study of 2,428 men in Finland (21). During 6 years of follow-up, those men with moderate and high levels of hopelessness had more than a two-fold increase in risk of mortality from cancer (of any site) (21). Stommel (1999) reported that cancer patients with a higher pre-morbid history of depression or functional limitations had a 2.6-fold greater hazard of dying than patients without these

problems, and the combination of these two risk factors increased cancer death risk by 7.6 times (22). These data suggest that chronic psychological or functional problems may convey a vulnerability for cancer development and aggressive tumor behavior. Watson and colleagues (23) found that among 578 women diagnosed with early stage breast cancer, high levels of depression predicted a 3.59 times increased chance of death (23). Depressed mood has been associated with poorer survival in studies of lung cancer and bone marrow transplant patients as well (24,25). In contrast, Tross and associates found no association between distress and disease progression in a cohort of 280 Stage II breast cancer patients followed over a 12–16 year period (26). Other researchers have also failed to find links between depression and survival in lung and in bone marrow transplant patients (27,28). Stressful events have not generally been linked to cancer recurrence or survival (16,29,30). Thus, there are several strong studies supporting a link between depression and cancer progression. Chronicity of negative affect, as manifested in depressed mood or hopelessness, appears to have stronger relationships to outcomes than do stressful events, suggesting that sustained activation of negative affective pathways may be the strongest links to cancer progression. Moderators of stress, such as social support, have been frequently studied with respect to cancer outcomes. Social support refers to an individual’s perceived satisfaction with social relationships, and is thought to play a major role in buffering psychological and biological stress responses (31). Several studies have linked high levels of social support to improved clinical outcomes in cancer patients. In breast cancer patients, social support has been related to longer survival in several large-scale studies (32–36), although negative findings exist as well (16,37). As summarized in Chapter 41, the strongest findings indicate that characteristics such as suppression of negative emotions, hopelessness, denial, and lack of social support are associated with poorer survival. These are generally long-standing trait-like individual difference factors rather than short-term characteristics such as mood or acute stress, and thus likely to exert sustained neurohormonal effects in the body. The biological pathways underlying the links between behavioral factors and cancer incidence or progression remain poorly defined in the human clinical setting, although animal studies have identified some potential mechanisms in the context of tumor cell growth processes and anti-tumor immune responses. The effects of stress on the immune system are reviewed elsewhere in this volume, and their relevance to cancer is considered in Chapter 12. In this chapter, we con-

11. Neuroendocrine Regulation of Cancer Progression: I. Biological Mechanisms and Clinical Relevance

sider in more detail the role of behavioral and neuroendocrine factors in directly regulating the activity of tumor cells. After reviewing basic processes involved in the initiation and progression of malignant cell growth, we consider how the neuroendocrine effects of behavioral dynamics might impact those processes to affect the incidence and progression of tumors. We conclude by considering the implications of those dynamics for the clinical treatment of cancer and the maintenance of health in cancer survivors.

II. OVERVIEW OF THE STRESS RESPONSE SYSTEM Biological responses to stress are mediated by CNS processes that evaluate circumstances as threatening and modulate peripheral neuroendocrine function to respond to that threat (38,39) Two major pathways by which CNS stress responses regulate peripheral function are the HPA and SNS (40,41), and the neuroendocrine mediators of these two systems can modulate cellular function in many of the peripheral tissue sites most relevant to cancer onset and progression. For example, there is emerging evidence that catecholamine neurotransmitters from the SNS (epinephrine and norepinephrine) play physiologically relevant roles in regulating the microenvironment of peripheral organs. The ovary provides one example highly relevant to cancers of the reproductive system. Overall concentrations of catecholamines are substantially higher in the ovary than in the plasma (42). Catecholamine levels in the ovary are known to be increased in response to stress due to increased sympathetic activity, which has been shown to result in appearance of precystic follicles (43–46). Similarly, catecholamines are present at substantially higher levels in the bone marrow microenvironment than in circulation and originate from nerve endings and bone marrow cells (47). Functionally, there is growing evidence that neuroendocrine factors can modulate hematopoiesis within the bone marrow microenviroment (47). The effects of catecholamines on cancer growth and progression are discussed below, but these studies suggest that stress-induced changes in catecholamines are robust enough to alter organ microenvironment and function. Both norepinephrine and epinephrine are known to be elevated in individuals with acute or chronic stress (48–50). Beta-adrenergic receptors (βAR) mediate many effects of catecholamines on target cells and have been identified on several cancer cell types, including breast cancer cells (51) and ovarian cancer cells (52), where the β2 subtype is the dominant adren-

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ergic receptor present. βARs are G-protein–coupled receptors, whose primary function is the transmission of information from the extracellular environment to the interior of the cell (53). Three distinct βAR subtypes have been identified (β1-AR, β2-AR, and β3-AR) (54– 56), and all three can signal by coupling to the stimulatory G-protein Gαs, leading to activation of adenylyl cyclase and accumulation of the second messenger cAMP. To verify the role of beta-adrenergic activation in mediating stress effects on tumor dynamics, some investigators have used agonists such as metaproterenol to demonstrate dose-dependent increases in, for example, the retention of tumor cells within the lung. Similarly, adrenaline injections have been shown to cause suppression of NK cell activity and promote tumor cell retention (57). Cortisol is a glucocorticoid hormone that is secreted by the adrenal cortex in response to stress (40,58,59). Social support and stress reduction are associated with lower cortisol levels (60,61). Glucocorticoids may have a bimodal effect on tumor cells. For example, Kawamura and colleagues have reported, at lower doses, dexamethasone can stimulate tumor growth, but at higher doses it inhibits tumor growth (62). In addition, cortisol may act in a synergistic fashion with catecholamines. For example, cortisol has been shown to potentiate the isoproterenol-induced increase in cAMP accumulation in lung carcinoma cells (63).

III. CANCER INITIATION Several lines of evidence indicate that tumorigenesis in humans is a multi-step process and that these steps reflect genetic alterations that drive the progressive transformation of normal human cells into malignant cells (64,65). The transformation of a normal cell into a cancerous state appears to require multiple ratelimiting steps including both genetic and epigenetic processes. Even in cultured rodent cells, tumorigenic potential requires at least two introduced genetic changes, and human cells are more difficult to transform (66). Hanahan and Weinberg (64) proposed that there are six essential alterations in cell physiology that dictate malignant growth: (1) self-sufficiency in growth signals, (2) insensitivity to growth-inhibitory (antigrowth) signals, (3) evasion of programmed cell death (apoptosis), (4) limitless replicative potential, (5) sustained angiogenesis, and (6) tissue invasion and metastasis. Each of these acquired capabilities reflects a breach in the normal host defenses against malignant cell growth. In some tumors such as Kaposi’s sarcoma and cervical cancer, viral infections contribute to the acquisition of some of these capabilities by delivering

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new genes into the cell (viral oncogenes). In other cases, physical damage to cellular DNA by carcinogens alters the expression or function of human genes. Regardless of whether tumors are initiated by viral infections or other carcinogens, the result is a somatic cell with enhanced growth capacity and a consequent increase in its ability to “evolve” within the body by acquiring new genetic alterations. Below, we focus on viral oncogenesis as a model for understanding biobehavioral influences on tumor initiation due to the well-characterized influences of behavioral stress and neuroendocrine factors on the activity of oncogenic viruses. However, other pathways for neuroendocrine influence clearly exist. For example, several studies have shown that stress can affect the cellular process involved in the repair of damaged DNA (67–70), providing a generalized mechanism by which behavioral factors might facilitate the establishment of oncogenic mutations. Behavioral factors can also increase tumor incidence via accelerated replication of hormoneresponsive cells, leading to increased opportunities for mitotic errors that may contribute to increased tumorigenesis in stressed animals (71–73). Several other fundamental processes of tumor initiation and evolution will be considered in the context of metastasis later (see also Chapter 12).

A. Viral Oncogenesis Some of the earliest experimental demonstrations that biobehavioral factors could influence cancer came from animal studies of virally induced tumors (74–76). As animal models of malignant cell growth emerged in the 1950s, 1960s, and 1970s, a raft of studies found accelerated growth of virally induced tumors in animals subject to experimental stress (4,5,77), as well as seemingly paradoxical protective effects of handling, fighting, and crowding (76,78–82). As the basic pathogenesis of these virally induced tumors became better understood, biological bases for these conflicting results became apparent in the diverse biology of distinct tumor types. Changes in neuroendocrine function were found to play a central role in stress effects on tumor growth, particularly elevated production of glucocorticoids (e.g., corticosterone, the rodent analogue of primate cortisol). Depending upon the particular viral model, glucocorticoids could modulate viral replication, activate viral oncogenes, enhance tumor metabolism, undermine cellular immune responses, or kill lymphoid cells (83–88). The latter dynamic was particularly significant in determining when stress would manifest paradoxically protective effects: Malignancies of hematopoietic and lymphoid

cells (leukemias and lymphomas) were generally inhibited by experimental stress because those cell types are especially vulnerable to glucocorticoidinduced programmed cell death (apoptosis). In fact, many chemotherapy protocols for lymphoid and hematopoietic malignancies include synthetic glucocorticoids as a central pro-apoptotic component. In contrast to leukemias and lymphomas, animal models of solid tumors were generally found to be exacerbated by stress due to the effects of glucocorticoids and catecholamines in undermining cellular immune responses (89–92) and enhancing tumor metabolism and viral oncogene expression (83–88). Several studies also found that stress effects on tumor growth varied depending upon whether the stress was applied before, during, or after tumor initiation, or depending on the gender or housing conditions of the animal (4,5,75–77). These results are consistent with the multi-step nature of cancer in suggesting that stress-related neuroendocrine dynamics might have different impacts at varying stages of tumor development or on different aspects of the malignant cell growth process. As evidence for viral contributions to human cancer emerged during the 1970s and 1980s (Table 1) (93–95), “stress hormones” have been found to influence the activity of all major human tumor viruses, including both RNA and DNA viruses (Table 2). Seminal studies from the laboratory of Glaser and colleagues found that prolonged psychological stress could induce subclinical reactivation of Epstein-Barr virus (EBV) in healthy people, and similar results have emerged from studies of astronauts and Antarctic research scientists (96–99). In many of these studies, HPA activity increased in parallel (100,101) and glucocorticoid hormones were subsequently found to enhance EBV gene expression in vitro (101,102). Subsequent studies have also shown that glucocorticoids can modulate the TABLE 1

Viral Oncology

• Viral infections contribute to approximately 15% of human cancers worldwide (95). Pathogenic mechanisms include expression of viral oncogenes (e.g., HTLV Tax, EBV EBNAs, and LMP1), inhibition of host cell tumor suppressors (e.g., HPV E6/p53 and E7/pRB), and genomic damage stemming from immune-mediated cell turnover (e.g., HBV, HCV) (93–95). • All human tumor viruses are sensitive to the intracellular signaling pathways activated by the HPA axis and ANS (see Table 2), which can reactivate latent tumor virus, stimulate oncogene expression, and inhibit host cell antiviral responses. • Vaccination is a major primary prevention strategy against viral tumors, and behavioral factors can influence the efficacy of this approach by modulating vaccine-induced immune responses (138–141).

11. Neuroendocrine Regulation of Cancer Progression: I. Biological Mechanisms and Clinical Relevance TABLE 2

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Human Cancer Viruses Influence by Stress Hormones

Human tumor virus Human Papilloma Viruses (HPV-16, HPV-33) Hepatitis B Virus (HBV) Hepatitis C Virus (HCV) Epstein-Barr Virus (EBV) Human T-cell Lymphotropic Virus (HTLV-I, -II) Kaposi’s Sarcoma-associated Herpesvirus (KSHV)

Malignancy

Sensitivity*

Cervical and head/neck cancer Hepatocellular carcinoma Hepatocellular carcinoma Lymphoma, Nasopharyngeal carcinoma Adult T-cell leukemia/lymphoma Kaposi’s Sarcoma, Primary Effusion Lymphoma

HPA HPA HPA HPA ANS ANS

*Presumptive based on in vitro studies.

activity of cancer-causing Human Papilloma viruses (HPVs) by enhancing viral gene expression (103–110), facilitating interactions between viral gene products and cellular proto-oncogenes (111,112), and undermining cellular immune responses by downregulating Class I MHC molecules (113). Glucocorticoid antagonists can inhibit HPV activity in vitro (114–116), providing a potential molecular mechanism for clinical data linking psychological risk factors to the progression of cervical dysplasia in HPV+ women (117,118). Hepatitis B and C viruses (HBV/HCV) come from completely different viral lineages, yet both respond to glucocorticoids by enhancing gene expression and replication (119–127). These dynamics are so pronounced that glucocorticoids have been employed clinically to activate HBV and HCV for eradication by replication-dependent antiviral drugs (125–128). Cancer-related viruses are also sensitive to catecholamines and the cAMP-linked PKA signaling pathway. Virologic mechanisms are especially well defined for Acquired Immune Deficiency Syndrome (AIDS)– associated malignancies. Catecholamines can accelerate HIV-1 replication by enhancing cellular susceptibility to infection (129,130), activating viral gene transcription (129), and suppressing anti-viral cytokines (131). People with high ANS activity show elevated plasma viral load and impaired response to anti-retroviral therapy (129), placing them at increased risk for AIDSassociated B-cell lymphomas (132,133). Catecholamines can also activate the Kaposi’s Sarcoma-associated Herpesvirus (KSHV) via PKA induction of the viral transcription factor Rta (134). The Human T-cell Lymphotropic Viruses (HTLV-I/-II) are sensitive to PKAmediated induction of the oncogenic Tax transcription factor (135). In addition to direct effects on viral gene expression, biobehavioral factors can also indirectly affect tumor viruses by modulating host immune responses. Antiviral vaccines will play a growing role in the primary prevention of cancer (136,137), and biobehavioral modulation of vaccine-induced immune responses

TABLE 3

Steps Involved in Metastasis

1. Proliferation and growth of primary tumor 2. Detachment or shedding of cells from primary tumor into circulation 3. Survival of cancer cells in circulation 4. Transport of cancer cells to new organs with subsequent arrest 5. Extravasation into surrounding tissues 6. Angiogenesis and growth of the metastatic tumor

becomes especially relevant (138–141). Neuroendocrine influences on immune response may also explain why oncogenic viruses so consistently acquire hormone-responsive replication dynamics. Viruses that coordinate their gene expression with periods of hormone-induced immunosuppression should enjoy a significant survival advantage. Similar selective pressures may also shape the evolution of non-viral malignancies (65), as genomic alterations are selected for their ability to evade immune clearance or capitalize on endocrine dynamics to optimize growth and metastasis.

IV. CANCER METASTASIS Cancer is a complex disease in which many basic processes including cell division, apoptosis, and motility are dysregulated. Metastasis is a process by which cancer cells spread from the primary tumor to distant organs followed by relentless growth at those sites (142). Despite improvements in diagnosis and therapy, most deaths from cancer occur due to metastases that are resistant to conventional therapy (143). The process of cancer metastasis consists of a series of sequential, interrelated steps, each of which can be rate limiting (Table 3). To produce clinically relevant lesions, metastatic cells must complete all the steps of this process (144). After the initial transformation and growth of these cells, vascularization must occur for a tumor mass to exceed 2 mm in diameter (145,146). Subse-

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quent steps in the pathogenesis of cancer metastasis include invasion, detachment, embolization, arrest in capillary beds of organs, extravasation into the organ parenchyma, evasion of host defenses, and progressive growth. The production of metastases, therefore, depends on continuous interaction of tumor cells with host homeostatic mechanisms (147,148). Failure to complete any of the sequential steps aborts the process of metastasis. It has long been recognized that some types of cancer show an organ-specific pattern of metastasis. In 1989, Stephen Paget published an influential paper describing the propensity of various types of cancer to form metastases in specific organs and proposed that these patterns were due to the “dependence of the seed (cancer cell) on the soil (the metastatic site)” (149). His analysis indicated a non-random pattern of metastasis, and he concluded that metastases formed only when the seed and soil were compatible. The regulation of metastasis can be considered in terms of a balance between growth-stimulating factors from both the tumor and host cells versus inhibitory signals. To produce metastasis, the balance is weighted toward the stimulatory signals. Even though Paget’s “seed and soil” hypothesis dates back to 1889, the mechanistic role of microenvironment has only recently begun to be defined. Tumor progression is a product of an evolving crosstalk between different cell types within the tumor and its surrounding supporting tissue, tumor stroma (150). The tumor stroma contains a specific extracellular matrix as well as cellular components such as fibroblasts, immune and inflammatory cells, and blood-vessel cells. The interactive signaling between tumor and stroma contributes to the formation of a complex multi-cellular organ (Table 4). For example, the expression of the epidermal growthfactor receptor coupled with the expression of growth factors in the host tissue such as transforming growth factor-α (TGF-α) plays a role in metastatic

TABLE 4

Molecular Regulators of Metastasis*

Biological process Survival Adhesion Migration/Invasion ECM remodeling Immune escape Angiogenesis Homing to target organs

Molecular factors IGF survival factors CAMs, cadherins, integrins c-Met/HGF signaling, FAK MMPs, IPA, heparanase MHC loss VEGF, bFGF, PDGF Chemokines/chemokine receptors, CD44

*This list is meant to be illustrative and not comprehensive with regard to molecular factors regulating metastasis.

growth of colon and other cancer cells in the liver (151). The organ microenvironment can markedly change the gene-expression patterns of cancer cells, and therefore their behavior and growth ability (150). Recent studies regarding chemokines and their receptors provide important clues regarding why some cancers metastasize to specific organs. For example, breast cancer cells frequently express chemokine receptors CXCR4 and CCR7 at high levels. The specific ligands for these receptors CXCL12 and CCL 21 are found at high levels in lymph nodes, lung, liver, and bone marrow, which are common sites for breast cancer metastasis (152,153). Emerging evidence suggests that factors related to certain biobehavioral states such as neuropeptides and neurotransmitters also influence various steps in the cascade of metastasis.

A. Animal Models for Studying Effects of Stress Appropriate animal models play a crucial role in understanding the effects of stress of cancer and other diseases. Restraint stress animal models, using a variety of restraining devices, have been widely used to study the effects of stress on immunity, course of infectious diseases, and cancer (154–158). Physical restraint using 50 ml conical tubes for varying lengths of time has been shown to effectively modulate norepinephrine, epinephrine, and glucocorticoid levels (154–158). Restraint systems have also been demonstrated to consistently result in elevations of IL-6 (159,160). Furthermore, restraint stress has been shown to reduce the anti-tumor efficacy of chemotherapy in C57BL/6 mice bearing Lewis lung carcinoma (161) and has been demonstrated to result in metallothionein induction, which may be responsible for chemotherapy resistance (162). The length of restraint in different studies has ranged from 1 hour to 12 hours daily (155–157,159,161,163,164). In C57BL/6 mice, within 3 hours, there is almost a 50% increase in corticosterone levels, and at 6 hours the corticosterone levels reach a maximal increase of over 100% (155, 157,159,161,164,165). Furthermore, restraint stress has been demonstrated to raise catecholamine levels in murine models by two- to five-fold (45,164,166,167). Other models used for stress studies include swim stress, surgical stress, social confrontation, and hypothermia—these models have also been shown to promote lung metastasis from injected breast cancer cells (57,168–170). Most of these models have been used in the context of effects on the immune system, and specific applications are described in detail in the accompanying Chapter 12. The effects of stress on other biological pathways are described below.


V. NEUROENDOCRINE EFFECTS ON CANCER-RELATED BIOLOGICAL PATHWAYS A. Proliferation and Growth of Primary Tumor and Metastases Tumor growth at the primary site depends initially on nutrient and oxygen diffusion. Subsequently, at metastatic sites, signals from autocrine, paracrine, and/or endocrine pathways influence tumor cell proliferation with growth dependent on the net balance of positive and negative signals (144). There are limited data regarding the direct effects of stress hormones on cell proliferation. Most data suggest that catecholamines suppress proliferation of normal cells such as keratinocytes (171), which may contribute to impaired wound healing in the context of stress. The effects of stress-related hormones on tumor cell proliferation may depend on the type of substance and tumor type. In mammary tumors, activation of βARs have been related to accelerated tumor growth (51,172,173). The cAMP responsive element-binding (CREB) protein is an important transcription factor activated by multiple signal transduction pathways in response to external stimuli including stress hormones (174,175). Several studies have revealed a role for the CREB family of proteins in tumor cell proliferation, migration, angiogenesis, and inhibition of apoptosis (174–176). However, in other models, catecholamines appear to inhibit tumor cell proliferation, which may be mediated by alpha-adrenergic receptors. Scarparo and colleagues tested the effects of the α1-adrenergic agonist phenylephrine and reported a dose-dependent decrease in melanoma cell proliferation, which was reversed by the α1-adrenergic antagonist prazosin (177). Similarly, Pifl and associates found that norepinephrine inhibited neuroblastoma cell growth, primarily in cells expressing the dopamine transporter (178). In cells with dopamine uptake, the share of G0/ G1 population of cells was significantly increased after treatment with norepinephrine. Bijman and associates examined the effects of isoproterenol and histamine on head and neck carcinoma cells and found that both agents resulted in a minor inhibition of clonogenicity, which could be reversed with propranolol and cimetidine, respectively (179). In prostate carcinoma, treatment with agents that induce cAMP, such as epinephrine, isoproterenol, forskolin, and dibutyryl cAMP, result in acquisition of neuroendocrine characteristics by epithelial prostate cancer cells (180). The neuroendocrine characteristics were manifested by dense core granules in the cytoplasm, the extension of neuron-like processes, loss of mitogenic activity, and

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expression of multiple neuroendocrine markers. The presence of these neuroendocrine cells has been linked to prognosis in prostate cancer patients (181,182). Interestingly, the neuroendocrine cells have minimal proliferative activity, but yet these cells provide paracrine stimuli for proliferation of surrounding carcinoma cells (183). Among the epithelial tumors, some decrease in proliferation may be reflective of a more invasive phenotype. Next, we will discuss the effects of glucocorticoid hormones on cancer cell proliferation. Zhao and colleagues determined the effects of glucocorticoids on prostate cancer cells (184). Cortisol, and its metabolite cortisone, stimulated the growth of prostate cancer cells in the absence of androgens and increased the secretion of prostate-specific antigen (184). These cells had a mutated androgen receptor. Simon and coworkers examined the effects of several steroid hormones on human mammary carcinoma cells and found that physiological concentrations of glucocorticoids enhanced proliferation by nearly two-fold (185). The role of glucorticoid hormones in the context of other neuroendocrine hormones remains to be studied with regard to effects on proliferation.

B. Adhesion Tumor cell adhesion to the extracellular matrix within tissues greatly influences the ability of a malignant cell to invade and metastasize to outlying tissues (186). The proteins of the extracellular matrix consist of type I and IV collagens, laminins, heparin sulfate proteoglycan, fibronectin, and other noncollagenous glycoproteins (187). Cell adhesion to these proteins is mediated in part by a group of heterodimeric transmembrane proteins called integrins, which are composed of a non-covalently associated α and β subunit that defines the integrin-ligand specificity (188). The upstream factors that regulate adhesion to matrix components are not completely understood, although cAMP has been shown to regulate the small GTPases RhoA and Rac in a protein kinase A (PKA)–dependent manner (189). Cyclic AMP is a common second messenger that regulates many cellular processes. Previously, PKA was thought to be the main target of cAMP in eukaryotic cells. However, recently Epac (exchange factor directly activated by cAMP), a widely expressed exchange factor for the small GTPases Rap1 and Rap2, has been shown to be a receptor for cyclic AMP as well (190,191). Importantly, Epac controls a number of cellular processes previously attributed to PKA. Cyclic AMP controls cell adhesion in many cell types, and recently a link between cAMP, Epac-Rap1, and regulation of cell adhesion has been established (192). Enser-

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ink and colleagues have demonstrated that isoproterenol promotes ovarian cancer cell spreading and adhesion to laminin-5 independent of protein kinase A but rather dependent on Epac 1 (193). Similarly, isoproterenol stimulated adhesion to fibronectin in a cAMPmediated Epac-Rap1 pathway (192). Treatment with isoproterenol induced both activation of Rap1 and phosphorylation of CREB. Isoproterenol-induced adhesion was insensitive to pretreatment with a PKA inhibitor. Thus, stress hormones may promote cellmatrix attachments of cancer cells. Such a mechanism would be particularly relevant for ovarian cancer where cancer cells slough off the primary tumor and then implant at multiple peritoneal sites.

C. Migration and Invasion To reach blood vessels or lymphatics, tumor cells must penetrate host stroma, including the basement membrane. The process of tumor cell penetration of the host basement membrane consists of attachment, matrix dissolution, motility, and then actual penetration (194). There is growing evidence that stress hormones may affect tumor cell motility and invasion. Drell and colleagues demonstrated that norepinephrine not only induces chemokinetic migration, but also attracts breast cancer cells chemotactically (195). Similarly, Masur and associates demonstrated that norepinephrine was a potent inducer of colon cancer cell migration, which was inhibited by beta-blockers (196). Furthermore, they demonstrate that norepinephrinestimulated migration was dependent on phospholipase Cγ and protein kinase Cα activity. Sood and colleagues have examined the direct effects of catecholamines and cortisol on the invasive potential (i.e., capacity of tumor cells to penetrate extracellular matrix) of ovarian cancer cells, and their production of key matrix metalloproteinases involved in tumor cell penetration of extracellular matrix (197). Stress levels of norepinephrine increased the in vitro invasiveness of ovarian cancer cells by 89–198% (198). Epinephrine also induced significant increases in invasion of ovarian cancer cells ranging from 64–76%, but cortisol did not significantly affect the invasive potential of cancer cells. The beta-adrenergic antagonist propranolol completely blocked the norepinephrine-induced increase in invasiveness. Norepinephrine also increased tumor cell expression of MMP-2 and MMP-9, and pharmacologic blockade of MMPs abrogated its effects on tumor cell invasive potential. These findings provide direct experimental evidence that stress hormones can enhance the invasive potential of ovarian cancer cells in vitro.

D. Angiogenesis Hormones associated with SNS activation may favor angiogenic mechanisms in human tumors. Norepinephrine has been shown to upregulate vascular endothelial growth factor (VEGF) in adipose tissue through beta-adrenoreceptor/cAMP/PKA pathway (199) and in two ovarian cancer cell lines (52). VEGF is a direct angiogenic molecule that plays an essential role in embryogenesis, physiologic angiogenesis, and neovascularization of malignancy (200). VEGF stimulates endothelial cell migration, proliferation, and proteolytic activity (201). The effect of norepinephrine with regard to VEGF stimulation was abolished by a beta-blocker, propranolol, and mimicked by isoproterenol and was thus thought to be mediated via βadrenoreceptors (52,199). The effects of stress on tumor angiogenesis have recently been examined using in vivo models. In orthotopic murine models of ovarian cancer, Thaker and colleagues demonstrated that chronic stress induced by daily periods of immobilization results in higher levels of tissue catecholamines, greater tumor burden, and a more invasive pattern of disease (202). Angiogenesis, as reflected by microvessel density counts, was significantly increased in the stressed compared to control mice tumor samples. Furthermore, VEGF mRNA and protein levels were significantly elevated in the stressed tumor samples. Continuous infusion of propranolol (non-selective beta-blocker) ameliorated the effects of stress on tumor burden and pattern of disease, thereby confirming the importance of beta-adrenergic receptors on ovarian cancer cells in an in vivo model (202). In line with these findings, recent clinical studies have shown links between higher levels of social support and lower serum VEGF levels in ovarian cancer patients (203). Furthermore, social support has also been linked to lower levels of interleukin-6 (IL-6), another proangiogenic factor, both in peripheral blood and in ascites from ovarian cancer patients (204). Tumor-produced angiogenic molecules may further contribute to cancer progression by impairing immune cell function. Factors such as VEGF and tumor necrosis factor (TNF) may impair both effector function and early stages of hematopoiesis. Mice receiving transplants of Lewis lung carcinoma cells develop thymic aptrophy (205). VEGF causes a defect in the functional maturation of dendritic cells (DCs) from early hematopoietic progenitor cells (206,207). In addition to DC defects, non-tumor–bearing mice treated with recombinant VEGF have a decreased number of T-cells and a decreased T-cell to B-cell ratio in their lymph nodes and spleen (207). Ohm and colleagues demonstrated


profound thymic atrophy accompanied by a marked reduction in CD4+/CD8+ thymocytes after exposure of mice to recombinant VEGF at concentrations similar to those observed in advanced stage cancer patients (208). There is also evidence that other cytokines, such as IL-6, could contribute to peripheral T lymphocyte dysfunction, enabling tumor cells to escape immune surveillance by preventing the anti-tumor TH1 immune responses (209). These findings suggest that stressinduced changes in tumor-associated factors may further promote tumor growth by compromising components of the immune system. There are limited data regarding the effects of cortisol on angiogenesis. In a rat glioma model, dexamethasone (synthetic glucocorticoid) treatment of cancer cells resulted in a 50–60% downregulation of VEGF mRNA, and this effect was dependent on the glucocorticoid receptor function (210). However, this inhibitory effect was markedly reduced by hypoxia, which is a potent VEGF inducer. In ovarian cancer cell lines, cortisol alone had some stimulating effects at lower doses, but inhibitory effects at pharmacologic doses (52). Because stress often involves elevations in both cortisol and catecholamines, costimulation experiments were also performed. While priming with cortisol blunted norepinephrine-induced VEGF production, significant increases were still seen (52). These findings suggest that catecholamine-induced effects are dominant in the context of angiogenic cytokine production.

E. Cell Survival Tumor cell survival or avoidance of apoptosis is essential during the metastatic process. Most of the data with regard to the effects of stress hormones on tumor cell survival have focused on glucocorticoid hormones. Glucocorticoids regulate a wide variety of cellular processes through glucocorticoid receptormediated activation or repression of target genes. Recent studies have demonstrated that while glucocorticoid hormones induce apoptosis in lymphocytes (211), these hormones activate survival genes that protect cancer cells from the effects of chemotherapy (212,213). Herr and colleagues examined the effects of glucocorticoids on human cervical and lung carcinoma cells and found downregulation of pro-apoptotic elements of the death receptor and mitochondrial apoptosis pathways (212). Similarly, Wu and associates found that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced apoptosis in a glucocorticoid receptor-dependent manner and is associated with the transcriptional induction of

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serum and GC-inducible protein kinase-1 (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP1) (213). Specific inhibition of these two proteins reversed the anti-apoptotic effects of GC treatment (213). Glucocorticoids such as cortisol may also act in a synergistic fashion with catecholamines to facilitate cancer growth. For example, cortisol potentiated the isoproterenol-induced increase in cAMP accumulation; increased βAR density; and markedly increased the effects of IL-1α, IL-1β, and TNF-α in lung carcinoma cells (63). It is thus plausible that stressful situations characterized by both elevated catecholamine and cortisol levels (e.g., uncontrollable stress) may have the greatest impact on cancer-related processes.

F. Other Stress Mediators Other hormones affected by stress include prolactin (214,215), oxytocin, and dopamine (216). Prolactin plays a functional role in tumor cell growth and promotes survival of breast and other cancer cells (217,218). A number of epidemiological studies have demonstrated a consistent correlation between prolactin levels and well-confirmed breast cancer risk factors such as parity and age at menarche (217). Most breast cancer cell lines express the prolactin receptor, and exogenously added prolactin has modest trophic effects on human tumor tissues and cells in vitro (219). Prolactin has been shown to stimulate proliferation in prostate and endometrial cancer cells as well (218). In addition to stimulation of proliferation, prolactin may also actively inhibit apoptosis of mammary tumor cells via stimulation of the Akt pathway (220,221). Oxytocin inhibits the growth of some epithelial cell (e.g., breast, endometrial) tumors and those of nervous or bone origin, but the hormone has a growthstimulating effect in trophoblast and other tumors (small cell lung tumors, Kaposi’s sarcoma) (222,223). The presence of oxytocin receptor has been described in breast cancer cells (224,225). Dopamine, an important member of the catecholamine family, is one of the major neurotransmitters in the mammalian brain and also has important roles in the periphery (226,227). Chronic exposure to stress leads to decreased dopamine release (228,229). Several studies have indicated a direct effect of dopamine in growth inhibition of tumor cells including neuroblastoma, breast, melanoma, and head and neck cancer cell lines (230–234). The growth inhibition may be mediated through dopamine receptors on the tumor cell surface or by auto-oxidation of dopamine, resulting in the generation of reactive oxygen species. However, emerging data suggest that dopamine may have other

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anti-tumor effects as well, such as anti-angiogenic properties. Dopamine is known to inhibit the growth of several types of tumors in in vivo experimental models (235,236). Basu and colleagues reported that at non-toxic levels, dopamine administration inhibited the vascular permealizing and angiogenic activities of VEGF (237). Dopamine acted through D2 dopamine receptors to induce endocytosis of VEGF receptor 2, which is critical for promoting angiogenesis, thereby preventing VEGF binding, receptor phosphorylation, and subsequent signaling steps (237). Similar results were found in gastric cancer models (235). Teunis and co-workers examined tumor growth and angiogenesis in Wistar rats with high or low dopamine reactivity (236). Both tumor size and vessel density were lower in rats with a hyperactive dopaminergic system, again suggesting a link between dopaminergic activity, angiogenesis, and tumor development.

ing neuroendocrine dynamics to undermine their support for the progression of malignant disease. Some biobehavioral intervention studies have shown promise for modifying neuroendocrine dysregulation, and immunologic functions that could potentially have implications for tumor progression (3). As discussed above, beta-blockers have been shown to block many of the deleterious effects of stress. Some clinical studies have shown lower cancer incidence among patients treated with beta-blockers (238,239), while in others the cancer risk was neutral (240–242). Some antidepressant medications also suppress the inflammatory response (243), which may be a beneficial property in cancer patients. While great strides have been made in understanding the influence of behavioral factors on cancer initiation and progression, further research is needed to fully understand the complex underlying mechanisms. However, such work holds promise for novel patient-specific and comprehensive intervention strategies.

VI. SUMMARY Cancer initiation and progression is a complex process that is dependent on multiple steps, including genetic alterations, growth/proliferation, vascularization, invasion, embolization, and survival/evasion of apoptosis. Once a tumor is established, its survival, growth, and metastatic dissemination depends on many interactions with homeostatic factors and mechanisms. The cumulative clinical and pre-clinical work reviewed in this chapter demonstrates the interrelationships between behavioral factors and cancer initiation and metastasis. The effects of behavioral factors on the immune system will be discussed in the accompanying Chapter 12. However, stress-induced changes in tumor factors such as VEGF and IL-6 may also play a role in compromising components of the immune system, thus further tilting the physiologic odds in favor of the tumor. The findings in the current chapter suggest that behavioral process and their endocrine consequences can have multiple and direct effects on the tumor microenvironment, with the potential to generate clinically significant downstream effects on tumor progression. Based on the data reviewed here showing direct effects of “stress hormones” on the function and activity of tumor cells, perhaps the word psychoneuroimmunobiology may be more reflective of the effects of behavioral factors on cancer. The effects of behavioral factors on tumor initiation and progression are likely to be complex and should be studied in the context of the relevant microenvironment. These studies may offer new opportunities for therapeutic intervention based on behavioral and pharmacologic approaches that target tumor-support-

Acknowledgments Portions of the work in this chapter were supported by NCI grants (CA11079301, CA10929801, and CA083639) to Dr. Sood; NCI grants (CA 1045-25 and CA 102515) and NCCAM (P20 AT-756) to Dr. Lutgendorf; and Dr. Cole is supported by NIH grant AI52737 and the Norman Cousins Center at UCLA.

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12 Neuroendocrine Regulation of Cancer Progression: II. Immunological Mechanisms, Clinical Relevance, and Prophylactic Measures ROI AVRAHAM AND SHAMGAR BEN-ELIYAHU

I. IMMUNE-CANCER INTERACTIONS AND THE POTENTIAL ROLE OF CELL-MEDIATED IMMUNITY (CMI) IN RESTRICTING TUMOR GROWTH AND METASTASIS 252 II. THE CRITICAL PERIOPERATIVE PERIOD IN CANCER PATIENTS 253 III. NEUROENDOCRINE MODULATION OF CELL-MEDIATED IMMUNITY AND CANCER DEVELOPMENT IN THE PERIOPERATIVE PERIOD 254 IV. POSTOPERATIVE SUPPRESSION OF NK ACTIVITY AND RESISTANCE TO TUMOR PROGRESSION: MECHANISMS AND PREVENTION BY PHARMACOLOGICAL INTERVENTIONS 254 V. IS THERE EVIDENCE IN CANCER PATIENTS THAT POSTOPERATIVE SUPPRESSION OF CMI CAN INDEED PROMOTE METASTASIS? 257 VI. IMMUNE STIMULATION AS A PROPHYLACTIC MEASURE IN THE PERIOPERATIVE PERIOD: A DOUBLE-EDGED SWORD AND THE POTENTIAL FOR PNI-BASED INTERVENTIONS TO IMPROVE IMMUNOTHERAPY 258 VII. DOES MALIGNANT TISSUE EXPLOIT STRESSFUL PERIODS TO FACILITATE ITS AUTO-EVOLUTIONARY PROCESS? 260 VIII. CLINICAL IMPLICATIONS 261

of a neoplasm within a patient that harbors this disease. In this chapter we focus on the interactions between the malignant tissue and the immune and neuroendocrine systems, considering the ramifications of this interaction to cancer evolvement, progression, and treatment. We start by reviewing evidence supporting the role of the immune system in shaping and controlling cancer progression and metastasis. We argue that, for various considerations, the perioperative period is critical in determining long-term survival, and that the immune system is presented with a unique opportunity to eradicate cancer. We then present clinical evidence indicating that cellular immunity is often limited by neuroendocrine modulation during the perioperative period. From our own studies, we give evidence that immunosuppression that follows surgery can directly promote metastasis. We point at several mechanisms, including local and neuroendocrine responses, by which various aspects of surgery can cause immunosuppression, and introduce possible prophylactic interventions that reduce immunosuppression. We further suggest that the potential beneficial effects of immunotherapy are also jeopardized by patients’ stress responses. Complementing immunotherapy with blockers to stress hormones may thus promote its efficacy. We hypothesize that along the autoevolutionary process of cancer development and metastasis, the malignant tissue capitalizes on neuroendocrine stress responses and synchronizes the

ABSTRACT Common approaches to cancer treatments focus on the malignant tissue itself, rather than on the existence PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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periods of maximal risk and exposure with periods of suppressed immunity. We conclude this chapter with clinical implications of these insights and of the suggested prophylactic anti-immunosuppressive interventions.

I. IMMUNE-CANCER INTERACTIONS AND THE POTENTIAL ROLE OF CELL-MEDIATED IMMUNITY (CMI) IN RESTRICTING TUMOR GROWTH AND METASTASIS The suggestion that neuroendocrine modulation of immune competence has biological and clinical significance to tumor development depends critically on the ability of the immune system to control malignancies. Whether and under what circumstances such control is implemented has been debated for decades, and we have recently reviewed this issue in detail (Shakhar and Ben-Eliyahu, 2003). Below we present the controversy, a contemporary theory that accounts for the apparently conflicting evidence, and our view that ascribes a significant role for CMI in controlling tumor metastasis under certain clinical conditions.

A. The Controversy over the Role of the Immune System in Controlling Cancer Development, and Its Resolution by Contemporary Theories The involvement of the immune system in controlling cancer has been debated for more than a century, since William Coley used extracts of pyrogenic bacteria to cause sporadic anti-tumor responses (Coley, 1893). Empirical evidence has been brought to support theories advocating immune surveillance, as well as theories negating immune control over malignant tumors. Today, state-of-the-art studies have provided ample evidence in favor of immune-cancer interactions and started to revive enthusiasm in immunotherapy. These studies can be categorized into several non-conclusive domains of research, including identification of immunogenic tumor-associated antigens (TAAs); activation of professional antigen-presenting cells (APCs) for the induction of effective T-cell–mediated immunity; the use of pattern recognition receptors to link innate to adaptive immunity via dendritic cells (DCs); and the role of suppressor or regulatory T lymphocytes in inhibiting responses against tumors. Nevertheless, the clinical significance of these interactions in controlling cancer progression has yet to be shown. Animal studies provided ample evidence for the involvement of the immune system in controlling tumor initiation, growth, and metastasis (Kikkawa

et al., 2000; McCoy et al., 2000; Smyth et al., 2001), and specifically implicated immune effectors such as CTL and NK cells (Brittenden, 1996; Khavari, 1987; Lindauer et al., 1998). Other leukocytes, including dendritic cells (Gilboa et al., 1998; Fong and Engleman, 2000), NKT (Brutkiewicz and Sriram, 2002; Smyth et al., 2002), and T-helper (Toes et al., 1999) cells also seem to be involved in supporting cytotoxic activity against tumors. Nevertheless, it is widely acknowledged that animal models often do not reliably simulate immune-malignant interactions in humans (Shakhar and Ben-Eliyahu, 2003). Models of experimental metastasis are often chosen based on their artificially high immunogenicity, and the course of natural tumor development and metastasis in rodents is markedly shorter and potentially less complex than in humans. Indeed, the evidence in humans is not conclusive. Undermining the suggested role of immunity are the findings that spontaneous remission in cancer patients is rare, immuno-stimulatory regimens have so far yielded limited success, and immuno-suppressed transplant patients hardly show increased frequencies of major cancer types (Penn, 1993). On the other hand, ample evidence in cancer patients supports a role for immunity. Autologous innate and adoptive immunocytes were shown in vitro to lyse tumor cells expanded from excised tumors (Rosenberg, 2001), and many molecular mechanisms of tumor recognition by immunocytes have been recently identified (Khavari, 1987; Long, 2002; Soloski, 2001). Moreover, patients’ leukocyte cytotoxicity (Uchida et al., 1990) or proliferative response (McCoy et al., 2000) to the autologous tumor were shown to be the best independent predictor of metastasis-free long-term survival, better than tumor stage and grade. When cancer appears in already immunosuppressed patients, its clinical course is commonly accelerated and more metastases appear (Barrett et al., 1993; Detry et al., 2000). Last, while it is now evident that the majority of cancer patients harbor micrometastases and circulating malignant cells following the removal of the primary tumor (Yamaguchi et al., 2000), recurrence occurs only in a minority of this population, suggesting a role for immunity in eradicating such minimal residual disease (MRD). This apparent conflicting evidence is reconciled by contemporary theories as follows. A newly transformed tissue is initially exposed to immunocytes only when it attracts blood capillary and emits various danger signals (Fuchs and Matzinger, 1996). By this stage, malignant cells have accumulated sufficient mutations to become immunogenic, and are recognized and destroyed by immunocytes. This starts an auto-evolutionary process through which selection pressure by the immune system, if not eradicating the developing tumor, increases the proportion of resis-

12. Neuroendocrine Regulation of Cancer Progression

tant malignant cells. Remaining malignant cells, which are known to intensively mutate, continue to acquire escape mechanisms that can eventually yield an immune-resistant malignancy. In cancer patients, the primary tumor has clearly outmaneuvered the immune system, but the prospect of a transformed cell in a healthy individual is unknown and may be dismal. Most impressive are the findings that human tumor cells can develop mechanisms to escape every known process used by the immune system to recognize or destroy malignant cells (Pawelec, 2004). These findings attest to a rigorous interaction between malignant cells and immunocytes, and to the substantial tumor destruction carried by immunocytes.

B. Cancer Metastases Are More Susceptible to CMI Than the Primary Tumor Although in cancer patients the immune system has clearly failed to eradicate the evolving primary tumor, it is believed to maintain an important role in controlling metastases, particularly after the primary tumor is removed and minimal residual disease (MRD) includes circulating single tumor cells and isolated micrometastases. Following detachment from the primary tumor, single tumor cells are targeted by unfamiliar populations of lymphocytes that employ recognition and destruction strategies that were not used against the primary tumor. Moreover, MRDs are not shielded by the primary tumor, and due to their insignificant mass cannot use escape mechanisms such as the release of immunosuppressive factors (e.g., prostaglandins, IL-10). There is evidence that metastases express more advanced escape mechanisms than the primary tumor, testifying to the strict selection pressure imposed by the immune system on metastasizing cells (Schirrmacher, 1985). Overall, the evidence suggests that immunity has an anti-malignant and anti-metastatic role in the natural course of human cancer, and that suppression of immune functions could thus worsen the prognosis of cancer patients. As indicated below (Section II), the perioperative period may constitute a setting in which immune competence is critical for restricting metastasis.

II. THE CRITICAL PERIOPERATIVE PERIOD IN CANCER PATIENTS A. Surgery as a Risk Factor in the Development of Metastases Surgery for the removal of the primary tumor, although essential, can increase the risk of postoperative metastases through several mechanisms. First, as

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malignant tissue is notoriously non-adhesive, the mechanical manipulation involved in excising the tumor and cutting its blood vessels has been shown to release tumor cells into the circulation (Eschwege et al., 1995; Yamaguchi et al., 2000). Second, growth factors released by the body to facilitate the healing of damaged tissue following surgery also facilitate the growth of pre-existing micrometastasis and the development of new metastases. Third, the removal of the primary tumor commonly terminates the release of anti-angiogenic factors, enabling dormant micrometastases to develop blood vessels and expand beyond a critical size (O’Reilly et al., 1994; O’Reilly et al., 1997). Fourth, stress hormones released perioperatively were shown in vitro to elevate the release of angiogenic factors by tumor cells (e.g., VEGF), potentially promoting the outbreak of dormant micrometastases (Lutgendorf et al., 2003). Last, surgery causes marked suppression of CMI, specifically if the procedure is large, invasive, and physiologically traumatic. These risk factors peak simultaneously during and immediately following surgery, and can act in synergy to promote the spread and seeding of new metastasis and the flare-up of pre-existing dormant micrometastases. At this critical setting, anti-metastatic immunity can become a critical factor restricting metastasis, and interference with its functioning can lead to the development of metastases that would have been prevented if immune competence was not impaired (Shakhar and Ben-Eliyahu, 2003).

B. A Window of Opportunity for Eradicating Cancer On the other hand, surgery opens a window of opportunity for eradicating cancer. The excision of the primary tumor eliminates the major source of mutating and metastasizing tumor cells, and the immune system is thus faced with a lesser challenge. Remaining malignant tissue in the form of MRD is also more susceptible to immunity, as indicated above, and the levels of immuno-suppressive factors often released by the primary tumor subside. Thus, the postoperative period presents the immune system with a second opportunity for eradicating cancer. This window of opportunity will be closed once potential metastases will grow beyond a critical size that enables the use of escape mechanisms originally employed by the excised primary tumor. Taken together, the postoperative period is characterized by a high risk for initiation and progression of metastases, but simultaneously presents an opportunity to eradicate cancer. Both prospects critically depend on immune competence, specifically immune anti-metastatic activity. Therefore, maintaining

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immune competence or/and boosting it during this critical period can impact long-term survival rates in cancer patients.

III. NEUROENDOCRINE MODULATION OF CELL-MEDIATED IMMUNITY AND CANCER DEVELOPMENT IN THE PERIOPERATIVE PERIOD Immunosuppression by surgery is caused by an intricate array of local and systemic physiological responses. As described below, many of these responses involve a CNS-mediated neuroendocrine feedback. Generally, whereas in the vicinity of the wound the predominant response is pro-inflammatory, in the periphery, where the fate of metastases is determined, an opposite picture emerges: There is a drastic reduction in pro-cellular Th1-type responses and suppression of CMI, with minor perturbation to humoral immunity.

A. The Potential Role of the Sympathetic Nervous System (SNS) The SNS innervates lymphoid organs and most leukocytes express adrenergic receptors (Elenkov et al., 2000). During the perioperative period and following psychological stress, catecholamines are released systemically, as well as locally by nerve endings that are believed to form “synapses” with leukocytes (Elenkov et al., 2000). In vitro studies have indicated that catecholamines can act directly to suppress many aspects of CMI, including NK, CTL, and macrophage activity (Elenkov et al., 2000). Catecholamines can also act indirectly to suppress CMI by reducing macrophages and Th production of type 1 cytokines (e.g., IL-12, TNF-α, and IFN-γ), and by stimulating the release of immunosuppressive factors including IL-10 and TGF-β (Platzer et al., 2000; Woiciechowsky et al., 1999).

B. The Hypothalmo-Pituitary-Adrenal (HPA) Axis Surgery activates the HPA axis via a spinal pathway, as well as through the release of IL-1 and IL-6 (Mastorakos et al., 1993). Increased levels of corticosteroids following surgery last for days and correlate with the severity of surgery and the degree of immunosuppression (Tashiro et al., 1996). Corticosteroids are established in vitro immunosuppressors and, when administered in pharmacological doses, cause potent immunosuppression. Indeed, the use of synthesis inhibitors or competitive antagonists of corticosteroids reduced T-cell apoptosis and metastasis following

surgery in rats (Deguchi et al., 1998), and attenuated the suppression of monocyte activity in injured mice (Cech et al., 1994). In addition, stress was shown to suppress various aspects of CMI and promote infection via activation of the HPA axis (Sheridan et al., 1994).

C. Local Factors and Cytokine Responses The response to tissue damage is initiated by local products of cell lysis, humoral factors released by macrophages and other resident cells, and by local neurogenic inflammatory agents. Prominent among these factors are prostaglandins (e.g., PGE2), which are potent in vitro suppressors of CMI and are known to be involved in the initiation of the cytokine response (Faist et al., 1996). In humans, cyclooxygenase inhibitors blunted the cytokine responses (Chambrier et al., 1996) and the suppression of CMI following surgery (Faist et al., 1990; Markewitz et al., 1996), and in rats they were also shown to reduce the promotion of metastasis by surgery (Colacchio et al., 1994). The local neurogenic pro-inflammatory response is initiated by nociceptive afferents. It involves local and spinal reflexes, and promotes erythema and edema around the surgical wound by releasing numerous compounds, including substance P (Rameshwar, 1997; Schaffer et al., 1998). The cytokine response to major surgery includes an immediate surge in systemic levels of pro-inflammatory cytokines (e.g., IL-6 and IL-8), and an increase in plasma levels of type 2 cytokines and other factors that interfere with CMI (e.g., IL-10, IL-1rA, sTNF-αr, and sIL-2r) (Faist et al., 1996; Lin et al., 2000). Additionally, in vitro studies indicated a marked decrease in the production of pro-CMI cytokines (such as IL-2, IL-12, IFN-γ, TNF-α, and IL-1β) by monocytes and Th1 cells, and an increase in the production of factors that interfere with CMI, such as IL10 and IL-1rA (Munford and Pugin, 2001). The cytokine response to surgery is intertwined with the neuroendocrine response described above. For example, IL-1 and IL-6 are critical for initiating the HPA response to surgery, while sympathetic activity was shown to trigger IL-10 release following surgery (Woiciechowsky et al., 1998; Woiciechowsky et al., 1999).

IV. POSTOPERATIVE SUPPRESSION OF NK ACTIVITY AND RESISTANCE TO TUMOR PROGRESSION: MECHANISMS AND PREVENTION BY PHARMACOLOGICAL INTERVENTIONS Major surgeries cause complex perturbations to various physiological processes, including the meta-

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bolic, cytokine, neuroendocrine, and immune systems. Various aspects of the surgical procedure are suspected to mediate the immunosuppressive effects of surgery. These include perioperative stress and anxiety, anesthetic and analgesic agents, tissue damage, blood loss and transfusion, hypothermia, and nociception and pain. The local paracrine mediators and the systemic cytokine and neuroendocrine responses that mediate the immunosuppressive effects of surgery are diverse and only partly understood. We have studied most of the aforementioned aspects of surgery with respect to their impact on rats’ NK activity and resistance to experimental metastasis, and searched for specific endocrine mediators of their impact. In these studies we made use of the mammary adenocarcinoma MADB106 tumor line and the CRNK-16 leukemia line, both syngeneic to the F344 rat. These tumor cells are sensitive in vivo to NK activity (Avraham et al., 2005; Barlozzari et al., 1983; Barlozzari et al., 1985; BenEliyahu and Page, 1992; Ben-Eliyahu et al., 1996), and thus we consider resistance to their metastases an in vivo index for NK activity (Ben-Eliyahu and Page, 1992; Ben-Eliyahu et al., 1996). These models also simulate some aspects of the more comprehensive metastatic process (e.g., extravasation of tumor cells and growth of metastases), or leukemia progression, but clearly suffers from some of the above-mentioned drawbacks of animal cancer models.

Garabal et al., 1993a; Freire-Garabal et al., 1993b). We have also shown that swim stress suppressed circulating NKA and promoted lung metastasis and leukemia progression by activating the SNS and releasing adrenal catecholamines (Figure 1) (Inbar et al., 2003) that activate β1 and β2 adrenoceptors (Ben-Eliyahu et al., 2000). Perhaps more related to human stress and anxiety, we have shown that social confrontation in rats can suppress NK activity and promote MADB106 experimental metastasis and that these effects were attenuated using a β-adrenergic blocker or diazepam (Abitri et al., 2005; Stefanski and Ben-Eliyahu, 1996; Stefanski and Engler, 1998). Interestingly, the coping style in the confrontation paradigm (e.g., type and frequency of submissive behavior) predicted susceptibility to metastasis (Stefanski and Ben-Eliyahu, 1996). Simulating the effects of SNS activation during stress, we also showed that the administration of physiologically relevant doses of a β-adrenergic agonist or adrenaline suppressed NK activity and consequently increased lung metastasis (Ben-Eliyahu et al., 2000; Shakhar and Ben-Eliyahu, 1998) and the progression

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Control Vehicle 0.8

1. Perioperative Stress and Anxiety and the Involvement of Catecholamines Most cancer patients experience emotional distress before and after surgery. Distress stems from loss of control, fear from the surgery and progression of the disease, the prospect of chemotherapy or radiotherapy, disfigurement, disability, and death (Lutgendorf and Costanzo, 2003). Psychological stress, especially when chronic, is associated with depressed CMI and increased susceptibility to infectious disease (Cohen and Herbert, 1996). Conducting animal studies, we and others have provided causal evidence that stress suppresses immunity (Moynihan and Ader, 1996) and consequently increases susceptibility to various types of metastasis and to leukemia (Ben-Eliyahu et al., 1991; Ben-Eliyahu et al., 1999; Ishihara et al., 1999). Correspondingly, anxiolytic drugs were shown to reduce postoperative suppression of CMI in mice (Freire-

Stress Nadolol Stress Vehicle

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A. Specific Aspects of Surgery That Contribute to Immunosuppression and Cancer Progression, and Their Mediating Immunological and Neuroendocrine Mechanisms

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0 15 25 35 45 55 65 75 Days FIGURE 1 The effect of swim stress on survival and its attenuation by the β-adrenergic blocker, nadolol. A miniscule number of 60 CRNK-16 leukemia cells were administered before subjecting rats to stress. Swim stress significantly increased mortality rates, and nadolol significantly attenuated this effect.

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of the CRNK-16 leukemia (Inbar et al., 2003). Although perioperative psychological factors may affect neuroendocrine responses less profoundly than intra- and postoperative physiological stressors, they last longer and may thus have a profound effect. 2. Causal Relationship between NK Suppression and Tumor Promotion It is often hard to show causal relationships between two in vivo processes. Strongly suggesting that the immunosuppressive effects of our above stressors underlie their tumor-promoting effects are the following findings: Different stress paradigms suppressed NKA for different durations that corresponded well with the time courses of their tumor promoting effects; metastasis of tumor lines that are sensitive to NKcontrol (MADB106, CRNK-16) were affected by stress, but not those that are not sensitive to NKA (C4047); the same interventions that attenuated the suppression of NKA reduced the promotion of metastasis and leukemia (Melamed et al., 2005); and selective depletion of NK cells eliminated the metastatic-promoting effects of swim-stress and β-adrenergic agonist, but not of surgery (Ben-Eliyahu et al., 1999; Ben-Eliyahu et al., 2000; Inbar et al., 2003; Shakhar and Ben-Eliyahu, 1998). Thus, suppression of NKA is sufficient to promote NK-sensitive metastasis under some stressful conditions, but additional mechanisms could play a role in more complex stressors, such as surgery. 3. Anesthetics and Analgesic Agents, and the Effects of Hypothermia Our studies in rats indicated that some anesthetics (i.e., ketamine, fentanyl, and thiopental) can alone suppress NK activity and promote tumor metastasis, while halothane and propofol have minor or no such effects (Melamed et al., 2003; Shavit et al., 2004). We also found that prolonged and severe hypothermia in rats, but not moderate hypothermia, causes similar effects, and that both the effects of hypothermia and of some anesthetics can be markedly reduced by a β-adrenergic antagonist (Ben-Eliyahu et al., 1999; Melamed et al., 2003). Thus, our surgical paradigm is based on the non–NK-suppressive general halothane anesthesia and is monitored to prevent severe hypothermia. 4. Tissue Damage Several studies in humans and rodents indicated that the severity of postoperative immunosuppression is associated with the degree of tissue damage (Carter, 2001; Lennard et al., 1985; Nelson and Lysle, 1998). Minimally invasive surgeries, such as laparoscopy,

limit postoperative infections in patients (Targarona et al., 2000) and the promotion of metastasis in operated animals (Da Costa et al., 1998). These beneficial effects could be related to attenuation of the local paracrine or systemic neuroendocrine responses, or to a lesser degree of hypothermia, blood loss, nociception, and pain. 5. Blood Loss and Transfusion Blood loss and transfusion were shown to interfere with several aspects of CMI, including cytokine levels, NK cell activity, and T-cell blastogenesis (Klein, 1999). Blood transfusion (BT) was suggested to be an independent risk factor for tumor recurrence in cancer patients (Klein, 1999), although the specific mechanisms are yet unclear. In an ongoing study in rats, we found that BT that is based on standard clinical procedures promotes MADB106 metastasis and the progression of the CRNK-16 leukemia. Surprisingly, our findings also indicate a critical role for red blood cells in mediating these effects of BT, rather than a role for leukocyte or soluble factors. We suspect that donors’ deteriorating red blood cells disrupt the normal host anti-tumor response, presumably through preoccupying host immunocytes (Atzil et al., 2004). 6. Nociception and Pain Pain and nociception are known to activate responses of the SNS, HPA, and the opioid systems via various mechanisms including the activation of spinal ascending and descending pathways. Because pain alleviation and spinal blockade are clinically feasible and were shown to reduce postoperative immunosuppression (Le Cras et al., 1998; Sacerdote et al., 2000), we tested their efficacy in reducing the tumor-promoting effects of surgery. We have established in rats that low analgesic doses of morphine or fentanyl (e.g., up to 5 mg/kg and 40 μg/kg respectively) can reduce the promotion of MADB106 metastasis by surgery (Page et al., 1998; Page et al., 2001). Preemptive analgesia, which is known to abrogate the nociceptive afferent barrage, was as efficient as prolonged postoperative treatment (Page et al., 1998). However, higher doses of narcotics (e.g., morphine 10 mg/kg) were detrimental, inducing immunosuppression and tumor promotion, as was also reported in human and animal studies (Saurer et al., 2004; Shavit et al., 2001; Shavit et al., 2004). Thus, a delicate balance between insufficient analgesia and too much narcotics must be maintained to clinically employ this approach. We therefore assessed another approach—perioperative spinal block by a combination of bupivacaine and morphine. This approach ensures low systemic levels of narcotic with


superb pain and nociception blockade. Our studies indicated that this approach was more efficient in reducing the tumor-promoting effects of surgery, most likely acting by blocking the HPA and SNS responses to surgery (Bar-Yosef et al., 2001; Page et al., 2001). 7. The Involvement of Prostaglandins in the Immunosuppressive Effects of Surgery Prostaglandins, in particular PGE2, are important local mediators of CMI dysfunction. These substances are potent immunosuppressants, quickly synthesized in damaged tissue by macrophages and other cells, and have been reported by some but not other studies to increase systemically following surgery (Lai et al., 2000; Vitoratos et al., 1996). The administration of cyclooxygenase inhibitors to surgical patients results in blunted cytokine responses (Chambrier et al., 1996) and attenuated suppression of CMI (Faist et al., 1990). We have shown that the administration of PGE2 in physiologically relevant doses to rats suppresses circulating NKA per NK cell. By selective depletion of NK cells, we showed that dysfunction of NK cells induced by PGE2 increased susceptibility to metastasis. Finally, by treating rodents with COX or by selective COX2 inhibitors, we and others were able to reduce the promotion of metastasis by surgery (Colacchio et al., 1994; Page et al., 2003; Yakar et al., 2003). 8. A Unique Role for Marginating Pulmonary-NK Cells in Resisting Syngeneic Tumor Cells Many autologous tumor cells excised from patients are considered NK-insensitive, as patients’ circulating lymphocytes fail to exhibit spontaneous in vitro NK cytotoxicity against them (i.e., no cytotoxicity in a 4 h 51 Cr release assay). However, several tumor lines that are clearly controlled by NK cells in vivo, including the MADB106 line (Barlozzari et al., 1985; Ben-Eliyahu and Page, 1992; Ben-Eliyahu et al., 1996), meet the above “clinical” criterion for being NK-insensitive. Thus, we suspected that there is a unique population of NK cells that act in vivo in a manner that LAK cells act in vitro— recognize a larger spectrum of tumor cells and exhibit greater cytotoxicity than circulating NK cells. Recently, we have found a leukocyte population that seems to contain such an NK population—marginating pulmonary (MP) leukocytes (leukocytes adhering to the lungs’ vasculature). We have established that this population exhibits markedly greater NK cytotoxicity per MP-NK cell against syngeneic and xenogeneic target cells, and has a greater subpopulation of large NK cells (between 16 and 25 μm in size (Melamed et al., 2005) compared to circulating NK cells (30% vs. 10% in the blood) (Melamed et al., 2005). Although MP-NK cells

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are relatively few (approximately 3–10 × 105 cells/rat), they are strategically located to physically interact with circulating malignant cells that have to force their way through the pulmonary capillary system. By purifying MP-NK cells, we are now studying whether their unique capacities are contained within MP-NK cells themselves (as we believe), or whether supporting cells (e.g., NKT cells) are involved. Similar populations of NK cells may reside in other immune compartments (e.g., hepatic vasculature). If such populations exist in humans, then autologous tumor cells that were considered NK-insensitive are actually controlled in vivo by such NK cells.

B. The Prophylactic Use of a Combination B-Adrenergic Antagonist and a PGSynthesis Inhibitor against the Deleterious Effects of Surgery We recently reported the efficacy of a combined use of a β-adrenergic antagonist and a PG-synthesis inhibitor in preventing surgery-induced suppression of circulating and MP-NK activity against the YAC-1 and the syngeneic MADB106 target cells. We also showed that this treatment abolished the MADB106 metastasis-promoting effect of surgery (see Figure 2) (Melamed et al., 2005). Such a combined use of antagonists may be a key to a clinical approach, as blocking only one pathway may be insufficient given that either catecholamines or PGs can alone suppress NK activity by increasing intracellular cAMP levels (Torgersen et al., 1997; Whalen and Bankhurst, 1990). In our study, the interventions were given only postoperatively, as non-selective COX inhibitors can cause complications during surgery. We have also started the use of a selective COX2 inhibitor and a β-blocker that were administered before and after surgery. This approach has already yielded promising results (Avraham et al., 2005) and can be advantageous in the clinical setting, as it can be expected to also alleviate pre-operative immunosuppression caused by potential release of PGs by the primary tumor, and to reduce the effects of SNS responses caused by psychological stress.

V. IS THERE EVIDENCE IN CANCER PATIENTS THAT POSTOPERATIVE SUPPRESSION OF CMI CAN INDEED PROMOTE METASTASIS? Studies in animals cannot be expected to simulate the much longer, different, and complex process of metastasis in cancer patients (Hewitt, 1983; Killion et al., 1998). Thus, evidence in support of the suggestion

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% Lung Tumor Retention (MADB106)

2 1.8 1.6 1.4

Saline Nadolol Indo

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Surgery

FIGURE 2 The effects of surgery and their attenuation by the β-adrenergic blocker, nadolol, by the prostaglandin synthesis inhibitor, indomethacin (indo), and by the combined use of these drugs (indo and nadolol) (mean ± SEM). Surgery increased lung tumor retention of the MADB106 tumor and increased the number of experimental MADB106 lung metastases counted 3 weeks later (not shown). Each of the blockers attenuated these effects, and their combined use almost completely abolished them. ¥ indicates a significant effect of surgery (difference between the control saline and surgery saline groups), and * indicates a significant attenuation of this effect by drug treatment (difference between the surgery saline group and the surgery drug group). The combined treatment was significantly lower than each treatment alone.

that postoperative suppression of CMI can promote metastasis in cancer patients should also be obtained in cancer patients. Because surgery is always conducted in patients presenting with operable tumors, potential evidence could only be based on correspondence between variations in the levels of postoperative immunosuppression and subsequent long-term recurrence rates. Studying the literature, we found such correspondence with respect to variations on surgical procedures that exacerbate or lessen postoperative immunosuppression. Specifically, local rather than general anesthesia was shown to reduce postoperative immunosuppression (Galley et al., 2000), and prospective studies showed that this approach also reduces long-term mortality rates in skin cancer patients (Schlagenhauff et al., 2000). Blood transfusion was shown to cause immunosuppression (Klein, 1999), as well as to be an independent risk factor for recurrence (Klein,

1999; Vamvakas and Blajchman, 2001). Repeated surgeries cause more immunosuppression than a single surgery and were reported to worsen prognosis in colon cancer (Fielding and Wells, 1974). Last, minimally invasive surgeries (MIS) are known to cause less immunosuppression (Carter, 2001; Sietses, 1999), and data are now accumulating to suggest that they can reduce recurrence rates (Kaseda et al., 2000; Sugi et al., 2000). Most importantly, local anesthesia, blood transfusion, repeated surgeries, and MIS were shown to predict recurrence rates independently of other factors associated with them, and in some studies subjects were randomly and prospectively allocated to the different studies’ groups. Thus, these positive and negative risk factors should be considered seriously when planning and conducting surgeries in cancer patients. The different levels of postoperative immunosuppression they induce could be the major factor mediating their impact on recurrence rates. Some of these correspondences have been simulated in animal models, and these studies provided more direct support for the mediating role of immunosuppression [for more details, see (Shakhar and Ben-Eliyahu, 2003)]. Because in cancer patients the evidence is still circumstantial, the most convincing evidence that one could strive to achieve in this population seems to be through interventions aiming at specifically blocking postoperative immunosuppression. We feel that our knowledge is now sufficient to start developing these interventions in patients undergoing major surgeries. Such strategies could be implemented in cancer patients once they are proven successful and provided that there are no contraindications.

VI. IMMUNE STIMULATION AS A PROPHYLACTIC MEASURE IN THE PERIOPERATIVE PERIOD: A DOUBLEEDGED SWORD AND THE POTENTIAL FOR PNI-BASED INTERVENTIONS TO IMPROVE IMMUNOTHERAPY In the context of tumor excision, residual circulating tumor cells are potentially immunogenic and are outnumbered by immunocytes outside the protective environment of the primary tumor. This presents an opportunity for immune-activation–based therapies to reduce long-term postoperative metastases. Unfortunately, stress and surgery often cause the release of stress hormones that could potentially interfere with immune responses to immunostimulatory agents. Also, the immunosuppressive effects of surgery could annihilate the beneficial effects of immunostimulation. We have conducted several studies to explore poten-


tial interactions between stress responses and immunotherapy, and the feasibility to integrate PNI-based interventions with immunotherapy in the perioperative period to reduce postoperative metastasis.

A. Are the Beneficial Effects of Immunostimulation Limited by the Deleterious Effects of Stress and Surgery? Marked postoperative immunosuppression could theoretically annihilate the beneficial effects of preoperative immunostimulation. We believe that while boosting baseline levels of immunity would prove beneficial under no-stress conditions, after severe stress, which can occur postoperatively, the beneficial effects of immunotherapy could be undermined. To explore this notion in the context of stress and surgery, we studied two different agents commonly used in immunotherapy: poly I-C and IL-12. Repeated pre-tumor administration of IL-12 was found in our in vivo studies to potently reduce metastasis in non-stressed rats. However, surgery and stress had the same deleterious effects in IL-12–treated rats as in saline-treated rats. Ex vivo studies indicated that IL-12 induced its favorable effects by increasing total numbers of circulating and marginating pulmonary NK cells, both in the stress and no-stress groups. Surgery caused a marked suppression in NK activity per NK cell, without affecting the numbers of circulating or marginating pulmonary NK cells. Corresponding with the in vivo findings, IL-12 did not protect NK activity per NK cell from suppression by surgery (Avraham et al., 2004). Thus, in our studies IL-12 acted by increasing baseline levels of immunity and tumor resistance, but did not protect the host from the deleterious effects of surgery. A similar paradigm of low doses in repeated administration of Poly I-C was not as effective as IL-12 in reducing metastasis in non-stressed rats. However, the metastasis-promoting effects of stress and surgery were dramatically decreased or completely absent in poly I-C treated rats. Our findings also indicate that poly I-C was not as effective as IL-12 in increasing numbers of circulating and marginating pulmonary NK cells. However, unlike IL-12, poly I-C significantly protected NK activity per NK cell from stress- and surgery-induced suppression (Shakhar et al., 2003). Moreover, we conducted further studies that directly confirmed these ex vivo results; in vivo treatment with poly I-C completely abolished the in vitro suppression of marginating pulmonary NK cells by corticosterone and prostaglandin E2. Taken together, these results suggest that boosting immune functions and protecting immunity from

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postoperative suppression are independent outcomes that seem to act via different cellular mechanisms. While boosting immune function acted in the current studies via increasing numbers of effector cells, protecting immunity from suppression acted at the single cell level. Hence, in cancer patients subjected to immunostimulatory regimens, complementary treatments should be considered.

B. Immunostimulation Can Also Suppress Immune Functions Immunostimulation in and of itself can induce various physiological stress responses that can suppress some immune functions or affect tumor progression via non-immunological mechanisms. For example, poly I-C was found to induce the release of corticosterone (Pruett et al., 2003). In our studies we found that while repeated administrations of poly I-C reduced experimental metastasis, a single dose causes detrimental effects during the first hours following administration. This may be attributed to a specific stress response caused by poly I-C, or may reflect the activation of the hypothalamic-pituitary-adrenal axis by one or more of the cytokines induced by poly I-C (e.g., IL6) (Turnbull and Rivier, 1999). Indeed, in a recent study we were able to significantly attenuate these detrimental effects of poly I-C by administrating a non-selective β-blocker, and to a lesser degree by administrating the non-selective corticosterone antagonist, RU486 (unpublished data). The notion that the sickness syndrome is stressful and potentially immunosuppressive has been previously reflected upon in the realm of PNI. Its biological significance has thus far been reported mainly with respect to psycho-depressive symptoms induced by cytokine therapy or by spontaneous immune responses to environmental challenges (Yirmiya et al., 2000). In the context of immune-stimulations in cancer patients, such effects may bear significant clinical consequences.

C. Immunostimulation Can Directly Promote Tumor Progression Cancer cells are transformed cells of the body, and as such they often express receptors and other surface molecules expressed by their pre-malignant progenitors. Consequently, malignancies that have originated from leukocytes might interact with immunostimulatory agents. In our studies with the CRNK-16 leukemia-line, we administered IL-12 or poly I-C before or after leukemia inoculation. These immunostimulatory agents were effective in reducing mortality rates from


this leukemia when administered before leukemia inoculation, but not after it. One explanation could be that CRNK-16 cells were triggered to proliferate by IL-12 or by cytokines induced by poly I-C. Given that CRNK-16 cells originate from LGLs (Ward and Reynolds, 1983), they are likely to express receptors to such factors. This phenomenon may present an obstacle to immunotherapy in tumor-bearing patients. The balance between boosting immune activity and stimulating tumor cells should be considered with respect to the specific malignant tissue and the context of treatment.

D. The Potential for Combining Immunostimulation with PNI-Based Interventions Given the above results and considerations, activation of the immune system in the perioperative setting may not suffice if not protected from the massive immunosuppression that follows major surgeries. To this end, we have studied the combination of PNIbased interventions in the form of a β-blocker and COX inhibitor with IL-12 immunotherapy. The in vivo results are summarized in Figure 3. The combined approach was most effective, boosting anti-tumor resistance and protecting it against suppression by surgery. Studying underlying cellular mechanisms, it followed that IL-12 increased the numbers of MP-NK, while the PNI-based intervention prevented NK suppression on a per NK cell basis. These results imply that the two approaches utilize different sets of molecular mechanisms that can complement each other in the postoperative period.

VII. DOES MALIGNANT TISSUE EXPLOIT STRESSFUL PERIODS TO FACILITATE ITS AUTO-EVOLUTIONARY PROCESS? Ample evidence reviewed above indicates that neuroendocrine stress responses can modulate immune competence against metastatic processes and that these modulations bear clinical significance. While metastasis is an advanced stage of tumor progression, the outbreak and growth of cancer is an auto-evolutionary process that is affected by numerous factors and various selection pressures along its course. It is our hypothesis that neuroendocrine perturbations impact the evolvement of the malignant tissue via diverse mechanisms, and that the malignant tissue itself senses neuroendocrine stress responses and uses them to coordinate processes that promote its evolvement and survival. Specifically, we believe that the malignant

900 Lung Tumor Retention (% of control)

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800 700

Control IL-12 Combined Nadolol & indo

600 500 400 300 200 100 0 Control

Surgery

FIGURE 3 The effects of surgery and their attenuation by IL-12, the use of a β-adrenergic blocker and a prostaglandin synthesis inhibitor (nadolol & indo), and by the combined use of all these interventions (combined) (mean ± SEM). * indicates a significant decrease in baseline LTR in the IL-12 treated groups. ¥ indicates a significant effect of surgery (difference between the control saline and surgery saline groups), and ** indicates a significant attenuation of this effect by drug treatment (difference between the surgery saline group and the surgery drug groups). The combined treatment was significantly lower than each treatment alone.

tissue synchronizes stages in which it is maximally exposed or endangered, with periods of minimal selection pressure. Indeed, fulfilling a prerequisite for this hypothesis, evidence from recent years indicates that malignancies express β-adrenergic and PGE2 functional receptors (Park et al., 1995; Wang and Dubois, 2004), which can enable them to detect periods of neuroendocrine perturbations and potential immune suppression. At least three circumstances along tumor evolvement can be considered as critical for malignancies to rely on neuroendocrine signaling to safely initiate their progression: (a) The first major emission of danger signals from hypoxic dividing malignant cells attracts immunocytes (Collingridge et al., 2001; Lewis and Murdoch, 2005) and exposes the tissue to massive selection pressure. Tumor cells were shown to accelerate their proliferative rate and increase resistance to apoptosis when subjected to increased levels of catecholamines and PGs (Park et al., 1995; Romano and


Claria, 2003; Wang and Dubois, 2004). Thus, tumor cells synchronize this high exposure period with elevated levels of immunosuppressive factors. Similar processes apparently occur with respect to the progression of viruses that were shown to activate gene transcription and to accelerate replication during periods of heightened epinephrine levels (Cole et al., 1998; Cole et al., 2001; Romero et al., 1992). (b) The creation of new blood vessels enables the malignant tissue to grow beyond a critical size, but also markedly exposes the tissue to interaction with newly arrived immunocytes. Tumors were shown to release factors (e.g., VEGF) to facilitate the angiogenic process (Folkman, 1990). Studies have shown that stress-related mediators (e.g., norepinephrine) can directly enhance the production of VEGF by cancer cell lines through tumor ß-adrenergic receptors (Lutgendorf et al., 2003). (c) The metastatic process is another critical stage in which cancer cells detach from the primary tumor and enter the circulation to invade remote organs. Outside the protective environment of the tumor, these single cells are more vulnerable to immune surveillance and are exposed to new populations of immunocytes in target organs. Studies have shown that catecholamines increase the invasiveness of tumor cells (Wang and Dubois, 2004) and tumor cell migration (Lang et al., 2004). Overall, these findings are in agreement with the notion that tumor cells respond to their neuroendocrine milieu in a fashion that synchronizes critical stages of their progression with periods of minimal selection pressure by the immune system. This ability of tumor cells is most likely acquired through random mutation and selection that have eliminated cells that failed to exploit such a potential survival advantage. This characteristic of tumor cells should be considered as an escape mechanism acting at the organism systemic level.

VIII. CLINICAL IMPLICATIONS Today, the medical perspective of cancer is focusing on the neoplasm, addressing its stage, metastatic status, etc. Accordingly, most therapies are directed at the malignant tissue itself (e.g., surgery, chemotherapy, immunotherapy targeting tumor-associated antigens), and the patient’s reaction to the therapy is merely a side effect. In this chapter we emphasize a point of view in which the patient’s natural biological processes have a central role, affecting the progression of the disease and the prognosis. This point of view embraces the notion that cancer evolves within the body through interactions with the immune and neu-

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roendocrine systems. The anti-cancer treatments and the malignant tissue itself can affect the immune and neuroendocrine systems, thereby impacting cancer progression. The following issues are central to this perspective and its implications. It is still controversial whether the theory of immunosurveillance holds and bears clinical significance. While animal studies of immune stimulation have yielded promising results, large-scale clinical trials employing various immunostimulatory agents were mostly inconclusive (Smyth et al., 2001), supporting the view that immune control over cancer progression might be dismal. However, our findings in rats (VI.a) indicate that stress and surgery can cause immunosuppression in immuno-stimulated rats, which might undermine the potential beneficial effects of immunotherapy. Also, the host response to immunostimulating agents may be jeopardized by neuroendocrine responses to stress. Importantly, psychological and physiological stress responses are common in patients, and were not addressed in most animal studies of immunostimulation. Therefore, it is our belief that the controversy over the significance of immune surveillance in cancer patients could be better resolved if clinical trials will assess approaches to prevent immunosuppression in critical periods, as opposed to relying on immunostimulatory approaches. This approach could be implemented by using blockers to stress hormones and PGs during the perioperative period. Such studies will also indicate, for the first time, not only whether the notion of immunosurveillance holds, but also the biological significance of immunosuppression in cancer patients. A few issues should be considered with respect to anti-immunosuppressive therapies: (a) As malignancies evolve and acquire more sophisticated escape mechanisms, immunosurveillance and its neuroendocrine regulation gradually become inconsequential. Encouragingly, early detection of the disease is a major focus of many studies, yielding profound advances in several types of cancer. An early detection may also provide an advantage to anti-immunosuppressive therapy. (b) Cancer patients are often in distress while undergoing immunostimulatory treatments, and the physiological responses to immunostimulation are often stressful in and of themselves. Stress hormones may cause perturbation to specific immune functions, jeopardizing the host response to an immunostimulatory agent. Therefore, stress-alleviating approaches may improve the outcome of immunostimulatory regimens. (c) Such stress-alleviating approaches may also prevent the tumor from capitalizing on neuroendocrine responses that directly accelerate its evolvement (Section VII). (d) In the context of surgery, anti-

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immunosuppressive approaches would, at best, preserve immune competence at baseline levels. Combining immunostimulatory approaches with antiimmunosuppressive approaches could potentially yield synergistic effects, as the two approaches utilize different sets of cellular mechanisms and can thus complement each other. Integrating such a combined approach with surgical treatment might improve longterm prognosis of cancer patients.

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II IMMUNE SYSTEM EFFECTS ON NEURAL AND ENDOCRINE PROCESSES AND BEHAVIOR ROBERT DANTZER

CYTOKINES IN THE BRAIN: AN INTRODUCTION Pro-inflammatory cytokines are soluble mediators that are produced by activated innate immune cells to serve cell-to-cell communication within the immune system. These same proteins are also messengers between immune and non-immune cells so as to enable development of an inflammatory response. The main pro-inflammatory cytokines are interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α), and IL-6. Shortly after their characterization in the innate immune system, pro-inflammatory cytokines have been found to be expressed in the central nervous system either constitutively or in an inducible manner, e.g., in response to injury or peripheral immune stimulation. The study of the production and action of cytokines in the brain has become a hot topic in psychoneuroimmunology due to the diversity of functions that are mediated by these molecules. During the course of an infection, brain pro-inflammatory cytokines relay the action of peripherally produced pro-inflammatory cytokines on neuroendocrine, metabolic, and behavioral endpoints. For instance, the febrile response that is the main feature of the organism’s response to invading pathogens is mediated by prostaglandindependent action of brain pro-inflammatory cytokines on thermoregulatory neurons located in the anterior part of the pre-optic area of the hypothalamus. Brain proinflammatory cytokines are also produced in pathological circumstances in the injured brain. Normally, the production and action of pro-inflammatory cytokines in the brain are tightly regulated by a wide variety of endogenous molecules termed cryogens. These mediators include anti-inflammatory cytokines such as IL-10, hormonal factors such as glucocorticoids and insulin-like growth factor I, and neuropeptides such as vasopressin and α-melanotropin. When pro-inflammatory cytokines are produced in excess or over a long period of time and endogenous cryogens are deficient, functional and structural

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alterations in cell targets of brain cytokines can develop and give rise to alterations in affect and cognition that culminate in mood and cognitive disorders. The eight chapters that are part of this section aim at reviewing the most recent developments in this new field at the interface between neurosciences and immunology. They specifically discuss the pathophysiological implications of the expression and action of cytokines in the central nervous system. In Chapter 13, Robert Dantzer provides an overview of the mechanisms that mediate the production and action of pro-inflammatory cytokines in the brain and discusses how these cytokines are involved in a variety of brain disorders. He starts with a presentation of the key elements of the brain cytokine system before discussing what is currently known concerning the mechanisms of action of cytokines in the brain. He then summarizes the evidence in favor of the involvement of cytokines in brain development and synaptic plasticity, and the role of these molecules in neuropathology and psychopathology. One of the clearest results of the action of cytokines in the brain is the development of sickness behavior. A sick individual displays decreased motor activity, reduced food and water intake, social withdrawal, and disinterest in the external environment. These changes, when prolonged, can be associated with alterations in mood and cognition. In human beings, these behavioral alterations are accompanied by subjective feelings of malaise and distress. Sick patients also express subjective health complaints including fatigue, reduced appetite, sleep disorders, and depressed mood. In Chapter 14, “Cytokines, Sickness Behavior, and Depression,” Dantzer et al. review the current evidence in favor of the organized character of this response of the host to infection and its relation to other motivational systems. These authors present at different levels of description (from the organism to the molecular level) the mechanisms that underlie the behavioral effects of cytokines, including the modalities of immune-to-brain communication, the receptor mechanisms of the sickness-inducing effects of cytokines and the way cytokine-induced sickness behavior can culminate in major depressive disorders in vulnerable individuals. In this last section, Dantzer et al. show that the mechanisms of cytokine-induced depression can be studied in a translational manner by going from the bench to the bedside and vice versa. Whether the actions of cytokines on brain functions are direct or mediated via the synthesis of intermediate molecules such as prostaglandins has long been difficult to determine, because of the relative non-specificity of the tools available for these studies. To address this problem, Michael Lazarus and Clifford B. Saper, in Chapter 15, describe the use of a combination of neuroanatomical approaches and micropharmacological administration of very specific agonists and antagonists of the different subtypes of prostaglandin receptors (EP1–4) that are expressed in the brain. They propose a fever model in which EP1 and EP4 receptors in the pre-optic area promote thermogenesis, whereas EP4 receptors promote hypothermia. At the level of the paraventricular nucleus of the hypothalamus, EP1 and EP3 appear to be required for synergistic activation of the hypothalamo-pituitary-adrenal response to peripheral immune stimulation. The role of the same receptors in the modulation of pain and sleep-wake cycles is also discussed.


The regulatory actions of pro-inflammatory cytokines in memory processes and neural plasticity are developed by Inbal Goshen and Raz Yirmiya, in Chapter 16. They present a dual model in which cytokines that are expressed constitutively in the brain, probably in the neuronal compartment, are necessary for the formation of hippocampal-dependent memory, whereas cytokines that are induced in the glial compartment of the brain in response to peripheral immune stimulation impair memory. Of course, the demonstration of the involvement of cytokines in learning and memory in an inverted U manner gives rise to another important question: What is the place of these immune mediators in the myriad of factors that have already been found to modulate learning and memory? Although this point is not addressed in Goshen and Yirmiya’s chapter, it is tempting to propose that many of the observed effects of cytokines could be indirectly caused via the regulation of brain temperature and metabolism. Aging is well known to be associated with an increased risk for alterations in cognition. Since the many physiological changes that develop during aging include a lowgrade inflammatory status, Rodney W. Johnson and Jonathan P. Godbout propose in Chapter 17 that inflammatory mediators might actually mediate the age-associated impairment in cognition. They demonstrate that while brain microglial cells are normally quiescent, they become activated or “primed” in aged individuals, therefore contributing to neuroinflammation in the brain of otherwise healthy animals. These primed microglial cells are hyper-responsive to a secondary stimulus from the peripheral innate immune system and can produce an exaggerated pro-inflammatory cytokine response that translates functionally in an increased sickness behavior. The same mechanisms probably account for the exacerbation of cognitive disorders and neurodegenerative processes that develop in elderly patients during episodes of peripheral infections. The emphasis in previous chapters on the role of pro-inflammatory cytokines in sickness behavior, learning and memory, and fever should not be detrimental to their well-recognized role in the modulation of pain. Pain is one of the four cardinal signs of inflammation and, as detailed extensively by Linda R. Watkins and her colleagues in Chapter 18, pain is powerfully amplified by activation of peripheral immune cells associated with peripheral nerves and by the activation of immune-like glial cells (microglia and astrocytes) within the central nervous system. After reviewing the basics of pain and pain modulation, Watkins et al. focus on chronic pain, which is poorly, if at all, controlled by currently available drugs. They propose that such pain arises as a consequence of immune and glial activation and discuss the very recent finding that glia and their pro-inflammatory cytokine products also oppose the effects of painrelieving drugs like morphine. All these findings provide new and important molecular and cellular targets for clinical pain control. Building on the repeated observation of increased expression of pro-inflammatory cytokines that is associated with brain injury, Barry McColl and colleagues make the point in Chapter 19 that besides the role of these mediators and especially IL-1 in acute damage during brain injury, there is growing evidence in favor of the involvement of

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cytokines in the post-acute phase. This effect most notably occurs in the resolution of acute inflammation and the impact on brain tissue repair and long-term functional recovery. Involvement of cytokines in chronic neurodegenerative disorders is also an area of expanding investigation in light of the discovery of cytokine gene polymorphisms which confer increased risk of developing Alzheimer’s disease. In Chapter 20, the final chapter of this series, Leigh M. Felton and V. Hugh Perry return to the issue of neuroinflammation that is implicit in all the previous chapters. They describe the biological mechanisms underlying central cytokine expression, discuss their physiological relevance in the context of systemic inflammation, and contemplate the recent evidence demonstrating that pro-inflammatory cytokines can exacerbate neuronal death and inflammation in rodent models of CNS disease. Felton and Perry also describe the bi-directional nature of communication between the CNS and peripheral immune system, placing particular emphasis on the acute phase response to brain injury, and the complex role of the liver. The range of immune mediators they consider encompass a number of pro- and anti-inflammatory cytokines, chemokines, and lipophilic mediators of inflammation. They show how the expression of these mediators may exacerbate tissue injury in acute brain injury, multiple sclerosis (MS), and chronic neurodegenerative disease (prion diseases, Alzheimer’s disease). They also describe the impact of repeated systemic infection on the immunological status of the mammalian central nervous system and summarize evidence demonstrating that priming of microglia and other glia by prolonged or repeated exposure to proinflammatory mediators can impact on the progression, severity, and clinical expression of subsequent CNS disease. Even a cursory reading of the chapters in this series on cytokines in the brain will show that this new information creates pioneering insights on a wide variety of clinical conditions affecting brain function and structure, which certainly accounts for the rapid progression of knowledge in this area at the interface between immunology and neurosciences. The elucidation of mechanisms of immune-to-brain communication has become relevant not only for neurology and psychiatry, but also for somatic diseases with an inflammatory component, including cancer, coronary heart disease, and obesity. In the case of these important systemic diseases, the question that is now crucial is whether the non-specific subjective health complaints of patients that have somewhat been neglected by physicians to the profit of the specific signs of the disease are not just another manifestation of their inflammatory status when it invades the brain. This would explain the comorbid nature of many of these symptoms, including fatigue and depression, and the fact that they are often associated with poor prognosis of the underlying pathological process.

C H A P T E R

13 Expression and Action of Cytokines in the Brain: Mechanisms and Pathophysiological Implications ROBERT DANTZER

The brain represents an important target organ for cytokines since it controls fever, another essential component of the host response to infection. Since cytokines are geared to act locally, they do not travel all the way from the injured body site to the brain. The brain actually reconstitutes a cellular and molecular image of the peripheral inflammation, using its own cellular machinery in the form of macrophage-like cells and microglia. The cytokines that are synthesized and released by these brain cells mirror the cytokine response at the periphery and mediate not only the brain response to immune and non-immune injury, but also the central component of the acute phase reaction. However, this is not their only role. In the brain, like at the periphery, cytokines are two-edged swords that in acute situations, or at low levels of expression, help to deal with the injury and ultimately promote healing. When chronically produced at high levels, cytokines can promote cell death and seriously damage the tissue in which they are expressed. In the brain, this is at the origin of functional and structural alterations that culminate in astrogliosis and neuronal death. Until a few years ago, the study of the production and actions of cytokines in the brain was a very restricted scientific area. Since then, the field has literally exploded. The chapters that are part of the present Part aim at reviewing the most recent developments in this new field at the interface between neurosciences and immunology, and discussing the pathophysiological implications of the expression and action of cytokines in the central nervous system.

I. KEY ELEMENTS OF THE BRAIN CYTOKINE SYSTEM 272 II. MECHANISMS OF ACTION OF CYTOKINES IN THE BRAIN 273 III. ROLE OF CYTOKINES IN BRAIN DEVELOPMENT AND SYNAPTIC PLASTICITY 274 IV. ROLE OF CYTOKINES IN NEUROPATHOLOGY 275 V. ROLE OF CYTOKINES IN PSYCHOPATHOLOGY 277 VI. CONCLUSION 277

Inflammation is a defense response of the body against traumatic, infectious, or toxic injury. This response begins at the site of injury but has the potential of leading to a body-wide response. It first mobilizes mast cells and macrophages stationed in the affected tissues before very rapidly recruiting leukocytes from the blood. These cells respond in an innate manner to highly conserved molecules that are associated with pathogens, the so-called pathogen-associated molecular patterns, by releasing a wide variety of inflammatory mediators including histamine, eicosanoids, proteases, chemokines, and newly synthesized cytokines. The local inflammatory response is geared to kill the invading infectious agents at the expense of tissue damage, before switching to a mode that promotes tissue repair and epithelial closure (Nathan, 2002). Cytokines function as paracrine and autocrine communication molecules between immune cells and between immunocytes and other peripheral cells, such as fibroblasts and endothelial cells. They also act at a distance to coordinate the host response to infection. PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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I. KEY ELEMENTS OF THE BRAIN CYTOKINE SYSTEM Innate immune recognition relies on a limited number of receptors that recognize pathogenassociated molecular patterns and are called Toll-like receptors (TLRs). Stimulation of these receptors by microbial products and, in some cases, by endogenous danger signals coming, for instance, from cells dying from necrosis, in the form of heat shock proteins or from oxidative stress (Gallucci and Matzinger, 2001), leads to the activation of signaling pathways that result in the induction of anti-microbial genes and proinflammatory cytokines (Janeway and Medzhitov, 2002). TLR4s that recognize LPS together with CD14 are constitutively expressed in macrophage-like cells that are present in the circumventricular organs (CVOs), choroid plexus, and leptomeninges (Rivest, 2003). CVOs are devoid of a functional brain barrier and are therefore well suited for responding to signals from the internal milieu. In response to circulating LPS, activation of TLR4 in CVOs leads to the synthesis of pro-inflammatory cytokines that can subsequently invade the brain parenchyma and diffuse toward key brain areas involved in the control of behavior and neurovegetative functions (Konsman et al., 2002). The diffusion of this message into the brain is demonstrated by the rapid transcriptional activation of genes encoding TLR2 in the brain parenchyma (Rivest, 2003). This relatively slow pathway of immune-to-brain communication is complemented by a more rapid pathway involving the neural afferents originating from the site of the body in which the inflammation takes place (Konsman et al., 2002). Circulating cytokines can also get access to the brain via specific saturable transporters (Banks, 2005). The brain inflammatory response to an intraparenchymal injection of LPS is quite different from that of other tissues. In particular, polymorphonuclear cells are largely excluded, and monocytes are recruited only after a delay of several days (Perry and Andersson, 1992). Furthermore, while interleukin-1 (IL-1) administered at the periphery triggers the synthesis of tumor necrosis factor-alpha (TNF-α) and vice versa, this is not the case in the brain parenchyma since IL-1 triggers only its own synthesis but not that of TNF-α (Blond et al., 2002). TNF-α has no effect on its own synthesis, whereas LPS elicits the synthesis of both cytokines. A question that has been disputed for a long time is whether microglial cells that are resident macrophages in the brain parenchyma are formed exclusively locally, are derived from myeloid progenitor cells in the bone marrow, or are actively recruited to

the central nervous system. In a modernized version of the work carried out originally by Hickey and Kimura on the bone marrow origin of perivascular microglial cells (Hickey and Kimura, 1988), the use of lethally irradiated bone marrow chimeric mice, of which the innate immune system was reconstituted with bone marrow stem cells expressing green fluorescent protein, allowed settlement of this issue. These cells were found to immigrate into the brain parenchyma in many regions of the brain and differentiate there in true microglial cells (Kokovay and Cunningham, 2005; Simard and Rivest, 2004). The relative contribution of resident and immigrating cells to brain inflammation was studied by generating bone marrow chimeras using wild-type and TLR4 mutant mice that were unresponsive to LPS (Chakravarty and Herkenham, 2005). In response to systemic LPS, TLR4expressing cells of hematopoietic origin were able to release pro-inflammatory cytokines at the periphery. However, transcription of pro-inflammatory genes in the brain was reduced in duration. Brain-resident TLR4-expressing cells were necessary for the late inflammatory response to LPS. In the normal healthy brain, the macrophage population that includes the microglia and perivascular and meningeal macrophages is switched off. This process that is particularly important for microglia is dependent on several signals including OX2 (CD200), MyD-1 (CD172, a member of the family of signal regulatory phosphatase-binding proteins), and CD47, an integrinassociated protein that binds to the inhibitory receptor signal regulatory protein alpha (Alblas et al., 2005; Hoek et al., 2000). Since the macrophage population in the brain plays a key role in the transduction of immune signals from the periphery to the brain, it is clear that the state of activation of these macrophages influences the severity of this transduction process. There is accumulating evidence showing that in chronic neurodegenerative disorders the macrophages become activated, in the sense that their morphology and phenotype change relative to the resting state. This process and its consequences on the reactivity of the brain to episodes of systemic infection are extensively described in the chapter by Felton and Perry. This is important from the viewpoint of neuropathology since the course of many inflammatory diseases of the brain is well known to be modulated by systemic infections, although this aspect has usually been neglected by pathologists (Holmes et al., 2003; Sibley et al., 1985). A contrario, the enhanced response of the brain to systemic or local immune activation can be taken as evidence of low grade inflammation affecting the brain either directly, such as during chronic neurodegeneration, or indirectly, via the influence of peripheral

13. Expression and Action of Cytokines in the Brain: Mechanisms and Pathophysiological Implications

inflammation on brain inflammation. Instances of such phenomena during aging are reported in the chapter by Johnson and Godbout, and during type I and II diabetes by Dantzer et al. in their chapter on cytokines, sickness behavior, and depression (Dantzer et al., 2006). A potent new tool for inhibiting microglial activation and therefore assessing the role of microglial activation in a given condition is the use of lipid-soluble semisynthetic tetracyclines like minocycline. This compound has been found to be neuroprotective in various animal models of central nervous system trauma and several neurodegenerative diseases (Stirling et al., 2005); to attenuate the production of pro-inflammatory cytokines and their consequences on nociception in the spinal cord (Ledeboer et al., 2005); and to reduce the severity of encephalitis, suppress viral load in the brain, and decrease the expression of neuroinflammatory markers in a simian immunodeficiency virus model of HIV (Zink et al., 2005). The microglia are not the only cell type that is involved in brain inflammation. Other glial cells including astrocytes, oligodendrocytes in the central nervous system, and Schwann cells in the peripheral nervous system are also important. These cell types are both the source and target of cytokines and other inflammatory mediators (Baumann et al., 1993; Dong and Benveniste, 2001). Endothelial cells of brain blood vessels play an essential role in inflammation since they respond to circulating cytokines by producing prostaglandin E2 that diffuses into the brain parenchyma and targets neuronal circuits involved in the control of thermoregulation of activity of the hypothalamic-pituitaryadrenal axis (Matsumura and Kobayashi, 2004). The presence of functional IL-1 receptors on brain endothelial cells, of which the activation induces the expression of COX-2, has been confirmed by immunohistochemistry (Konsman et al., 2004). The latest developments in this field are discussed in the chapter by Lazarus and Saper, with a particular emphasis on the subtypes of prostaglandin E2 receptors that mediate the effects of prostaglandins on neurons. Brain mast cells are often forgotten in the list of cell types that can be at the origin of significant amounts of pro-inflammatory cytokines in the brain (Cocchiara et al., 1998). This is unfortunate since mast cells contain high levels of pre-formed IL-1 and TNF-α that can be released upon degranulation (Moller et al., 1998). In one of the rare studies examining the contribution of mast cells to the production of pro-inflammatory cytokines, intraperitoneal LPS was found to lower visceral pain threshold (allodynia) through a mechanism involving mast cell degranulation and resulting in IL1β and TNF-α release (Coelho et al., 2000).

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An important aspect of the expression and action of cytokines in the brain is its temporal dimension. As illustrated in the chapter by Dantzer et al., the expression of cytokines in the brain in response to peripheral activation of the innate immune system is not an instantaneous phenomenon but a gradually developing one that involves several immune-to-brain communication pathways and various cellular and molecular cascades (Dantzer et al., 2006). This is also true when cytokines are expressed in the brain in the absence of peripheral immune signals. For instance, although IL-1β is rapidly synthesized in the rat brain by glial cells after the induction of seizures, its action is conditioned by the expression of type I IL-1 receptors. IL-1RI expression takes place first in hippocampal neurons before spreading to astrocytes localized in limbic and extra-limbic areas (Ravizza and Vezzani, 2006). Communication between the brain and the innate immune system does not flow only from the immune system to the brain, but also from the brain to the innate immune system, as elegantly demonstrated by Tracey (Pavlov and Tracey, 2005). The efferent branches of the vagus nerves originating from the dorsal motor nucleus of the vagus inhibits release of proinflammatory cytokines at the periphery via α7 nicotinic receptors located on peripheral innate immune cells. This communication pathway utilizes probably choline diffusing from the synaptic cleft rather than acetylcholine. Although the relevance of this pathway has mainly been studied in the context of sepsis, it could play a physiological role by being activated by dietary fat (Tracey, 2005).

II. MECHANISMS OF ACTION OF CYTOKINES IN THE BRAIN Fever had long been known to be elicited by endogenous pyrogens from leukocytic origin rather than from microbial products (Dinarello and Wolff, 1982). However, it took a long time to characterize which molecular factors elaborated by leukocytes were responsible for this response. In the mid 1980s, recombinant technology allowed to produce murine and human IL-1β in sufficient quantities for injection to naïve experimental animals (Dinarello et al., 1986; Tocco-Bradley et al., 1986). The demonstration of the pyrogenic effects of IL-1β was followed by that of the pituitary-adrenal activating effects of this cytokine (Besedovsky et al., 1986) and its ability to induce sickness (Tazi et al., 1988). Adriano Fontana and his colleagues in Switzerland were the first scientists to

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propose that IL-1β was produced in the brain in response to peripherally administered LPS as an intermediate step in fever induction (Fontana et al., 1984). They showed that brain extracts from LPS-injected mice stimulated thymocyte proliferation, induced the production of prostaglandin E2 by synovial cells, and caused fever when injected to LPS-naïve mice. They postulated that astrocytes were the cellular source of this bioactive IL-1β. However, this concept remained dormant until molecular biologists were able to use sufficiently sensitive techniques to demonstrate that LPS induces cytokines in the brain and that cytokine receptors are expressed in the brain. The exact mechanisms of the pyrogenic effects of IL-1 and other cytokines that share its action, including TNF-α and IL-6, have not yet been fully elucidated. In addition to endogenous pyrogens, fever can also be caused by direct activation of TLR receptors by circulating pathogen-associated molecular patterns. The final common pathway for these diverse triggering stimuli is claimed to be the generation of prostaglandin E2 (PGE2) intermediates via the induction of cyclooxygenase-2 and the activation of neuronal PGE2 receptors in the pre-optic area of the hypothalamus (Dinarello, 2004). There are, however, many variants to this mode of action. Cytokines could act directly on neurons of the pre-optic area of the hypothalamus. IL-1 receptors have been identified on hypothalamic neurons including vasopressinergic neurons of the paraventricular nucleus (Diana et al., 1999). However, electrophysiological recordings of vasopressinergic neurons showed that their response to IL-1 is mediated by prostaglandins rather than by a direct neuronal action of IL-1 (Ferri et al., 2005). In the same manner, hyperpolarization induced by IL-1 in the dorsal motor nucleus of the vagus was blocked by inhibition of prostaglandin synthesis (Mo et al., 1996). Several studies indicate that IL-6, rather than IL-1, is the cytokine that is responsible for fever in the brain. In particular, IL-1 did not induce a fever in IL-6 knock-out mice (Kagiwada et al., 2004). The role of prostaglandins in the pyrogenic effects of IL-6 is unclear. Prostaglandins have been proposed to mediate the synthesis of IL-6 in response to IL-1 (Kagiwada et al., 2004) or to be responsible for the effects of IL-6 on its neuronal targets (Li et al., 2003). In another system, the paraventricular nucleus of the hypothalamus, neuronal IL-6 receptors have been proposed to mediate the LPS-induced activation of the HPA axis in a prostaglandin-independent manner (Vallieres and Rivest, 1999). Prostaglandin-amplified thermosensitivity of neurons of anterior hypothalamus could still be another possibility for explaining

the fever response to cytokines (Tabarean et al., 2004). Whatever the ultimate mechanism(s), a role for prostaglandins assumes the presence of prostaglandin receptors on target cells of cytokines. The nature of the receptors that mediate the effects of prostaglandindependent responses to cytokines is extensively discussed in the chapter by Lazarus and Saper. Much progress has been achieved during the recent years on the intracellular signaling pathways that mediate the effects of pro-inflammatory cytokines on their target cells (Aggarwal, 2003; O’Neill et al., 2003; Shishodia and Aggarwal, 2002; Wajant, 2003). However, this knowledge has hardly been used for understanding the mechanisms of action of cytokines in the brain. In the case of IL-1, there is evidence that brain IL-1 receptors are identical to those characterized on peripheral immune and nonimmune cells and that, for the generation of sickness behavior, their activation at the blood-brain interface likely involves a nuclear factor-kappaB (NFκB) signaling pathway in contrast to mitogen-activated protein kinases (MAP-kinases) and especially Erk1/2 that mediate the neuronal effects of IL-1 (Nadjar et al., 2005a; Nadjar et al., 2005b; Parnet et al., 2002). The role of MAP kinases on the neuronal effects of IL-1 has mainly been studied in the model of IL-1– induced impairment of long-term potentiation (see section “Role of Cytokines in Neuropathology”). The induction of COX-2 by IL-1β in the brain vasculature is associated with NFκB activation, as demonstrated by co-localization experiments (Konsman et al., 2004; Nadjar et al., 2005c). Activation of NFκB is necessary for induction of COX-2 since antagonism of NFκB in vivo abrogates the synthesis of inducible COX-2 in brain endothelial cells without altering constitutive neuronal COX-2 (Nadjar et al., 2005c). The observation that IL-1 is still able to exacerbate ischemia-induced neuronal damage in the brain of mice deficient in the gene for the type I IL-1 receptor provides indirect evidence for the role of additional IL-1 receptors in the brain (Touzani et al., 2002), as presented in their chapter by McColl et al.

III. ROLE OF CYTOKINES IN BRAIN DEVELOPMENT AND SYNAPTIC PLASTICITY Besides their role in the elaboration of the central component of the acute phase response, cytokines are also involved in brain development and neuronal plasticity. Programmed neuronal cell death is an essential


factor in the normal development of the brain, since excess neurons that do not form synaptic connections need to be eliminated early in life (Roth and D’Sa, 2001). This process appears to regulated by proinflammatory cytokines such as TNF-α (Bessis et al., 2005). Cytokines are expressed early in brain development and may serve specific roles during the migration of neurons to their targets and the establishment of synaptic connections, for instance, by regulating the expression of adhesion molecules. Cytokines can also regulate the fate of stem cells in neuronal and nonneuronal lineages. For instance, there is evidence that cytokines of the IL-6 family and bone morphogenetic proteins act in concert to promote astrocyte differentiation (Nakashima and Taga, 2002). Chemokines also play a role in the migration, differentiation, and proliferation of glial and neuronal cells (Bajetto et al., 2001). There is evidence, for instance, that the chemokine CXCL12 that binds to the CXCR4 is involved in migration of neural and oligodendrocyte progenitors in the developing brain (Lazarini et al., 2003). Plasticity in the central nervous system is often studied via the model of long-term potentiation (LTP). In this model of memory, a particular pattern of activation of incoming excitatory fibers, presented as mimicking the learning experience, induces long-lasting enhancement of the communication between the presynaptic and post-synaptic elements (representing the memory). As discussed in the chapter by Goshen and Yirmiya, there is evidence that cytokines modulate LTP, with divergent results according to the authors. In the case of IL-1 for instance, endogenous IL-1 has been shown to be necessary for the establishment of LTP (Schneider et al., 1998), whereas exogenous IL-1 impairs LTP (Murray and Lynch, 1998). A possible mechanism for the modulatory effects of IL-1 on LTP is the involvement of prostaglandins E2 in hippocampal synaptic plasticity. Prostaglandins E2 are synthesized in a COX-2–dependent manner in the post-synaptic dendritic spines and function as a retrograde messenger via pre-synaptic EP2 receptors (Sang et al., 2005). A likely explanation for the observation of divergent effects of IL-1 on LTP is the nature of the cellular compartment in which cytokines are expressed and active. Using NFκB as a ubiquitous signaling pathway for the effects of cytokines on their brain targets, Mattson proposed that the expression of this transcription factor in neurons is responsible for plasticity and survival, whereas its expression in glial cells (and probably endothelial cells of blood vessels) is necessary for inducing the production and release of pro-inflammatory cytokines, reactive oxygen molecules, and excitotoxins, contributing to neuronal degeneration (Mattson, 2005). This perspec-

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tive is reinforced by the dual cellular localization of cytokines. For instance, IL-1β is expressed constitutively in neurons and in an inducible manner in the glial compartment (Bartfai and Schultzberg, 1993). Another model of plasticity is represented by sensitization of nociceptive pathways in the dorsal horn of the spinal cord. Cytokines released by activated glial cells sensitize peripheral pain signaling sensory neurons, as presented by Watkins et al. in their chapter.

IV. ROLE OF CYTOKINES IN NEUROPATHOLOGY Cytokines can cause cell death by several processes including apoptosis, necrosis, and autophagy. Apoptosis is a form of programmed cell death that is usually initiated by a stimulus or by removal of a repressor agent and results in self-destruction of the cell. Necrosis, in contrast, is a form of cell death that is caused by injury and involves special enzymes that are released by lysosomes. Autophagy is a dynamic process that occurs in response to nutrient deprivation and involves the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled (Klionsky and Emr, 2000). A role for autophagy in the response of brain cells to pro-inflammatory cytokines has not yet been investigated, despite the fact that the net result of the effect of pro-inflammatory cytokines on many cell types is to interfere with the growth signal transduction that is normally required to direct the utilization of exogenous nutrients to maintain cell functioning and viability, as proposed by Kelley et al., in this book. In a situation of apparent scarcity of nutrients, autophagy is essential for maintaining cell survival, and this process can sustain viability for several weeks. During this time, cells respond to growth factor re-addition by rapid restoration of their ability to take up and metabolize glucose and by subsequent recovery of their original size and proliferative potential (Lum et al., 2005). Rather than directly causing autophagy, proinflammatory cytokines such as IL-1β could actually precipitate this process. Pre-treatment with IL-1β was found to potentiate neuronal death caused by depriving of oxygen and glucose-mixed cortical cell cultures containing neurons and astrocytes (Fogal et al., 2005). AIDS and neurodegenerative disorders like Alzheimer’s or Parkinson’s disease represent the most widely studied group of disorders in which apoptosis has been implicated. For example, the programmed

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cell death of neurons, astrocytes, and endothelial cells that occur in the brain of patients infected with the human immunodeficiency virus has been imputed to neurotoxic substances, including pro-inflammatory cytokines, shed by infected glia and macrophages (Li et al., 2005). In the case of TNF-α, major steps in the cytotoxicity cascade induced by this cytokine include activation of phospholipases, generation of free radicals, and damage to nuclear DNA by endonucleases, leading ultimately to DNA fragmentation and cell death (Larrick and Wright, 1990). There is evidence in favor of an early role of neuroinflammation in the pathophysiology of Alzheimer’s disease (AD) (Benveniste et al., 2001; Eikelenboom et al., 2002; Minagar et al., 2002). The amyloid plaques in AD brains are co-localized with a broad variety of inflammation-related proteins (complement factors, acute-phase proteins, proinflammatory cytokines) and clusters of activated microglia. Amyloid beta (Aβ) deposits in AD brains appear to be closely associated with a locally induced, non-immune–mediated, chronic inflammatory response. Microglial activation precedes the process of neuropil destruction in AD. Increased neuronal COX2 expression also appears to be involved in the cellular changes that take place in pyramidal neurons early in AD. Epidemiological studies indicate that polymorphisms of certain cytokines and acute-phase proteins that are co-localized with amyloid-β plaques are genetic risk factors of AD. In the autosomaldominant inherited forms of AD, the primary factor is the increased production of amyloid-β1-42, resulting into fibrillar amyloid-β deposition that elicits a brain inflammatory response. This state of microglial activation in AD has important functional consequences since animal models show that when activated microglia are further activated by a subsequent systemic infection, this results in significantly raised levels of pro-inflammatory cytokines within the CNS, which may in turn potentiate neurodegeneration, as pointed out in the chapter by Felton and Perry. In particular, a prospective pilot study in AD subjects showed that cognitive function can be impaired for at least two months after the resolution of a systemic infection and that cognitive impairment is preceded by raised serum levels of interleukin 1beta (Holmes et al., 2003). These relations were not confounded by the presence of any subsequent systemic infection or by baseline cognitive scores. Although microglial activation is closely associated with neuronal death, this reaction is not always responsible for neuronal death. It can be protective. In an experimental situation in which microglial cells were repeatedly challenged with lipopolysaccharide or co-

cultured with healthy, apoptotic, or necrotic neuronal cells, microglial cells released immunoregulatory and neuroprotective agents (prostaglandin E[2], transforming growth factor-beta, and nerve growth factor), whereas the synthesis of pro-inflammatory molecules (tumor necrosis factor-alpha and nitric oxide) was inhibited (Minghetti et al., 2005). These findings indicate that signals that are relevant to chronic diseases lead to a progressive downregulation of proinflammatory microglial functions. However, as pointed out by Felton and Perry in their chapter using the example of prion disease, this does not mean that the microglia cannot be re-activated in situations of systemic or local inflammation and actually contribute to neuronal damage (Felton and Perry, 2006). The neurodegenerative effects of pro-inflammatory cytokines are most often indirect via the generation of radical oxygen species and peroxides. Another important mechanism is the potentiation of excitotoxicity. Cytokines can potentiate the effects of glutamate receptor antagonists and, reciprocally, excitotoxicity can lead to the production of pro-inflammatory cytokines. Furthermore, as illustrated in the chapter by McColl et al., cytokines can promote neuronal death at a distance from where they are produced (McColl et al., 2006). The effects of cytokines in the brain involve interactions between many different cellular and molecular events, of which the combination and outcome vary with time. Interactions between the tissue-type plasminogen activator (tPA), N-methyl-daspartate (NMDA) receptors, transforming growth factor-beta1 (TGFβ1), and the type-1 plasminogen activator inhibitor provide a good example of this complex process. The only approved therapy for stroke is early reperfusion by intravenous injection of the thrombolytic agent, tPA. This treatment improves neurological outcome in a great proportion of patients, provided it is performed within the recommended therapeutic time window. However, tPA has the paradoxical effect of enhancing the NMDA receptormediated signaling in neurons, which results in increased excitotoxicity (Benchenane et al., 2004). This effect is mediated by the formation of a direct complex of tPA with the amino acid terminal of the NR1 subunit of the NMDA receptor and the cleavage of this subunit at arginine 260. Transforming growth factor-beta1 is strongly upregulated in the central nervous system following ischemia-induced brain damage. This anti-inflammatory cytokine attenuates ischemia-induced neuronal death in vivo (Buisson et al., 2003). In vitro, TGF-beta1 protects neurons against excitotoxicity by inhibiting the tPApotentiated NMDA-induced neuronal death through a mechanism involving the upregulation of the type-1


plasminogen activator inhibitor in astrocytes (Buisson et al., 2003).

V. ROLE OF CYTOKINES IN PSYCHOPATHOLOGY Besides their role in neuropathology, cytokines have also been implicated in psychopathology. The macrophage theory of depression proposes that major depressive disorders are actually the consequence of the brain effects of pro-inflammatory cytokines due to the activation of the innate immune system that prevails in depressed subjects (Leonard, 2001; Maes, 1999). This process would lead not only to functional changes in brain functions but also to structural alterations, in the form of death of hippocampal neurons. Neuronal death would be due to the neurotoxic effects of tryptophan derivatives produced as a consequence of the activation of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) by immune stimuli (Wichers et al., 2005). Whatever the attractiveness of this hypothesis, it is unlikely that all instances of major depression are due to activation of the innate immune system. It is certainly more realistic to position this event as another precipitating factor for depression rather than a true causal factor (Capuron and Dantzer, 2003). Instances of the involvement of activation of the peripheral immune system in mood disorders are numerous and range from aging subjects in the general population to patients treated with cytokines for cancer or hepatitis C. As illustrated by Capuron et al. in their chapter, this last condition provides a unique opportunity to assess the clinical features of depressive symptoms associated with activation of the immune system and study the mechanisms that are involved in the relationship between cytokines and depression. In Chapter 14, Dantzer et al. describe the progression from sickness behavior to depression, the problems associated with modeling this condition in experimental animals and the possible mechanisms (Dantzer et al., 2006). It is important to point out that IDO activation by cytokines such as IFNγ and TNF-α is probably not sufficient to explain all the symptoms of depression that develop in response to cytokines, and that other mechanisms are at work, including alterations in dopaminergic and glutamatergic neurotransmission. Cytokines have been proposed to play a role not only in mood disorders but also in psychosis. In schizophrenia for instance, findings from clinical studies and postmortem neuropathology point to disturbances of the blood-brain barrier, presence of auto-antibodies or antibodies against various microbial pathogens, acute

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phase proteins, immunocompetent cells, and activation markers of immunocompetent cells that can be explained either by the infectious hypothesis or the autoimmune hypothesis. The epidemiological findings in favor of an increased risk of psychosis in children born from mothers exposed to influenza virus during early pregnancy have been recently confirmed by a group of American psychiatrists based on measure of influenza antibodies in a nested case-control study of a large birth cohort, born from 1959 through 1966, and followed up for psychiatric disorders 30 to 38 years later (Brown et al., 2004). Animal models confirm that exposure to infectious agents during the perinatal period leads to permanent changes in physiology and behavior that are associated with changes in synaptic regulatory genes (Beraki et al., 2005). Intranasal inoculation of influenza virus to pregnant mice at embryonic day 9.5 that corresponds to the time of neural tube closure produced behavioral and neuropathological alterations in the progeny that mimic those observed in autistic children (Fatemi et al., 2002; Shi et al., 2003). Microarray analysis shows that several genes associated with schizophrenia and autism are altered in the brain. These alterations are associated with the maternal immune response to pathogens since they can be reproduced by administration of double-stranded RNA or IL-6.

VI. CONCLUSION In conclusion, the discovery of cytokines and their receptors, the identification of the molecular mechanisms that mediate their effects, and the characterization of the genes of which they modulate the expression have put neuroinflammation at the front stage in neuroscience and biomedical research. The diversity of disciplines involved in this research (from molecular and cellular neuroscience to neuropathology and psychiatry) is a clear testimony in favor of the interdisciplinary nature of the research on cytokines in the brain. The increasing sophistication of techniques and use of post-genomic approaches will certainly accelerate the rate of acquisition of new knowledge in the field and its relevance to clinical preoccupations. In particular the ability to simultaneously quantify multiple cytokines in the biological fluids, tissues, and cell-culture supernatants thanks to the commercial availability of bead-based immunoassay technology will certainly help to clarify the complex interactions that take place within the cytokine network under different circumstances (Hulse et al., 2004).

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Acknowledgments Supported by INRA, CNRS, University of Bordeaux 2, and the National Institute of Health (MH 71349).

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14 Cytokines, Sickness Behavior, and Depression ROBERT DANTZER, ROSE-MARIE BLUTHÉ, NATHALIE CASTANON, KEITH W. KELLEY, JAN-PIETER KONSMAN, SOPHIE LAYE, JACQUES LESTAGE, AND PATRICIA PARNET

I. II. III. IV. V. VI. VII. VIII.

IX.

X.

XI. XII. XIII.

metabolism that is necessary for the fever response. At the subjective level, a sick person experiences malaise, fatigue and apathy, and sometimes mental confusion. Hyperalgesia, or enhanced sense of pain, is another important component of sickness (Watkins and Maier, 2000). The symptoms of sickness can be elicited in healthy subjects and experimental animals by systemic injection of the cytokine inducer lipopolysaccharide (LPS) or the pro-inflammatory cytokines that are released by activated monocytes and macrophages during the host response to infection (Dantzer and Kelley, 1989). Similar symptoms can be induced by direct administration of LPS or pro-inflammatory cytokines into the brain of experimental animals. During the last 15 years, the mechanisms of cytokine-induced sickness behavior have been the subject of intense research, carried out at several levels of analysis, from the molecular to the behavioral and clinical levels. The purpose of the present chapter is to review the results that have been obtained in this field. At the behavioral level, cytokine-induced sickness behavior has been demonstrated to be the expression of a central motivational state that reorganizes the organism’s priorities in order to cope with infectious microorganisms. At the organ level, this motivational aspect of sickness behavior is mediated by the action of endogenous signals of sickness that are represented by pro-inflammatory cytokines on neuronal networks integrating the subjective, behavioral, and physiological components of sickness. Cytokine receptors are present in the brain, and they are activated in most cases not by peripheral cytokines that enter the brain, but by cytokines that are produced locally in the brain in response to peripheral cytokines. At the cellular level,

INTRODUCTION 281 THE CYTOKINE NETWORK 282 BEHAVIORAL EFFECTS OF CYTOKINES 282 SICKNESS BEHAVIOR IS THE EXPRESSION OF A CENTRAL MOTIVATIONAL STATE 285 MODES OF ACTION OF CYTOKINES ON THE BRAIN 286 CELLULAR ORGANIZATION OF THE CYTOKINE NETWORK IN THE BRAIN 291 MECHANISMS OF ACTION OF CYTOKINES ON THEIR BRAIN TARGETS 293 SPECIFICITY OF THE INVOLVEMENT OF PERIPHERAL AND CENTRAL CYTOKINES IN DIFFERENT COMPONENTS OF SICKNESS BEHAVIOR 297 MOLECULAR FACTORS OPPOSING THE EXPRESSION AND ACTION OF PROINFLAMMATORY CYTOKINES IN THE BRAIN 298 MODULATION OF CYTOKINE-INDUCED SICKNESS BEHAVIOR BY DIETARY AND OTHER ENVIRONMENTAL FACTORS 303 SENSITIZATION OF THE BRAIN CYTOKINE SYSTEM 303 CYTOKINES, DEPRESSION, AND ANXIETY 304 CONCLUSION 309

I. INTRODUCTION Sickness is a common if not trivial experience that occurs during the course of an infectious process (Dantzer, 2001). At the behavioral level, sick individuals typically display depressed activity and little or no interest in the environment. Body care activities are usually absent, and food intake is profoundly depressed despite the increased PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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brain cytokines are produced by macrophage-like cells in the meninges and around the blood vessels and by microglial cells in the brain parenchyma. These locally produced cytokines act on other glial cells and ultimately on neurons. At the molecular level, the brain effects of cytokines are mediated by receptors that are similar to those characterized at the periphery. Sickness behavior is normally a reversible process. At the molecular level, this corresponds to the fact that the brain production and action of pro-inflammatory cytokines are downregulated by potent endogenous factors. These factors have been termed cryogens, to contrast them with pyrogens, and they include a wide variety of bioactive molecules, from glucocorticoids to anti-inflammatory cytokines. When the cytokine network remains activated over time, the non-specific symptoms of sickness can become chronic and culminate in mood and cognition disorders. Structural changes can also occur in the brain, in the form of astrogliosis, degeneration of oligodendrocytes that form the myelin sheath, and neuronal death.

II. THE CYTOKINE NETWORK The main pro-inflammatory cytokines that are responsible for the innate immune response to infectious agents are interleukin-1 (IL-1) alpha and beta, interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα). At the periphery, these cytokines are synthesized and released by activated monocytes and macrophages in response to pathogen-associated molecular patterns, and they play a pivotal role in inflammation. A convenient way to induce expression of these cytokines in healthy subjects is to inject lipopolysaccharide (LPS), the active fragment of endotoxin from Gram negative bacteria. Like other pathogen-associated molecular patterns, LPS activates innate immune cells by binding to Toll-like receptors (TLR). Members of the TLR family recognize conserved microbial structures such as Gram negative bacterial LPS for TLR-4, lipoproteins and peptidoglycans for TLR-2, and viral-double stranded RNA for TLR-3. Binding of pathogenassociated molecular patterns to TLR activates a common signaling pathway of which the proximal part involves various adaptor proteins and the distal part activation of nuclear factor-kappa B (NFκB), as well as several mitogen-activated protein kinases (MAPKs) (Barton and Medzhitov, 2003). Although the biological actions of the various proinflammatory cytokines widely overlap, each cytokine also has its own specific properties. IL-1 can be considered as a prototypical pro-inflammatory cytokine (Dinarello, 1998). It exists in two molecular forms, IL-

1α and IL-1β, which are encoded by two different genes. Another member of the IL-1 family is the IL-1 receptor antagonist (IL-1ra). This last cytokine behaves as a pure endogenous antagonist of IL-1 receptors and blocks most biological effects of IL-1α and IL-1β in vitro and in vivo. IL-6 is mainly responsible for the synthesis of acute phase proteins by hepatocytes. This cytokine also plays a pivotal role in the induction of the fever response. TNF-α is a key factor in the pathogenesis of the septic shock syndrome. IL-1α and β bind to two types of receptors belonging to the superfamily of Toll-like and interleukin-1 receptors, the type I and the type II IL-1 receptors. Upon binding with IL-1, the type I IL-1 receptor forms a heterodimeric complex with the IL-1 receptor accessory protein (IL-1RAcP), which activates a cascade of adaptor proteins and kinases that ultimately transduce the signal to the cell nucleus via the activation of NFκB and MAP kinases. This results in the induction of several genes, including the genes for the inducible form of the nitric oxide synthase (iNOS) and the inducible form of cyclo-oxygenase (COX-2). In contrast, the type II IL-1 receptor does not transduce the signal and functions as a “decoy” receptor that binds the excess of IL-1 to downregulate its actions. IL-1ra behaves as an antagonist of IL-1 receptors by preventing the type I IL-1 receptor from associating with IL-1RAcP. Several anti-inflammatory cytokines downregulate the production and action of pro-inflammatory cytokines. They include IL-10, IL-4, IL-13, and transforming growth factor-beta (TGF-β), although many of these cytokines have activities on their own. Other endogenous factors that oppose the production and action of pro-inflammatory cytokines but do not belong sensu stricto to the cytokine network include glucocorticoids, neuropeptides such as vasopressin and alphamelanotropin, and growth factors such as insulin-like growth factor I (IGF-I).

III. BEHAVIORAL EFFECTS OF CYTOKINES Peripheral and central administration of IL-1α, IL-1β, and TNF-α to healthy laboratory animals induces fever (Kluger, 1991), activation of the hypothalamic-pituitaryadrenal axis (Besedovsky et al., 1986), and behavioral symptoms of sickness (Kent et al., 1992b). Depressed activity and apathy are commonly observed, together with decreases in food intake and the adoption of a curled posture. The two behavioral end points that have been mostly used to investigate the mechanisms of cytokine-induced sickness behavior are social exploration and food intake (Figure 1). In the first case, the effects of cytokines are assessed by measuring decreases

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FIGURE 1 Behavioral effects of recombinant IL-1β in rats. Rats were injected intraperitoneally with various doses of recombinant human IL-1β or physiological saline and tested at different times after the injection in a fixed ratio schedule of food reinforcement or in a test of social exploration. In the first case, rats had been previously trained to press an operant lever 10 times consecutively for a food reward (a 45 mg food pellet) and were maintained at 85% of their free feeding body weight for keeping them sufficiently motivated. They were tested for 5 minutes at different time intervals post treatment, and their performance, measured by the number of lever presses, was expressed as percent of response rate just before treatment. In the second case, rats were presented with a juvenile conspecific introduced into their home cage for 5 minutes. Different juveniles were presented on the different occasions of test. Social exploration was measured as the duration of time spent sniffing and following the juvenile. (From Kent et al., 1992b.)

in the duration of exploration of a conspecific juvenile that is temporarily introduced into the home cage of the animal under test. The presentation of this social stimulus normally elicits a full sequence of close following and olfactory investigation that is profoundly depressed in a time- and dose-dependent manner in mice and rats injected with LPS and IL-1 (Kent et al., 1992b). TNF-α is less potent than IL-1 to decrease social exploration, but the effects of IL-1 are potentiated by TNF-α (Bluthe et al., 1994a). Despite its potent pyrogenic effect, IL-6 has no behavioral effect on its own although it is able to potentiate the effects of IL-1 (Lenczowski et al., 1999). Changes in ingestive behavior that develop in sick animals can be measured by the disruption of food intake in animals provided with free food, or the decrease in operant responding in animals trained to press a lever or to poke their nose in a hole for a food reward (Kent et al., 1996; Plata-Salaman, 1995; Plata-Salaman et al., 1996). In both cases, LPS and re-

combinant pro-inflammatory cytokines have profound depressive effects on ingestive behavior whether injected peripherally or centrally. Anorexigenic cytokines include IL-1, IL-6, IL-8, TNF-α, and IFN-α. Based on computerized analysis of meal pattern, low doses of IL-1β were found to reduce meal size and meal duration, and decrease feeding rate. Higher doses of IL-1β also decreased meal frequency and prolonged intermeal intervals (Plata-Salaman and Turrin, 1999; Plata-Salaman, 1999). In contrast, IFN-α induced anorexia by reducing meal size and meal duration but had no effect on meal frequency. In rats trained to eat a meal from separate sources of proteins, fats, and carbohydrates, LPS and IL-1β decreased caloric intake, but increased the relative intake of carbohydrates while decreasing protein intake and keeping fat intake constant (Aubert et al., 1995). This pattern of macronutrient intake differs from what is observed in rats exposed to cold, which increase their caloric intake

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mainly by consuming more fat. This difference between cytokine-treated rats and cold-exposed rats is consistent with the metabolic consequences of the cytokine treatment. Cytokines impair lipid metabolism by decreasing lipoprotein lipase activity and lipolysis (Grunfeld and Feingold, 1996; Spurlock, 1997). They also increase glycogenolysis and glucose oxidation and utilization, and enhance protein breakdown and synthesis of acute phase proteins (Spurlock, 1997). In addition to their direct effects on food and water intake, pro-inflammatory cytokines can alter eating and drinking by inducing conditioned taste aversions. More specifically, if the taste of a new solution is associated with sickness induced by immune activation, subsequent presentation of the taste solution leads to its reduced intake. This occurs whether LPS, peptidoglycan, muramyl dipetide (Goehler et al., 1995), TNFα, or IL-1β are used as sickness-producing agents (Langhans et al., 1990; Mormede et al., 2004; Tazi et al., 1988). However, conditioned taste aversions in animals made sick by immune activation apparently differ from conditioned taste aversions in animals made sick by a toxin (Cross-Mellor et al., 2004). In the former case LPS-treated animals displayed only decreased appetitive taste reactions to the solution paired with sickness. In the second case animals treated with lithium chloride displayed both decreased appetitive and increased aversive taste reactions (Cross-Mellor et al., 2004). However, the possibility of a bias due to the use of an intra-oral perfusion of the taste solution in this experiment cannot be discarded since the amount of sucrose ingested in this way did not vary in LPS-treated rats in contrast to what was observed in lithium chloride– treated rats (Cross-Mellor et al., 2003). Sexual behavior is also disrupted by systemic administration of LPS and IL-1β, and this effect is more marked in female than in male rats (Avitsur et al., 1997a; Avitsur et al., 1997c; Avitsur and Yirmiya, 1999). All components of sexual behavior are affected, including sexual motivation, proceptive behavior (soliciting), receptivity, and attractivity. Although most of the work on the behavioral effects of cytokines has concentrated upon LPS and proinflammatory cytokines, a few studies have addressed the action of other immune stimuli. Bacterial T-cell antigens like staphylococcal enterotoxins A and B induce mainly IL-2, IFNγ, TNF, and IL-6 in a T-cell dependent manner. They activate the HPA axis and reduce food intake, possibly by inducing anxiety since their effect is mainly observed when mice are presented with new food or food in a new environmental context (Kusnecov and Goldfarb, 2005). This aspect is discussed further in “Cytokines, Depression, and Anxiety.” In contrast to LPS, of which the behavioral

effects appear to be mainly dependent on IL-1, the sickness-inducing effects of staphylococcal enterotoxin are mainly mediated by TNF-α (Rossi-George et al., 2005). It is important to note that decreased behavioral activities are not always observed in cytokine-treated animals. For instance, IL-2 and IL-6 can have behavioral-activating effects in mice, in the form of an increase in digging, rearing, exploration of a new object, and, for IL-6, locomotion and grooming (Zalcman et al., 1998). IL-2 was actually able to sensitize mice to behaviorally activating effects of a dopamine agonist (Zalcman, 2001). Conversely, IL-2 induced climbing in mice was blocked by dopaminergic receptor antagonists (Zalcman, 2002). Mice transgenic for a cytokine or the receptor of a cytokine have been used to assess the way cytokines affect brain function. Mice overexpressing pro-inflammatory cytokines in astrocytes or in neurons often develop severe neuropathological alterations (Campbell et al., 1998), which make the study of behavioral activities not very pertinent, except when focusing on early signs of disease progression. Mice in which the gene for a given pro-inflammatory cytokine or its receptors has been deleted by homologous recombination do not show any obvious behavioral alteration, which is in agreement with the redundancy characterizing the biological activity of these proteins. However, subtle behavioral alterations can sometimes be observed. For example, IL-6 knockout mice were found to display a higher degree of aggressive behavior and a lower frequency of affiliative interactions (Alleva et al., 1998). Although the results of behavioral observations in knockout mice are not easy to interpret due to the fact that the expressed phenotype varies according to the genetic background, the fact that in this study mice overexpressing IL-6 specifically in the central nervous system displayed more intense affiliative interactions than their wild-type counterparts was interpreted to suggest that IL-6 might be involved in the regulation of agonistic behavior. In a different experiment controlling for the effect of the genetic background, IL-6(−/−) mice were observed to be less emotional than wild-type mice in the holeboard and the elevated plus-maze, indicating that IL-6 could be involved in the control of emotionality (Armario et al., 1998). Mice overexpressing TNF-α in neurons of the central nervous system displayed alterations of exploration in the hole-board and black/white box, and increased grooming when exposed to highly unfamiliar environmental stimuli (Fiore et al., 1998).


IV. SICKNESS BEHAVIOR IS THE EXPRESSION OF A CENTRAL MOTIVATIONAL STATE Most of the symptoms of cytokine-induced sickness behavior resemble the expression of physical debilitation and general weakness (Figure 2). During an infection episode, sick individuals usually remain prostrated and engage in little physical activity. They do not care for their physical appearance and bodily condition and refrain from engaging in social interactions. There is accumulating evidence in favor of the adaptive nature of the phenotypic changes that occur in sick individuals. A typical example is provided by the changes in bill color that develop in male blackbirds during immune activation (Faivre et al., 2003). Male blackbirds normally display a black plumage with a yellow to orange bill that serves as a secondary sexual trait of health and vigor. The color of the bill is dependent on the availability of carotenoids, but these compounds are also important for immune defenses. During the course of an immune response, the allocation of carotenoids to immune functioning is detrimental to their role in sexual signaling, so that bill color fades off during immune activation (Faivre et al., 2003). In male zebra finches, manipulation of dietary carotenoids supply invoked parallel changes in cell-mediated immune function and sexual attractiveness (Blount et al., 2003). These results show that immune activation has a cost that impacts on phenotype. In mammals, Hart (Hart, 1988) has convincingly argued that sickness behavior supports the metabolic and physiological changes occurring in the infected organism and

FIGURE 2 Motivational aspects of sickness behavior. The behavioral signs of sickness that develop during an infectious episode can be seen as the result of physical debilitation and weakness (upper pathway) or the expression of a reorganization of the organism’s priorities in face of the immune challenge (lower pathway). The evidence collected during recent years supports this last interpretation.

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increases their efficiency. The increase in body temperature that is needed for mounting a fever leaves little room for expensive activities that are of no use in fighting the infectious pathogens. The reduced behavioral activities that occur in ill individuals would therefore be the expression of a highly organized strategy that is critical to the survival of the organism. A typical example of such a reorganization of behavioral activities is provided by the effect of IL-1 on agonistic behavior in mice. When confronted to an adult conspecific, mice typically engage in offensive behavioral patterns, and this phase of attack is followed by defensive behavior if they are likely to lose the fight. In contrast, IL-1–treated mice displayed no offensive behavioral elements, but still responded to the opponent’s attacks with defensive elements such as upright defensive posture, upright submissive posture, crouching, and fleeing (Cirulli et al., 1998). This reorganization of behavioral activities under the influence of pro-inflammatory cytokines is characteristic of what psychologists call motivational changes (Figure 2). A motivation can be defined as a central state which reorganizes perception and action (Bolles, 1970). In the case of fear, for instance, the individual confronted with a threat focuses his attention on all potential sources of danger, and at the same time, gets ready to engage in the most appropriate defensive behavioral pattern that is available in his behavioral repertoire. In other words, a motivational state does not trigger an unflexible behavioral pattern in response to an internal or external stimulus. Instead of this, motivation enables the subject to uncouple perception from action, and therefore to select the appropriate strategy in relation to the eliciting situation (Bolles, 1970). The first evidence that sickness behavior is the expression of a motivational state rather than the consequence of weakness was provided by Miller in an obscure review paper (Miller, 1964). While he was initially searching for endogenous signals of the thirst motivational system, Miller observed that endotoxin administration made rats stop bar pressing for water. However, this motivational effect was not specific of thirst since the same endotoxin treatment also reduced bar pressing for food and for intracranial selfstimulation in the lateral hypothalamus. This decrease in response rate was not generalized since rats placed in a rotating drum that they could stop for brief periods by pressing a lever increased their bar pressing rate in response to endotoxin instead of decreasing it. The mere fact that endotoxin treatment decreased or increased behavioral output depending on its consequences was for Miller a strong indication in favor of a motivational effect of this treatment (Miller, 1964).

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An important characteristic of a motivational state is that it competes with other motivational states for behavioral output. As a typical example, it is difficult if not impossible to search for food and at the same time court a sexual partner, as the behavioral patterns of foraging and courtship are not compatible with each other. The normal expression of behavior therefore requires a hierarchical structure of motivational states which is continuously updated according to variations in the external and internal milieux. When an infection occurs, the sick individual is at a life or death juncture, and he needs to adjust his physiology and behavior so as to overcome the disease. However, this is a relatively long-term process which must make room for more urgent needs when necessary. If a sick person lying in his bed hears a fire alarm ringing in his house and sees flames and smoke coming out of the basement, he must be able to momentarily overcome his sickness behavior to escape danger. Translated into motivational terms, this means that fear competes with sickness, and fear-motivated behavior takes precedence over sickness behavior. To show that sickness does not interfere with the subject’s ability to adjust his behavioral strategies with regard to his needs and capacities, Aubert and colleagues (Aubert et al., 1997b) assessed the effects of LPS on food hoarding and food consumption in rats that did or did not receive a food supplement in addition to normal amounts of daily food. Briefly, rats were trained to obtain food for 30 minutes in an apparatus consisting of a cage connected to an alley with free food at its end. In this apparatus, rats normally bring back to their home cage the food that is available at the end of the alley, and the amount of food they hoard is lower when they receive a food supplement than when they have no supplement. In response to LPS, food intake was decreased to the same extent whether rats received a food supplement or not. However, food hoarding was unaffected in LPS-injected rats that did not receive a food supplement compared to rats provided with the food supplement. These results are important because they indicate that the internal state of sickness induced by LPS is more effective in suppressing the immediate response to food than the anticipatory response to future needs. Another example of the motivational aspects of sickness behavior is provided by the effects of cytokines on maternal behavior (Figure 3). Rodent neonates are poikilothermic, and they need the thermoregulatory protection of a nest. Maternal behavior is therefore critical for survival of the progeny. In accordance with the motivational priority of maternal behavior, lactating mice that were made sick by an appropriate dose of LPS remained able to retrieve their

pups, which had been removed from the nest, but did not engage in nest building when tested at 22°C. However, when the dams and their litters were placed in cold temperatures (6°C) to increase the survival value of nest building, this last behavior was no longer impaired (Aubert et al., 1997a). Collectively, all these data support the view that sickness behavior is not simply the result of a debilitated state. Instead, sickness behavior represents a motivational state that is shaped by both the internal and external needs of the organism.

V. MODES OF ACTION OF CYTOKINES ON THE BRAIN The fact that sickness is a central motivational state implies that the endogenous signals of sickness act on the brain. However, cytokines are relatively large protein molecules, about 15 kDa for IL-1, and their hydrophilic nature does not allow them to easily cross the blood-brain barrier. It has therefore been proposed that cytokines, like many other centrally acting peptides, enter the brain at the sites at which the bloodbrain barrier is deficient. These privileged sites for communication with the internal milieu are known as the circumventricular organs, because of their anatomical location around the brain ventricles. Based on results of lesion and knife cut experiments, the organum vasculosum of the lamina terminalis (OVLT) has long been considered as the site of action of peripherally released cytokines (Blatteis, 1990). Bloodborne cytokines were initially assumed to diffuse into the brain side of the blood-brain barrier through fenestrated capillaries in these sites, and act on parenchymal astrocytes to induce the synthesis and release of prostaglandins of the E2 series. Prostaglandins would then freely diffuse to nearby target brain areas, such as the median pre-optic area of the hypothalamus in the case of fever. However, there are at least two problems with this proposed mechanism. First, most cytokines have a very limited range of diffusion, and are more tuned to act locally in an autocrine and paracrine manner than to act at distance in a hormonal manner. Second, fully functional cytokine receptors are expressed in the brain (see below), and they mediate the effects of endogenously produced or exogenously injected cytokines. For example, blockade of brain IL-1 receptors has been found to abrogate the effects of peripheral immune stimuli on behavior (Kent et al., 1992a), corticotropin-releasing hormone gene expression in the hypothalamus (Kakucska et al., 1993), and body temperature (Klir et al., 1994). The simplest interpretation for these findings is that


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FIGURE 3 Interactions between LPS-induced sickness behavior and maternal behavior in lactating mice. Schematic representation of the experimental design allowing to test the hypothesis of motivational properties of sickness behavior. If the behavioral alterations that develop in lactating mice are only due to changes in their internal state (the socalled medical hypothesis), their nature and intensity should be solely a function of the changes in internal state. If these behavioral alterations are the expression of a motivational state (the so-called motivational interpretation), their nature and intensity should be a joint function of the changes in internal state and the environmental contingencies. Lactating mice were injected with LPS at a sickness-producing dose (400 μg/kg IP) or apyrogenic saline. Their maternal behavior was assessed before and 2 hours after LPS by measuring their ability to build a nest after replacement of the original nest by surgical cotton and to retrieve their pups after pups had been removed from the nest and aligned along the wall on the opposite side from the nest. LPS-treated dams overcame their sickness to retrieve their pups but did not build a nest when tested at 24°C, whereas they built a functional nest in addition to engaging in pup retrieval when tested at 6°C. (From Aubert et al., 1997a.)

cytokines are actively transported into the brain, as proposed for other peptides. For example, insulin enters the brain by a receptor-mediated, saturable transport process across brain capillary endothelial cells (Baura et al., 1993; Wu et al., 1997). Pharmacokinetic data with radiolabeled cytokines have revealed the existence of specific saturable transport systems for a number of pro-inflammatory cytokines (Banks, 2005). In the case of insulin, the transporter is nothing else than the insulin receptor. However, the molecular identity of cytokine transporters is still unknown, and the efficacy of the transport system varies according to cytokines, brain regions, and physiological conditions. Although such a transporter has been shown to mediate learning impairment induced by an intravenous injection of IL-1α (Banks et al., 2001), it is important to note that the existence of a transport system alone is not sufficient to explain how peripheral

cytokines act on the brain. There is still the problem that in order to be transported, cytokines need to be present in the general blood circulation, or synthesized and released close to their transporter systems. Furthermore, a transport process is apparently not necessary for central effects of peripheral cytokines, since independent experiments have provided evidence for the existence of a brain compartment of cytokines that is inducible by peripheral cytokines (Gatti and Bartfai, 1993; Laye et al., 1994). Systemic administration of LPS induces the coordinated expression of IL-1β, TNF-α, IL-6, and IL-1ra in various brain structures at the mRNA and protein levels (Gatti and Bartfai, 1993; Laye et al., 1994) (Figure 4). The same effect is obtained when IL-1β is injected in place of LPS (Hansen et al., 1998), which is consistent with the concept that IL-1β induces its own synthesis and the synthesis of other cytokines in cascade.

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FIGURE 4 Systemic administration of LPS induces the expression of IL-1β and IL-1ra in the brain of mice. Mice were injected intraperitoneally with LPS (10 μg/mouse) and killed just before or at different times after injection. Total RNA was extracted from the hypothalamus and submitted to comparative RT-PCR to determine transcripts for IL-1β and IL-1ra, using beta2-microglobulin (β2mgl) as an internal standard. Levels of IL-1β were measured in the hypothalamus (pg/mg protein) and plasma (pg/ml) using a validated ELISA (mean of 5 different experiments). (From Laye et al., 1994.)

The same cascade of cytokines can be activated when LPS or pro-inflammatory cytokines are injected directly into the lateral ventricle of the brain (Plata-Salaman, 1999). In order to produce cytokines in a coordinated manner in response to peripheral cytokines, brain cells require a communication pathway for transmission of immune information from the periphery to the brain (Dantzer et al., 2000; Kent et al., 1992b). Neural afferents are good candidates for this role, since they already transmit two sensory components of inflammation, calor and dolor (heat and pain) (Kent et al., 1992b). When LPS or cytokines are injected into the abdominal cavity, an inflammatory response develops locally. The main neural pathway from the abdominal cavity to the brain is represented by the afferent branches of the vagus nerves. The role of vagus nerves in the transmission of the immune information from the periphery to the brain has first been demonstrated based on c-fos mapping experiments. The immediate early gene c-fos is differentially expressed in many regions of the central nervous system following various physiological challenges and can be used as a marker of neuronal activation. Intraperitoneal administration of LPS to rats induced the expression of c-Fos, the protein product of the c-fos gene, in the nucleus tractus solitarius that corresponds to the primary projection area of the vagus nerves, and in the parabrachial nuclei,

the paraventricular nucleus and the supraoptic nuclei of the hypothalamus, the central amygdala, and the bed nucleus of the stria terminalis that all correspond to secondary projection areas of the vagus nerves (Wan et al., 1994). The specificity of the relationship of this labeling to vagally mediated transmission was demonstrated by its disappearance following subdiaphragmatic section of the vagus nerves (Wan et al., 1994). Subdiaphragmatic vagotomy also blocked the depressing effects of LPS and IL-1β on social exploration and food intake in rats and mice (Figure 5) (Bluthe et al., 1994b; Bret-Dibat et al., 1995). Blockade of the effects of peripheral cytokines on the brain by vagotomy was not due to an impaired peripheral immune response since vagotomy had no effect on the LPS-induced increases in the levels of peripheral IL-1β measured in peritoneal macrophages or in the bloodstream (Bluthe et al., 1994b; Hansen et al., 2000b; Laye et al., 1995). The involvement of the vagus nerves in the behavioral effects of cytokines is restricted to cytokines that are released in the abdominal cavity and sensed by afferent vagal terminals since vagotomy did not block sickness behavior induced by subcutaneous and intravenous injections of IL-1β (Bluthe et al., 1996b), and had no effect on sickness behavior induced by central injection of IL-1β (Bluthe et al., 1996a). Based on these results, Romeo et al. proposed that immune stimuli from the posterior oral cavity are sensed by the central nervous system via glossopharyngeal afferents (Romeo et al., 2001; Romeo et al., 2003). In accordance with this hypothesis, they showed that bilateral transection of the glossopharyngeal nerves attenuated the febrile response elicited by injection of LPS or IL-1β into the soft palate of rats but had no effect when LPS and IL-1β were injected intraperitoneally. In the same manner, the febrile response caused by injection of a small dose of LPS into an artificial subcutaneous chamber in guinea pigs was blocked in part by injection of a local anesthetic into the subcutaneous chamber, indicating a participation of cutaneous afferent nerve signals in immune-to-brain communication (Roth and De Souza, 2001). According to the hypothesis of a neural immune-tobrain communication pathway, sectioning of vagal afferents should abrogate induction of brain cytokines in response to intraperitoneal administration of LPS and IL-1β. In accordance with this prediction, vagotomy abrogated LPS- and IL-1β–induced increases in IL-1β mRNA in the hypothalamus and hippocampus but not in the pituitary (Hansen et al., 1998; Laye et al., 1995). Results at the protein level are less clear since vagotomized rats continued to respond to intraperitoneal LPS by an increase in brain IL-1β that was identical to that observed in sham-operated rats (Hansen et al.,


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FIGURE 5 Vagotomy abrogates the depressing effects of systemic LPS on social exploration. The experiment was carried out on rats that had been submitted to subdiaphragmatic vagotomy or sham surgery 4 weeks before the test. Physiological saline (SAL) or LPS (1.25 mg/kg) was injected IP immediately after the first behavioral session that took place at time 0 (baseline), and the same animals were tested again with different juveniles 2, 4, and 6 hours later (n = 4 for each experimental group except LPS-treated sham animals for which n = 3). Each behavioral test lasted 4 minutes. (From Bluthe et al., 1994b.)

2000c). The same phenomenon was observed in rats injected with LPS in the soft palate, since the increase in IL-1β protein levels was the same in LPS-treated rats independently of the transection of the glossopharyngeal nerves (Romeo et al., 2003). One of the reasons for this discrepancy between mRNA and protein levels of IL-1β could be the important contribution of extraparenchymal IL-1β to the amount of IL-1β that is measured in the brain by ELISA, in the form of IL-1β derived from meningeal and perivascular macrophages or that transported across the blood-brain barrier. A potential problem with all the vagotomy experiments is that the vagotomy procedure eliminates vagal afferent as well as efferent fibers. Furthermore, the vagotomy surgical procedure often includes stripping the esophagus from all its nerves, which is likely to remove non-vagal, myenteric nerve fibers that are at the origin of the splanchnic visceral nerves. Although surgical transection of the celiac superior mesenteric complex failed to attenuate the hyperalgesic effects of LPS in contrast to section of the vagus nerves (Watkins et al., 1995), the effects of this surgical manipulation on the inhibitory effects of LPS and IL-1β on behavior have received little attention. In the only experiment that specifically addressed the

question of the role of vagal afferents in the behavioral effects of cytokines, selective vagal rootlet deafferentation of the left vagus nerve associated to subdiaphragmatic section of the right vagus nerve did not alter the suppressive effects of LPS and IL-1β on food intake in rats (Schwartz et al., 1997). Negative results were also obtained when vagal deafferentation was combined with celiac superior mesenteric ganglionectomy (Porter et al., 1998). However, in the absence of a positive control for the effects of conventional vagotomy on the action of LPS and IL-1β, these last results are difficult to interpret. Marvel et al. used another approach to assess the role of vagus nerves in the communication pathway from the immune system to the brain by reversibly inactivating the dorsal vagal complex with a longacting local anesthetic (Marvel et al., 2004). The dorsal vagal complex is a brain stem structure that integrates visceral signals and comprises a circumventricular organ, the area postrema, and the primary projection area of the vagus nerves, the nucleus tractus solitarius. The local injection of bupivacaine totally abrogated LPS-induced reduction of social exploration and attenuated LPS-induced Fos expression in various parts of the brain.

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The involvement of vagal afferents in the transmission of information from the peripheral cytokine compartment to the brain is not restricted to the behavioral effects of cytokines, since vagotomy also attenuated LPS-induced hyperalgesia, fever, and elevated plasma ACTH levels (Watkins et al., 1995). Vagotomy was also found to attenuate activation of locus coeruleus neurons by intraperitoneal injection of LPS and other immune activating substances [peptidoglycan and poly(I):(C) (Borsody and Weiss, 2005)]. The same effect was obtained when transection was limited to the dorsal trunk of the vagus nerve. However, it is important to note that the results concerning the effects of vagotomy on fever and pituitary-adrenal axis activation are not always as clear as the effects of this procedure on cytokine-induced sickness behavior. In an experiment comparing the effects of LPS on social exploration and body temperature in the same rats, subdiaphragmatic vagotomy was found to block LPS-induced social withdrawal but not LPS-induced increased body temperature (Konsman et al., 2000a; Luheshi et al., 2000). Since higher doses of LPS are necessary to induce sickness behavior than fever, these results are consistent with the observation that fever induced by high doses of LPS is resistant to the effects of vagotomy (Hansen et al., 2000a; Romanovsky, 2000; Szekely et al., 2000). Mice have been suggested to be less susceptible than rats to the effects of vagotomy (Wieczorek et al., 2005) although a direct comparison between species is still lacking. Vagal signaling of immune activation occurs fairly rapidly since c-Fos is expressed within 60 minutes of intraperitoneal LPS administration. Sensory structures associated with the abdominal vagus (vagal paraganglia) express binding sites for the IL-1 receptor antagonist (Goehler et al., 1997). Furthermore, IL-1β levels in dendritic cells and macrophages within connective tissue associated with the abdominal vagus nerves increased by 45 minutes after LPS administration, a time frame consistent with signaling of immune activation (Goehler et al., 1999). These cells certainly function as immune chemosensory elements (Goehler et al., 2000) that signal vagal neurons via prostaglandindependent and independent mechanisms. Using neuroanatomy techniques, Ek et al. showed that specific labeling for mRNAs encoding the type I IL-1 receptor and the EP3 subtype of the prostaglandin E2 receptor was detected in situ over neuronal cell bodies in the rat nodose ganglion (Ek et al., 1998). Moreover, intravenously applied IL-1 increased the number of sensory neurons in the nodose ganglion that express the cellular activation marker c-Fos, which was matched by an increase in discharge activity of vagal afferents arising from gastric compartments (Ek et al., 1998). This

response to IL-1 administration was attenuated in animals pre-treated with the cyclooxygenase inhibitor indomethacin, suggesting partial mediation by prostaglandins (Ek et al., 1998). The observation that subdiaphragmatic section of the vagus nerves attenuates the brain effects of systemic cytokines, together with the demonstration of an inducible brain cytokine compartment, has shifted the attention from circumventricular organs to neural pathways in the transmission of immune messages to the brain. Since then, neuroanatomical studies have confirmed the existence of a fast route of communication from the immune system to the brain via afferent nerves innervating the site of the body where the inflammatory response takes place (Figure 6). However, this neural pathway is complemented by a humoral pathway that involves a relay at the level of structures outside the blood-brain barrier, the circumventricular organs and the choroid plexus. These neural structures contain dendritic cells and macrophage-like cells that respond to circulating pathogen-associated molecular patterns by producing pro-inflammatory cytokines (Figure 6). These locally produced cytokines diffuse from their site of production to the adjacent brain parenchyma and from there propagate in the brain parenchyma in a manner that is typical of volume transmission, i.e., by using the extracellular space and following specific routes along the blood vessels and fiber tracts (Konsman et al., 2000b; Vitkovic et al., 2000). The recruitment of microglial cells and perivascular macrophages “en passant” is certainly at the origin of a second wave of cytokines produced in the brain parenchyma. Depending on their source, these locally produced cytokines can activate neurons that project to specific brain areas via prostaglandinindependent or dependent mechanisms. The way the neural pathway of immune-to-brain transmission interacts with this humoral pathway remains to be elucidated, but a likely possibility is that the neural pathway sensitizes the brain structures involved in the production and action of cytokines participating in the humoral pathway (Dantzer et al., 2000). In conclusion, the results of neuroanatomy and vagotomy experiments support the hypothesis that multiple routes of communication transmit the immune message from the periphery to the brain. Neural and humoral pathways together with specific saturable transporters are involved in this communication. This multiplicity of pathways points out to the importance of the involvement of the central nervous system in the host response to infection, not only for triggering a febrile response but also to adapt behavior to the new priorities required by the fight against infectious pathogens.


FIGURE 6 Schematic drawing of the neural immune-tobrain communication pathway. The upper portion of the figure represents a sagittal section of the rat brain, whereas the lower-right portion of the figure represents the liver. Intraperitoneally administered LPS induces the production of IL-1β (represented by asterisks) by Kupffer cells in the liver, resulting in the activation of vagal afferents. This neural message is transmitted to the NTS, and from there to the parabrachial nuclei (PB), the ventrolateral medulla (VLM), and the forebrain nuclei that are implicated in the metabolic, neuroendocrine, and behavioral components of the systemic host response to infection (Medial Preoptic area, MPO; ParaVentricular Nucleus, PVN; SupraOptic Nucleus of the hypothalamus, SON; Central nucleus of the Amygdala, CeA; Bed nucleus of the Stria Terminalis, BST). Neural activation of these structures is apparent from c-Fos expression. IL-1β, represented by asterisks, is first synthesized and released in the circumventricular organs (Organum Vasculosum of the Lamina Terminalis, OVLT; Area Postrema, AP; SubFornical Organ, SFO; Median Eminence, ME) and the choroid plexus (ChP). In the AP, IL-1β is synthesized and released by perivascular phagocytic cells and then diffused throughout the interstitium to act on IL-1 type I receptors that are present on AP neurons and project to the Nucleus Tractus Solitarius (NTS). This results in a second wave of FOS expression in the NTS and its projection structures. The NTS therefore plays a pivotal role in the integration of peripheral and central IL-1β. A second wave of IL-1β expression occurs on the neural side of the blood-brain barrier and corresponds to the sequential activation of ramified microglial cells. Not represented in this figure is the fact that the actions of IL-1β on many of its target structures are mediated by intermediates such as prostaglandins and NO. (From Konsman et al., 1999.)

VI. CELLULAR ORGANIZATION OF THE CYTOKINE NETWORK IN THE BRAIN The concept that peripheral immune activation induces expression of cytokines at the mRNA and

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protein levels in the brain was initially based on the observation that intraperitoneal administration of the cytokine inducer, LPS, induces IL-1β synthesis in the brain (Dantzer, 2001; Konsman et al., 2002). The brain cells at the origin of this synthesis are meningeal and perivascular macrophages, and ramified microglial cells (Buttini and Boddeke, 1995; van Dam et al., 1992; van Dam et al., 1995). However, IL-1 is not the sole cytokine to be induced in the brain by peripheral immune stimuli. TNF-α is also expressed at the mRNA and protein levels (Breder et al., 1994; Gatti and Bartfai, 1993), and its cellular sources are represented by perivascular cells and neurons. IL-6 mRNA is also induced in the brain in response to peripheral LPS but not IL-1, with a delayed expression in comparison to that of IL-1 (Laye et al., 1994; Vallieres and Rivest, 1997). IL-1β presents the peculiarity of being produced in the form of a biologically inactive precursor, known as proIL-1β, that needs to be clived at an aspartate residue by a specific enzyme, named interleukin-1β converting enzyme (ICE) or caspase-1, to provide biologically active IL-1β. Despite ICE being co-localized with IL-1β in microglial cells, proIL-1β is the predominant form that is secreted by stimulated microglial cells (Chauvet et al., 2001). Like in macrophages, the release of mature IL-1β from microglial cells requires high amounts of ATP that binds to a low-affinity P2X7 purinergic receptor on the membrane of microglial cells (Ferrari et al., 1997). This results in activation of ICE, and maturation and release of IL-1β. ATP derives from dying cells in case of brain injury or from astrocytes, in the absence of cell damage. The mechanism of release of microglial IL-1β in co-cultures of astrocytes and microglia has been elucidated by the use of a biochemical approach integrated with video microscopy experiments in response to astrocyte-derived ATP (Bianco et al., 2005). ATP induced the formation and the shedding of membrane vesicles containing IL-1β. At the periphery, LPS induces first the expression of TNF-α that is responsible for the synthesis of IL-1. IL-1 itself triggers the synthesis and release of IL-6. Using the technique of polymerase chain reaction after reverse transcription (RT-PCR), studies of brain transcripts for mRNA for these different cytokines at various intervals following intraperitoneal administration of LPS revealed that the same sequence of events occurs in the brain. TNF-α and IL-1β mRNAs appeared first and were followed by IL-1ra and IL-6 mRNAs (Laye et al., 1994). The importance of IL-1β in this cascade was apparent from the observation that intracerebroventricular i.c.v. administration of IL-1ra to LPS-treated mice blocked the induction of expression of IL-1β, IL-6, and TNF-α in the hypothalamus and attenuated it in the hippocampus (Laye et al., 2000). Based on in situ

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hybridization studies, IL-1ra mRNA appears to be mainly expressed in the meninges, choroid plexus, and perivascular cells (Wong et al., 1997). At the cellular level, IL-1ra is produced by microglial cells, although it is also present in astrocytes and neurons, perhaps secondary to an internalization process, the nature of which remains to be elucidated (van Dam et al., 1998). The exact cellular targets of pro-inflammatory cytokines in the brain are still elusive. IL-1 receptors have been identified in brain tissues, using radioligandbinding studies, in situ hybridization, and immunohistochemistry. Equilibrium binding of 125I-IL-1α by brain membrane homogenates and brain slices was the first technique that was used to demonstrate the presence of binding sites for IL-1 in the mouse brain (Ban et al., 1991; Parnet et al., 1994; Takao et al., 1990). These binding sites are located almost exclusively in the anterior pituitary, choroid plexus, and dentate gyrus of the hippocampus. However, these findings are inconsistent with proposed sites of action of IL-1 receptor agonists in such areas as the hypothalamus and brain stem. Autoradiography techniques have been unable to reveal the presence of IL-1 binding sites within the rat brain, in contrast to the mouse (Marquette et al., 1995). However, in situ hybridization identified the type I IL-1 receptor mRNA in several regions of the rat brain, including the anterior olfactory nucleus, medial thalamic nucleus, posterior thalamic nucleus, basolateral amygdaloid nucleus, ventromedial hypothalamus nucleus, arcuate nucleus, median eminence, mesencephalic trigeminal nucleus, motor trigeminal nucleus, facial nucleus, and Purkinje cells of the cerebellum (Ericsson et al., 1995). Immunohistochemistry with an antibody directed against the extracellular domain of IL-1RI revealed a predominant vascular localization of this receptor (Konsman et al., 2004). IL-1R1–immunoreactive perivascular cells were mostly found in choroid plexus and meninges. IL-1R1–immunoreactive vessels were seen throughout the brain, but concentrated in the pre-optic area, subfornical organ, supraoptic hypothalamus, and to a lesser extent in the paraventricular hypothalamus, cortex, nucleus of the solitary tract, and ventrolateral medulla. Vascular IL-1R1– immunoreactivity was associated with an endothelial cell marker that was not found in arterioles, corresponded to the induction patterns of phosphorylated c-Jun and inhibitory-factor kappa B mRNA upon IL-1 beta stimulation, and co-localized with peripheral IL-1 beta- or LPS-induced COX-2 expression (Nadjar et al., 2005). These observations indicate that functional IL-1R1s are expressed in endothelial cells of brain venules and suggest that vascular IL1R1 distribution is an important factor determining

prostaglandin-dependent activation of brain structures during infection. The accessory protein of the IL-1 receptor is expressed at high levels in the mouse and rat brain at a much larger extent than IL-1RI, which indicates a possible role of this accessory protein independently of its association with IL-1RI (Gabellec et al., 1996; Greenfeder et al., 1995; Liu et al., 1996). However, such a role has not yet been elucidated. Upon binding of IL-1, IL-1RI forms a dimer with IL-1RAcP, which ultimately leads to the transcription of NFκB and MAP kinases. It is therefore possible to identify the cellular targets of IL-1 by determining in which cell types NFκB is activated. This can be done using either induction of IκB mRNA or nuclear translocation of NFκB (Figure 7). As mentioned above, endothelial cells of blood vessels not only express IL1R1, but they also respond to intraperitional LPS and IL-1β by enhanced expression of IκB transcripts (Nadjar et al., 2003). These cells are supposed to be at the origin

FIGURE 7 The peripheral IL-1 message relays in circumventricular organs before diffusing into the brain parenchyma. Rats were injected intraperitoneally with 20 μg recombinant rat IL-1β before being killed 1–2 hours (upper line) or 3–4 hours (lower line) after. Coronal sections of the brain at the level of the dorsal vagal complex containing the area postrema (AP) and nucleus tractus solitarius (NTS) were made and labeled with an antibody against p65-NFκB subunit (left column) or rat IL-1β (right column). Note that NFκB labeling overlapped with IL-1β labeling and diffused with time from the AP into the NTS. Although not shown, NFκB labeling was seen in all cases in the nuclear compartment of cells present in the defined structures. (From Konsman et al., 1999; Nadjar et al., 2003.)


of prostanoids in response to IL-1 in the brain (van Dam et al., 1996). However, a careful analysis of the relative importance of endothelial cells and perivascular macrophages revealed that in response to low doses of IL-1 and LPS, perivascular macrophages were the first cells to respond by expressing the inducible enzyme COX-2, whereas endothelial cells responded only to higher doses of LPS (Schiltz and Sawchenko, 2002). Induction of IκB and activation of NFκB have been used further to delineate the neural circuits that are involved in the brain effects of cytokines, as outlined in the next section.

VII. MECHANISMS OF ACTION OF CYTOKINES ON THEIR BRAIN TARGETS The exact mechanisms of action of cytokines on their brain targets are still elusive. There are several aspects to this issue. The first one concerns the brain circuitry that is involved in the response to proinflammatory cytokines. The second one concerns the respective role of brain cytokines and intermediate molecules, such as prostanoids and nitric oxide (NO), in the activation of this circuitry.

A. Functional Neuroanatomy The neuronal circuits that underlie the response to immune stimuli have mainly been studied with regard to the activation of the hypothalamic-pituitary-adrenal axis, the fever response, and the alterations in food intake that develop in animals injected with LPS and IL-1β. Ericsson and colleagues have examined the pathways that mediate activation of the hypothalamicpituitary-adrenal axis in response to an intravenous injection of IL-1β (Ericsson et al., 1994). Using c-Fos immunohistochemistry, they found that c-Fos was induced in C1 noradrenergic neurons in the ventrolateral medulla. These neurons project to the CRH containing neurons of the paraventricular nucleus (PVN) of the hypothalamus. Interruption of the input from the C1 cells to the PVN by knife cuts prevented the CRH response to IL-1β. Quan et al. have proposed another mechanism independent of any neural pathway (Quan et al., 2003). In their model, activation of the PVN is secondary to prostaglandin synthesis by IL-1 activated endothelial cells in the PVN. In the case of fever, Saper and colleagues observed that intravenously injected LPS activated c-Fos in the ventromedial pre-optic area of the hypothalamus (VMPO) (Elmquist et al., 1996). Using retrograde tracing, they found that the VMPO has both direct and indirect (via the anterior perifornical area and

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parastrial nucleus) projections to the autonomic parvicellular divisions of the PVN. They proposed that activation of VMPO efferents following LPS disinhibits PVN neurons that are involved in thermogenesis by inhibition of warm sensitive neurons that are located in the anterior perifornical area, therefore raising the thermostatic set-point for thermoregulation (Elmquist et al., 1996; Plata-Salaman, 1998). In the case of food intake, Plata-Salaman and colleagues showed that IL1β suppressed the neuronal activity of the glucosesensitive neurons in the lateral hypothalamic area, while activating glucose-sensitive neurons in the hypothalamic ventromedial nucleus (VMH) (PlataSalaman, 1998; Plata-Salaman et al., 1988). More modern views on the regulation of food intake and energy expenditure emphasize the role of adiposity signals on food intake and regulation of fat metabolism (Schwartz et al., 2000). Leptin and insulin are adiposity signals, secreted in proportion to body fat content, which act in the hypothalamus to inhibit anabolic and stimulate catabolic effector pathways. These pathways have opposing effects on energy balance (the difference between calories consumed and energy expended) that in turn determines the amount of body fuel stored as fat. A first order brain structure in this process is the arcuate nucleus that is situated adjacent to the floor of the third ventricle and innervates a number of second-order structures directly involved in the regulation of metabolism and/or food intake. The arcuate nucleus transduces the afferent input from circulating leptin and insulin into a coordinated neuronal response thanks to the intervention of two different neuronal populations. One population contains neuropeptide Y (NPY) and agouti-related protein (AGRP) and activates an anabolic pathway, whereas the other population contains pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) and regulates a catabolic pathway. The arcuate nucleus appears to be a target brain structure for IL-1–induced anorexia since it contains IL-1RI (Ericsson et al., 1995) and is activated in response to systemic IL-1 challenge, as shown by enhanced c-Fos expression (Herkenham et al., 1998) that is apparent in both POMC- and NPY-containing neurons (Reyes and Sawchenko, 2002). However, lesion of this structure by various techniques did not impair IL-1– induced anorexia but enhanced it instead (Reyes and Sawchenko, 2002). A possible explanation for this somewhat discrepant finding is the priming effect of brain lesions on microglial reactivity to systemic immune stimuli, as discussed in “Sensitization of the Brain Cytokine System.” Although many studies used cytokine-induced social withdrawal as a characteristic indicator of

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sickness behavior, the neuronal basis of this effect remains unknown. One region of the brain that might be involved in generating social withdrawal is the amygdala (Phelps and Ledoux, 2005). A detailed analysis of the effects of systemic IL-1β on c-Fos immunolabeling in rats in the rat amygdala using retrograde tracing revealed that the central amygdala receives a distinct population of IL-1β–activated afferent inputs that are primarily situated in two nuclei, the parabrachial nucleus and the paraventricular thalamus (Buller and Day, 2002). The parabrachial nucleus is a secondary projection area of the vagus nerves via the nucleus tractus solitarius. The paraventricular thalamus is part of the non-specific thalamus that projects to the amygdala and is also activated by anxiogenic stimuli (Kurumaji et al., 2003). Both the central amygdala and the bed nucleus of the stria terminalis form part of the extended amygdala that projects to the periacqueductal gray. This last neural structure is formed of longitudinal neuronal columns that play key roles in the elaboration of neurovegetative and motor components of what some authors call passive and active emotional coping, especially in relation to painful stimuli (Bandler et al., 2000). Immobility and decreased responding to the environment can be considered as passive emotional coping strategies. The periacqueductal gray has also been involved in defensive recuperative behavior and freezing (Jhou, 2005). The exact role of these brain structures in cytokine-induced social withdrawal remains to be tested. Rather than using functional neuroanatomy techniques to identify the neural structures that mediate the behavioral effects of cytokines, some investigators have microinjected minute amounts of cytokines in specific brain areas. Bilateral infusion of IL-1β into the ventro-medial hypothalamic area of rats depressed food-motivated behavior in rats (Kent et al., 1994). These effects required a lower dose than that injected into the lateral ventricle of the brain. However, the same effect was observed when the cannula did not hit the VMH. In a different experiment, IL-1β was found to decrease locomotor activity, and food and saccharin consumption when injected into the PVN, whereas administration into adjacent thalamic locations had no effect (Avitsur et al., 1997b). These limited findings indicate that the techniques of micropharmacology are certainly useful for dissecting out the sites of action of cytokines in the brain. In the cat, microinjection of IL-1β into the medial hypothalamus facilitated the defensive rage response induced by stimulation of the periacqueductal gray, indicating that IL-1 sensitizes the activity of this last brain structure (Hassanain et al., 2005). However, more systematic investigations are clearly needed.

B. Molecular Intermediates Whether cytokines directly act on the abovedescribed neural pathways or activate them via intermediates such as prostaglandins and nitric oxide (NO) is still a matter of controversy. There is no doubt that cytokines are powerful inducers of the expression of cyclooxygenase-2 (COX-2) in the brain (Breder and Saper, 1996; Cao et al., 1996). COX-2 is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. This enzyme is induced in brain vascular- and perivascular-associated cells in response to systemic administration of LPS and IL-1β, but not IL-6 (Cao et al., 1996; Konsman et al., 2004; Lacroix and Rivest, 1998). Cytokines also induce NO synthase in the brain (Wong et al., 1996). The inducible form of NO synthase (iNOS), also called type II NOS, is synthesized by macrophages in response to inflammatory stimuli. Using in situ hybridization, Wong et al. reported that there is no detectable expression of iNOS in the brain in basal conditions, but that this enzyme is rapidly induced in vascular cells, glia, and neurons of the rat brain in response to intraperitoneal LPS. The induction of this enzyme in the hypothalamus was confirmed by Satta and colleagues, using RT-PCR (Satta et al., 1998). Since many of the functional neuroanatomical studies on the brain effects of immune stimuli were carried out using the intravenous route, and endothelial cells of the brain microvasculature respond to intraluminar cytokines by producing prostaglandins, Elmquist et al. proposed that these intermediates mediate the CRH and fever responses to immune stimuli (Elmquist et al., 1997). These actions of prostaglandins would take place in different regions of the brain, the C1 noradrenergic neurons for the CRH response, and the VMPO neurons for fever. Stimulation of COX-2 in the brain microvasculature of the MPOA results in an increased local production of prostaglandins. These intermediates would bind to specific receptors expressed on nearby neurons to promote the fever in response to peripheral immune stimuli. In the case of a local inflammation, prostaglandins released at the site of inflammation would activate local sensory fibers, resulting in the transmission of the peripheral immune message to the brain. The postulated association between IL-1 receptor activation, NFκB activation, and COX-2 expression in endothelial cells (Quan et al., 1997) has recently been confirmed by visualization of NFκB translocation together with COX-2 induction in response to intraperitoneal IL-1β (Konsman et al., 2004; Nadjar et al., 2003). IL-1β induced a robust translocation of NFκB in the brain vasculature that was associated with COX-2 induction in the same cells and abrogated by


pre-treatment with a cell permeant NFκB antagonist peptide administered directly into the lateral ventricle of the brain (Nadjar et al., 2005). The role of prostaglandins in cytokine-induced sickness behavior has been examined by inhibiting the synthesis of these compounds with various nonsteroidal anti-inflammatory drugs. Intraperitonal pretreatment with the non-selective COX inhibitor indomethacin or the COX-1 inhibitor piroxicam blocked the effects of intraperitoneal IL-1β on social exploration and food-motivated behavior in rats, whereas aspirin was ineffective (Crestani et al., 1991). Indomethacin or ibuprofen was also found to attenuate LPS-induced anorexia in rats (Hellerstein et al., 1989; Langhans et al., 1989; Uehara et al., 1989a) and LPS-induced reduction in sweetened milk intake (Dunn and Swiergiel, 2001). Diclofenac, another COX-1 inhibitor, attenuated LPSinduced reduction in response rate in rats trained to press a lever for a food reward (De La Garza et al., 2004). In an attempt to dissect the respective role of COX-1 and COX-2 in the reduction of sweetened milk intake induced by intraperitoneal LPS, Swiergiel and Dunn used mice in which the gene coding for one or the other enzyme had been deleted, together with administration of specific COX inhibitors in normal mice (Swiergiel and Dunn, 2001; Swiergiel and Dunn, 2002). In response to intraperitoneal IL-1β, COX-2 knockout mice were initially not affected but showed less of a decrease in their intake of sweetened milk 90 to 120 minutes after, whereas the reverse was true for COX-1 knockout mice. The COX-2–selective inhibitor, celecoxib, failed to alter the response to IL-1β 30 minutes after administration, but low doses antagonized the effects of IL-1β at 90 to 120 minutes. The COX-1– selective inhibitor, SC560, attenuated both the early and late responses, but with a larger effect at 30 minutes than at 90 minutes. These results indicate that COX-1 is involved in the early stage response to IL-1β, whereas COX-2 becomes more important at later times. Using a similar approach but with measurements of food intake and body weight for longer times, Johnson et al. found that COX-2 inhibition during LPS-induced inflammation resulted in preserved food intake and maintenance of body weight, whereas COX-1 inhibition resulted in augmented and prolonged weight loss (Johnson et al., 2002). The role of nitric oxide in cytokine-induced sickness behavior was examined using pharmacological approaches aiming at blocking nitric oxide synthase. Inhibition of nitric oxide synthase activity by N omega nitro-L-arginine-methyl-ester potentiated the depressive effects of IL-1β on social investigation in mice. This effect was attenuated by L-arginine but not by D-arginine, confirming the protective action of nitric

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oxide in sickness (Bluthe et al., 1992c). Similar effects were obtained in rats when IL-1β was replaced by LPS (Connor et al., 2002), and these effects were obtained only with an antagonist targeting the endothelial isoform of inducible nitric oxide synthase but not the other isoforms. However, administration of the same antagonist into the lateral ventricle of the brain abrogated the impairing effects of intravenous LPS and ICV TNF-α on drinking behavior induced by water deprivation (Calapai et al., 1994), indicating that production of nitric oxide in the brain could mediate some of the sickness-inducing effects of pro-inflammatory cytokines. In conclusion, there are multiple pathways and several intermediates that are involved in the immuneto-brain communication both at the periphery and in the brain itself (Figure 8). The exact pathway and intermediates that are involved depend on the component of the host response to infection and the time course of the response.

FIGURE 8 Schematic drawing of the mechanisms of cytokine-induced sickness. Pathogen-associated molecular patterns induce the production of pro-inflammatory cytokines at the periphery and in macrophage-like cells of circumventricular organs (CVOs) and choroids plexuses. Peripheral cytokines induce the expression of cytokines in the brain via neural afferents or a relay at the level of circumventricular organs and brain vasculature. Prostaglandins of the E2 series are produced locally and diffuse to brain targets to alter the set point for various regulatory processes. Alternatively, brain cytokines propagate by volume diffusion to their neural targets. The relative importance of humoral and neural pathways differs according to the specific component of the host response to infection. The vertical dotted line in the middle of the figure represents the blood-brain barrier, whereas the other dotted lines represent neurally transmitted actions of cytokines to distant targets. (From Konsman et al., 2002.)

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C. Role of Neurotransmitters and Neuropeptides Administration of LPS or cytokines to laboratory animals induces profound alterations in neurotransmitter metabolism in various areas of the brain. LPS, IL-1, and IL-6 stimulate brain noradrenaline (NA) metabolism, and increase tryptophan concentrations and serotonin (5-HT) metabolism (Dunn et al., 1999). Dopamine levels are elevated by LPS in some brain areas such as the paraventricular nucleus and arcuate nucleus (MohanKumar et al., 1999). IL-2 modulates veratrine-evoked release of endogenous dopamine from striatal slices in a biphasic pattern, increasing release at low concentrations and decreasing it at high concentrations (Petitto et al., 1997). TNF-α affects noradrenaline and tryptophan but only at high doses, whereas INFα has no effect. While the neurochemical response to physical stressors is normally distributed in a relatively uniform manner throughout the brain, the neurochemical response to cytokines is more localized and observed mainly in the hypothalamus (Dunn et al., 1999). Most of these effects have been studied in response to single injections of cytokines. Repeated administration of these soluble mediators can have potent effects on brain neurotransmission. For instance, repeated but not single administration of interferon-α to mice drastically reduced the brain levels of dopamine and its major metabolite, 3-4 dihydroxyphenylacetic acid (Shuto et al., 1997), which could account for the Parkinson-like symptoms observed in patients treated with this cytokine (Capuron and Miller, 2004). In view of the various biological effects of cytokines, it is not easy to relate the changes in neurotransmitter metabolism to specific behavioral effects of cytokines. An attempt to do so has been made by Linthorst and colleagues in an elegant series of studies carried out with in vivo microdialysis techniques in animals of which the core temperature, corticosterone, and behavior were monitored continuously (Linthorst et al., 1995a; Linthorst et al., 1995b). These studies focused on 5-HT and NA neurotransmission in the hippocampus and the hypothalamic preoptic area of rats injected intraperitoneally with LPS. LPS induced a dose-dependent increase in hippocampal extracellular levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). In contrast to what was observed in the hippocampus, intraperitoneal LPS had no effect on preoptic extracellular levels of 5-HT. Intraperitoneal LPS also increased hippocampal extracellular levels of NA and its major metabolite, 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) (Linthorst et al., 1996),

but this effect was small compared to the huge increase of NA and MHPG that was observed in the preoptic area. ICV administration of IL-1β mimicked the effects of LPS on 5-HT neurotransmission in the hippocampus, whereas ICV TNF-α had no effect on behavior and neurotransmitters. ICV IL-2 affected hippocampal 5-HT neurotransmission only at doses that depressed locomotor activity (Pauli et al., 1998). Based on these findings and the neuroanatomical connectivity of these brain structures, Linthorst and colleagues proposed that the increase in preoptic NA is involved in fever and/or activation of the hypothalamic-pituitary-adrenal axis, whereas the rise in hippocampal 5-HT is associated with the development of sickness behavior (Linthorst and Reul, 1999). This hypothesis still needs to be tested using local infusion of specific agonists and antagonists of 5-HT and NA. Studies on the role of neuropeptides in the behavioral effects of cytokines have mainly concentrated on corticotropin releasing hormone (CRH), cholecystokinin (CCK, a feeding inhibitory factor), neuropeptide Y (NPY, a feeding stimulatory factor), and leptin (a feeding inhibitory factor). Since IL-1 is a potent activator of the hypothalamic-pituitary-adrenal axis, the possibility that CRH mediates some of the effects of this cytokine on anxiety-like behaviors has been assessed, using immunoneutralization of CRH by ICV administration of CRH receptor antagonists and neutralizing antibodies directed against CRH. The results of these experiments have not been very conclusive, and the same applies to the depressing effects of IL-1 on food intake. In one experiment, immunoneutralization of endogenous CRF in the brain attenuated the anorexic effect of intraperitoneal IL-1β (Uehara et al., 1989b), whereas in another experiment, this treatment did not alter the disrupting effect of IL-1β administered intraperitoneally or into the lateral ventricle of the brain on food-motivated behavior (Bluthe et al., 1992a). The anorexic effects of the bacterial T-cell superantigen, Staphylococcal enterotoxin A, were attenuated by intracerebroventricular pre-treatment with α-helical CRH9-41, a non-selective CRH receptor antagonist, but not by astressin-2B, a selective CRH receptor 2 antagonist (Kaneta and Kusnecov, 2005). Discrepant results were also obtained with CCK receptor antagonists since administration of a CCK-A receptor antagonist blocked the effects of IL-1α on food intake and gastric emptying in rats in one experiment (Daun and McCarthy, 1993), whereas in another experiment antagonism of peripheral or CCK-A or CCK-B receptors had no effect on the behavioral changes caused by IL-1 (Bluthe et al., 1997b). The observation that both leptin and IL-1 are able to inhibit the potent orexigenic


effects of NPY, and conversely that NPY can reverse IL1–induced anorexia (Sonti et al., 1996), led some authors to speculate that a disregulation of leptin-NPY-cytokine interactions might play a pivotal role in the pathophysiology of feeding disorders (Plata-Salaman et al., 1996). IL-1β and other pro-inflammatory cytokines act on adipocytes and induce the synthesis of leptin (Faggioni et al., 1998; Francis et al., 1999; Sarraf et al., 1997). The in vivo effects of LPS on leptin are likely to be mediated by TNF-α since LPS had no effect on leptin secretion by adipocytes, whereas TNF-α increased it (Finck et al., 1998). The first test of the hypothesis of a role of leptin in cytokine-induced anorexia was carried out on leptindeficient (ob/ob) and leptin-receptor–deficient (db/db) mice. Contrary to expectations, these mice were found to remain sensitive to the anorexic effects of LPS (Faggioni et al., 1997). However, the interpretation of these last results is certainly confounded by the lowgrade inflammation that is associated with obesity and sensitizes the cytokine system both at the periphery and in the brain to immune stimulation (O’Connor et al., 2005). Immunoneutralization of circulating leptin decreased the inhibitory effect of LPS on food intake and attenuated LPS-induced expression of brain cytokines (Sachot et al., 2004). Conversely, the inhibitory effect of leptin on food intake was found to be mediated by IL-1 since it was blocked by central administration of the IL-1 receptor antagonist and was no longer present in mice deficient for the type I IL-1 receptor (Luheshi et al., 1999). These results are consistent with the observation that leptin increases hypothalamic IL-1β expression (Hosoi et al., 2003; Wisse et al., 2004). Because the leptin receptor is closely related to glycoprotein 130 (gp130), a common signal transducer among receptors for cytokines of the IL-6 family (IL-6, ciliary neurotrophic factor [CNTF], leukemia inhibitory factor [LIF]), the possibility that CNTF-induced anorexia is mediated by leptin was investigated in rats treated with intraperitoneal leptin and CNTF (Xu et al., 1998). Both molecules induced anorexia and suppressed NPY-induced feeding. However, CNTF decreased leptin mRNA in adipocytes and had a depressing effect on hypothalamic NPY gene expression only at high dosages, suggesting that the effects of CNTF are not mediated by interactions with these endogenous signals.

VIII. SPECIFICITY OF THE INVOLVEMENT OF PERIPHERAL AND CENTRAL CYTOKINES IN DIFFERENT COMPONENTS OF SICKNESS BEHAVIOR Although most of the work on the behavioral effects of pro-inflammatory cytokines has concentrated on

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IL-1, the different components of the behavioral syndrome that develops in sick individuals do not necessarily have an identical molecular basis. Moreover, the role of a given cytokine in sickness is likely to depend on the compartment, peripheral or central, in which it exerts its effects. The evidence that supports this interpretation is based on time course studies of the effect of IL-1β on social exploration and food-motivated behavior, on experiments examining the behavioral effects of LPS in animals pre-treated with various cytokine and cytokine receptor antagonists, and on investigation of the effects of immune stimulation in mice in which the gene coding for a specific cytokine or its receptor has been deleted by the technique of homologous recombination. Time course studies of the behavioral effects of IL-1β show that the changes in social exploration gradually develop within 2 hours following peripheral administration of this cytokine, whereas the changes in food-motivated behavior reach a maximum by 1 hour following treatment (Kent et al., 1996). The behavioral alterations that are induced by central administration of IL-1β usually dissipate faster than those induced by peripheral administration of this cytokine. The identification and cloning of IL-1ra provided a powerful pharmacological tool to attempt to resolve the role of IL-1 in sickness behavior. This cytokine blocks the in vivo biological effects of IL-1 when coadministered at a 100- to 1,000-fold excess dose with IL-1. To determine the importance of IL-1 in the behavioral effects of LPS, rats were injected intraperitoneally with IL-1ra before being administered LPS intraperitoneally. This treatment abrogated the depressing effect of LPS on social behavior (Bluthe et al., 1992b), but not on food-motivated behavior (Kent et al., 1992c) nor on female sexual behavior (Avitsur et al., 1997c). IL-1ra did not antagonize the decrease in milk intake induced by LPS administration in mice (Swiergiel et al., 1997). These results can be interpreted to suggest that peripheral IL-1 is a key cytokine in the network of proinflammatory cytokines that is induced by LPS when it comes to social activities, but not to food intake. To determine the importance of brain IL-1 in the depressing effects of peripherally administered IL-1 on social behavior and food-motivated behavior, rats received an intracerebroventricular injection of IL-1ra before being injected intraperitoneally with IL-1β. The depression of social exploration of a conspecific juvenile that normally occurs within 2–6 hours following IL-1β injection was totally blocked in IL-1ra pre-treated rats. However, in rats trained to press a bar for a food reward in a Skinner box, the same treatment only partially attenuated the decrease in bar pressing that

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normally occurs within 1–8 hours following IL-1β injection (Kent et al., 1992a). This did not appear to be a dose-dependent effect since increasing the dose of IL1ra by nearly nine-fold did not augment this partial blockade. These results indicate that among the different cytokines that are induced in the brain in response to peripheral IL-1, IL-1 is certainly the predominant molecular signal for the depression in social behavior but not for the decrease in food-motivated behavior. This effect of IL-1 appears to be mediated by the type I IL-1 receptor since intracerebroventricular administration of a neutralizing antibody directed against this receptor blocked the decrease in social behavior that was induced in mice by IP IL-1β (Cremona et al., 1998). In accordance with this last result, mice in which the gene for the type I IL-1 receptor or the accessory protein of the IL-1 receptor had been deleted by homologous recombination no longer responded to ICV IL-1β (Bluthe et al., 2000a). The importance of a given cytokine depends not only on the compartment, peripheral or central, and on the behavior under consideration, but also on the immune stimulus. As described above, brain IL-1 mediates the effects of IP IL-1 on social behavior. However, when LPS was injected in place of IL-1 to suppress social behavior, intracerebroventricular administration of IL-1ra was no longer able to abrogate the depressing effect of LPS (Bluthe et al., 1992b), whereas it still attenuated the LPS-induced reduction in food intake (Laye et al., 2000). The involvement of brain IL-1β in the depressing effects of LPS on food intake was confirmed in an experiment using mice in which the gene coding for ICE had been deleted (ICE(−/−)). These mice do not produce bioactive IL-1β. Both wild-type and ICE(−/−) mice responded to peripheral and central IL-1β injection by a decrease in food intake, indicating that the effector mechanisms of this cytokine were not affected by the mutation. However, ICE(−/−) mice were less sensitive than wild-type mice to the depressing effects of central LPS on food intake and food-motivated behavior, whereas they did not differ in their response to intraperitoneal LPS (Burgess et al., 1998). In general, the presence of a network of cytokines acting in a cascade fashion does not make easy the task of assessing the relative importance of individual molecules in the induction of the behavioral symptoms that are characteristic of sick individuals. Since cytokines act in the context of other cytokines, which have agonist and antagonist activities, the conclusions that can be drawn from a particular combination of cytokines induced by a specific immune stimulus does not necessarily apply to a different combination of the same and possibly other cytokines induced by a

different immune stimulus. Even apparently straightforward pharmacological experiments in which the concentration of a given cytokine is artificially modified can lead to erroneous conclusions. For example, peripheral and central administration of recombinant rat IL-6 to rats failed to cause any alteration in social behavior, whereas the same treatments reliably induced fever and activation of the pituitary-adrenal axis (Lenczowski et al., 1999). These results could easily be interpreted to suggest that IL-6 plays no role in sickness behavior. However, intracerebroventricular administration of IL-6 was found to potentiate the effects of a subthreshold dose of IL-1β administered via the same route on social behavior in rats. In accordance with their attenuated sensitivity to sickness associated with influenza pneumonitis (Kozak et al., 1997a), mice which are deficient in IL-6 were found to be less sensitive to the depressing effects of both IL-1 and LPS on social behavior when these molecules were administered either IP or ICV (Bluthe et al., 2000b). These results indicate that IL-6 is behaviorally active only in the context of other pro-inflammatory cytokines. The problem of identifying a major molecular signal for sickness behavior mirrors the difficulties experienced by pathologists in the selection of an appropriate molecular target for developing potential therapies for the treatment of septic shock and other pathological conditions in which cytokines play a critical role. This does not imply that pro-inflammatory cytokines are not important for the condition under examination; it just indicates that the redundant nature of the cytokine network imposes the choice of alternative strategies, based on the investigation of the mechanisms that are responsible for the commonality of action of pro-inflammatory cytokines, e.g., postreceptor mechanisms, and processes that regulate cytokine expression and actions.

IX. MOLECULAR FACTORS OPPOSING THE EXPRESSION AND ACTION OF PRO-INFLAMMATORY CYTOKINES IN THE BRAIN Pro-inflammatory cytokines are often viewed as a two-edged sword. At low concentrations, they have potent biological effects that play a key role in the development of the host response to infection. However, at high concentrations, they can lead to cell death by necrosis or apoptosis. The activity of proinflammatory cytokines therefore needs to be tightly regulated. Two main categories of molecules oppose the expression and action of pro-inflammatory


cytokines, molecules that are part of the cytokine network and are called anti-inflammatory cytokines, and molecules that are exogenous to the cytokine network and are called cryogens (Kluger, 1991) (Figure 9). Cryogens include neuropeptides, such as vasopressin and alpha-melanotropin, and glucocorticoids.

A. Anti-inflammatory Cytokines Anti-inflammatory cytokines belong to a very heterogeneous family of secreted signaling molecules that all have in common the property of inhibiting to some extent the expression and/or action of proinflammatory cytokines. These cytokines include IL-4, IL-10, IL-13, and transforming growth factors-beta (TGF-β). Although the biological activity of these molecules was initially defined on peripheral immune and non-immune cells, there is evidence that they are also able to regulate the effects of pro-inflammatory cytokines in the nervous system. Most of the studies on anti-inflammatory cytokines in the nervous system have been carried out in the context of neuropathology (Owens et al., 1994; Vitkovic et al., 2001). During the course of an immune response in the brain, antiinflammatory cytokines are mainly produced by T helper 2 lymphocytes, and they downregulate pro-

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inflammatory cytokines produced by T helper 1 lymphocytes (IFN-γ and TNF-α). However, antiinflammatory cytokines can also be produced by macrophages, monocytes, and glial cells, and therefore play a physiological role in the regulation of sickness behavior. TGF-β is constitutively expressed in the brain and is certainly an important factor for the maintenance of the immune privilege of this organ. Whether it is the same for other anti-inflammatory cytokines is still uncertain. Although IL-10, for example, is expressed at the mRNA level in the human pituitary and hypothalamus (Rady et al., 1995), its expression and that of IL-13 do not appear to be very sensitive to the inducing effects of systemic LPS, in comparison to that of IL-1β (Wong et al., 1997). This does not mean that IL-10 has no effect on the brain action of cytokines. In accordance with its ability to protect from the lethal effects of endotoxemia, systemic administration of IL10 was found to abrogate the fever induced in mice by low doses of LPS (Leon et al., 1999). In addition, IL-10 knockout mice developed an exacerbated and prolonged fever and a more marked suppression of food intake in response to a low dose of LPS, when compared to their wild-type counterparts. Central administration of IL-10 abrogated the depressing effects of intraperitoneal LPS on locomotor activity but not on

FIGURE 9 Regulation of the expression and action of pro-inflammatory cytokines in the brain. The production and action of pro-inflammatory cytokines in the brain are regulated by anti-inflammatory cytokines, glucocorticoids, neuropeptides, and growth factors such as IGF-I. Proinflammatory cytokines activate the hypothalamic-pituitary-adrenal axis at the level of the paraventricular nucleus (PVN). The resulting increase in glucocorticoid levels feeds back in a counter regulatory manner not only on the pituitary and hypothalamus, but also on activated immunocytes. Solid arrows represent positive effects, whereas dotted arrows represent inhibitory influences.

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water intake in rats (Nava et al., 1997). Intracerebroventricular administration of IL-10 also abrogated the depressing effects of LPS administered intraperitoneally or into the lateral ventricle of the brain on social exploration in rats (Bluthe et al., 1999a). The reduction of endotoxin-induced hyperalgesia by intraperitoneal IL-10 in mice was associated with a downregulation of LPS-induced increases in levels of TNF-α and IL-1β in the skin of injected hind paws (Kanaan et al., 1998), which indicates that IL-10 might act by inhibiting production of pro-inflammatory cytokines. Centrally administered IL-10 was also able to block sickness behavior induced by intracerebral administration of gp120, a glycoproteic component of the capsid of the human immunodeficiency virus that has potent inflammatory effects in the brain (Barak et al., 2002). Since many anti-inflammatory cytokines play an important role in growth and development, it is possible that more traditional growth factors actually behave as anti-inflammatory cytokines. This was found to be the case for insulin-like growth factor I, which is abundantly expressed in the brain and, when administered centrally, attenuates the behavioral depression induced by intracerebroventricular injection of LPS (Dantzer et al., 1999). Chronic administration of IGF-I into the lateral ventricle of the brain also attenuated the impairing effects of kainate on spatial memory, probably by interfering with TNF-α since the effects of IGF-I were mimicked by pentoxifylline, an inhibitor of TNF-α synthesis (Bluthe et al., 2005). The generality of the anti-inflammatory action of IGF-I and its mechanisms remains to be elucidated.

dependent pathway is also activated during fever (Dantzer and Bluthe, 1992). Central administration of VP attenuated the depressing effects of centrally injected IL-1β on social exploration. Conversely, central injection of an antagonist of vasopressin receptors, which has no biological activity on its own but prevents endogenously released vasopressin to reach its receptors, sensitized rats to the behavioral effects of IL-1 (Dantzer et al., 1991). These last results are important since they suggest that endogenous VP plays a physiological modulatory role in the behavioral effects of IL-1. To determine whether this phenomenon is mediated by an androgendependent or independent pathway, castrated male rats were compared to intact male rats for their sensitivity to the modulatory role of the VP receptor antagonist. Castration by itself potentiated the depressing effects of IL-1β on social exploration. Central administration of VP was more effective in attenuating the behavioral effects of IL-1 in castrated than in intact male rats and, conversely, ICV administration of the VP receptor antagonist was no longer active in potentiating the behavioral effects of IL-1 in castrated male rats lacking vasopressinergic innervation of the lateral septum (Dantzer et al., 1991). Pro-inflammatory cytokines such as IL-1 stimulate the release of vasopressin by acting on a non-specific cationic conductance on vasopressinergic neurons via a prostaglandindependent mechanism (Ferri and Ferguson, 2005; Ferri et al., 2005). Conversely, VP appears to be able to suppress the production of pro-inflammatory cytokines by glial cells via a cAMP response elementbinding protein-element (Zhao and Brinton, 2004).

B. Vasopressin Vasopressin (VP) plays a key role in the regulation of water metabolism. It is produced by hypothalamic magnocellular neurons and accumulates in the terminals of these neurons in the posterior pituitary. It is released in the general circulation and acts as an antidiuretic hormone in response to water deprivation. The release of VP is enhanced during the febrile response, and this enhanced release can be of such a degree that miction does not occur before defervescence. VP also acts as a neurotransmitter in the brain. It is particularly present in neurons whose cell bodies are located in the bed nucleus of the stria terminalis, and whose terminals project to the lateral septum. This vasopressinergic pathway is highly sensitive to circulating androgens in rodents. Castration leads to a dramatic reduction in the content of VP mRNA in the neuronal cell bodies of the bed nucleus of the stria terminalis and to a reduction in immunoreactive VP in the terminal areas of the septum. This androgen-

C. Glucocorticoids The potent activating effects of pro-inflammatory cytokines on pituitary-adrenal activity have already been mentioned. Glucocorticoids feed back on activated immunocytes to downregulate the production and action of pro-inflammatory cytokines (Besedovsky and del Rey, 2000). Glucocorticoids act by binding to the glucocorticoid receptor (GR) that, upon activation, translocates to the nucleus and either stimulates or inhibits gene expression. GR inhibition of many proinflammatory response genes occurs through induction of the synthesis of anti-inflammatory proteins as well as through repression of pro-inflammatory transcription factors, such as NFκB or activator protein-1 (AP-1) (Smoak and Cidlowski, 2004). The downregulatory effects of glucocorticoids on the synthesis and secretion of IL-1, IL-6, and TNF-α by activated monocytes and macrophages (Lee et al., 1988) are physiologically relevant, as demonstrated by adrenalectomy


(ADX) experiments. LPS-induced increases in plasma levels of TNF-α and IL-6 were much higher in adrenalectomized than in sham-operated animals (Zuckerman et al., 1989). Adrenalectomy sensitized experimental animals to the septic shock syndrome, whereas glucocorticoid supplementation had the opposite effect (Bertini et al., 1988; Ramachandra et al., 1992). To assess the possible influence of endogenous glucocorticoids on cytokine expression in the brain, ADX mice and sham-operated mice were injected with saline or LPS, and the levels of transcripts for IL-1α, IL-1β, IL-1ra, IL-6, and TNF-α were subsequently determined in the spleen, pituitary, hypothalamus, hippocampus, and striatum, using comparative RTPCR (Goujon et al., 1996). Levels of IL-1β were also measured by a specific ELISA in the plasma and tissues of experimental animals. LPS-induced expression of cytokines was potentiated by adrenalectomy in the plasma and other tissues, including several brain structures (Figure 10). Conversely, an increase in

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circulating glucocorticoids induced by a 15-minute restraint stress decreased LPS-induced cytokine gene expression both at the periphery and in the brain (Goujon et al., 1995c). It is important to note that the effects of stress are more complex than this experiment indicates, since increases in IL-1β mRNA levels in the hypothalamus of rats (Minami et al., 1991) and IL-6 levels in the plasma (LeMay et al., 1990; Zhou et al., 1993) have also been reported in stressed animals. Stress can actually increase cytokine expression via a β2 adrenergic receptor mechanism (Hetier et al., 1991). However, this effect is masked by endogenous glucocorticoids when they are released in sufficient quantities, as revealed by adrenalectomy experiments. Inescapable electric shocks increased cytokine expression at the periphery and in the brain in ADX, but not in control rats (Nguyen et al., 2000). As already mentioned, IL-1β is synthesized as an inactive precursor which must be cleaved to be secreted in an active form. The enzyme which is responsible for this cleavage is a protease, called interleukin-1β

FIGURE 10 Adrenalectomy increases expression of IL-1α mRNA in mice. Adrenalectomized (ADX) and sham-operated mice were injected subcutaneously with saline or LPS (10 μg/mouse) and killed 2 hours later. Total RNA was extracted from the hypothalamus, and IL-1α mRNA, expressed as a percentage of beta2-microglobulin mRNA, was measured by comparative RTPCR. The figure represents the mean results of three different experiments. Note that LPS increased IL-1α mRNA in various brain structures, and adrenalectomy increased the expression of this cytokine in saline- and LPS-treated mice. * p < 0.05, ** p < 0.01, *** p < 0.001 with respect to control values. (From Goujon et al., 1996.)

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converting enzyme (ICE). To determine whether glucocorticoids that regulate the expression of IL-1 in immune and non-immune tissues are also able to regulate the expression of ICE, mice were injected with LPS and the levels of ICE mRNA in the spleen, pituitary, and brain of experimental animals were measured by comparative RT-PCR. ICE mRNAs were more abundant in the spleen and hippocampus than in the pituitary and hypothalamus, but they were not significantly altered by LPS treatment. In another experiment, mice were submitted to adrenalectomy or a 15-minute restraint stress and injected with saline or LPS. ADX mice had significantly higher ICE mRNA levels, whereas stressed mice had significantly lower ICE mRNA levels than their respective controls. These results can be interpreted to suggest that endogenous glucocorticoids regulate the expression of ICE in peripheral and brain tissues, in a manner which is commensurate to their effects on IL-1β gene expression (Laye et al., 1996). This regulation of ICE by glucocorticoids was confirmed in vitro using primary cultures of murine microglia cells and a transformed murine microglial cell line (Yao and Johnson, 1997). In addition to their action on the synthesis of proinflammatory cytokines at the periphery and in the brain, glucocorticoids are also able to modulate the expression of IL-1 receptors. Although glucocorticoids induce the expression of IL-1 receptors in various immune and non-immune tissues, mixed results have been reported in the brain (Ban et al., 1993; Betancur et al., 1994). The problem with the radioligand-binding techniques which have been used for these studies is that they do not discriminate between type I and type II IL-1 receptor involvement and do not allow an accurate description of what is going on at the molecular level, since the same effect, in the form, for instance, of decreased binding, can be obtained as a result of very different processes, e.g., downregulation of receptors, or activation of receptors with internalization of receptor-ligand complexes. Binding techniques must therefore be complemented by other techniques, e.g., RT-PCR or western blot. By using specific blocking antibodies against type I and type II IL-1 receptors, in vitro studies on primary cultures of murine astrocytes showed that the synthetic glucocorticoid dexamethasone increased the expression of the IL-1RII decoy receptor at the protein and transcript level without affecting the affinity for IL-1β (Pousset et al., 2001). The downregulatory effects of glucocorticoids on the pro-inflammatory cytokine network at the periphery and in the brain should lead to an increased sensitivity of adrenalectomized animals to the brain actions of these cytokines. This appears to be the case since adrenalectomy increased the febrile response to LPS,

whereas administration of glucocorticoids had the opposite effect (Coelho et al., 1992; Morrow et al., 1993). In the same manner, adrenalectomy enhanced the depression of social exploration induced by peripheral injection of IL-1β or LPS (Goujon et al., 1995a). This effect was mimicked by administration of the glucocorticoid type II receptor antagonist, RU 38486. Chronic replacement with a 15-mg corticosterone pellet which yielded plasma corticosterone levels intermediate between baseline and stress levels, abrogated the enhanced susceptibility of ADX mice to the lower dose of IL-1β but had only partial protective effects on the response to a higher dose of IL-1β and to LPS. Taken together, these findings can be interpreted to suggest that the phasic response of the pituitaryadrenal axis to cytokines has an important regulatory role on the neural effects of cytokines. However, they do not reveal at which level (peripheral or central) glucocorticoids are acting. Central sites of action appear to be involved in the regulatory effects of glucocorticoids on the neural effects of cytokines, since central injection of RU 38486 potentiated fever induced by a peripheral injection of LPS to rats (McClellan et al., 1994). In the same manner, adrenalectomy sensitized mice to the depressing effects of intracerebroventricular administration of IL-1β on social exploration, and this effect was attenuated by corticosterone compensation (Goujon et al., 1995b). Central administration of RU 38486 mimicked the effect of adrenalectomy. The negative feedback provided by glucocorticoids on the production and action of pro-inflammatory cytokines in the brain is not specific of the sickness response since similar effects can be observed on the neurotoxic effects of LPS infused into the striatum of rats (Nadeau and Rivest, 2003). The limiting factors for the negative feedback of glucocorticoids on the production and action of cytokines are not necessarily the amount of glucocorticoids produced by the adrenal cortex or their bioavailability. Another important factor is the development of cortisol resistance. Cortisol resistance can be evidenced by the lower ability of synthetic or natural glucocorticoids to attenuate pro-inflammatory cytokine production by innate immune cells, e.g., monocytes. Cortisol resistance occurs during aging in males in part because of a decrease in testosterone since it can be eliminated by testosterone supplementation (Rohleder et al., 2002). Cortisol resistance can also occur in response to social disruption stress (Quan et al., 2001). The effects of glucocorticoids on pro-inflammatory cytokines in response to immune activation are not always inhibitory. They can also be permissive. This was found to be the case in a murine model of herpes simplex virus encephalitis (Ben-Hur et al., 2001).


Intracerebroventricular inoculation of HSV-1 in rats induced fever, motor hyperactivity, and aggressive behavior. These clinical signs were not apparent in adrenalectomized rats, although the mortality rate was not altered. Furthermore, despite the fact that adrenalectomy enhanced the expression of brain IL-1β, it was not further increased in response to HSV-1.

X. MODULATION OF CYTOKINE-INDUCED SICKNESS BEHAVIOR BY DIETARY AND OTHER ENVIRONMENTAL FACTORS Dietary supplementation with anti-oxidants and n-3 fatty acids has anti-inflammatory properties. Antioxidants scavenge radical oxygen species and therefore limit the effects of oxidative stress associated with inflammation on target cells. Omega-3 fatty acids are taken up by virtually all body cells and affect membrane composition, eicosanoid biosynthesis, cell signaling cascades, and gene expression. Intraperitonal administration of α-tocopherol for 3 days to mice attenuated LPS-induced reduction in social behavior (Berg et al., 2004). However, the dietary incorporation of this compound together with selenium was less effective in protecting mice against LPS-induced sickness behavior perhaps because the systemic administration of α-tocopherol better targets the brain than the oral route. This effect of α-tocopherol on sickness behavior is consistent with the demonstration that treatment with antioxidants reduces the production of pro-inflammatory cytokines and radical oxygen species by Kupffer cells or peritoneal macrophages stimulated with LPS (Bellezzo et al., 1998; Haddad et al., 2002; Kheir-Eldin et al., 2001). A fish oil diet rich in n-3 fatty acids was found to attenuate the reduction of food intake induced by intraperitoneal IL-1β and this effect was associated with a reduced production of prostaglandins (Hellerstein et al., 1989). The effects of such a regimen are apparently a function of the nature of the inflammatory response (Kozak et al., 1997b). When inflammation was induced locally by subcutaneous injection of turpentine, fever, lethargy, anorexia, and body weight decrease were all inhibited in mice fed the fish oil diet. In contrast dietary n-3 fatty acids prevented fever and attenuated the decrease in body weight caused by LPS but did not affect the LPS-induced lethargy and anorexia. In another series of experiments, mice fed a diet supplemented with n-3 fatty acids for 4 weeks were less sensitive to LPS-induced reduction in food-motivated behavior and social exploration than mice fed a diet supplemented with n-6 fatty acids (Watanabe et al., 2004). However, this protective effect

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disappeared when LPS was injected into the lateral ventricle of the brain rather than intraperitoneally, indicating that dietary supplementation affected only the peripheral cytokine compartment. The way polyunsaturated fatty acids modulate cytokine-induced sickness behavior is far from clear since rats fed with the n-3 fatty acid, eicosapentaenoic acid, displayed reduced behavioral response to ICV IL-1β (Song et al., 2004). In the same manner, eicosapentaenoic acid inhibited the detrimental effects of LPS on long-term potentiation both in vitro and in vivo (Kavanagh et al., 2004), confirming that n-3 fatty acids can act in the brain to regulate the production and action of cytokines. Because of its strong association with energy requirement, sickness behavior is likely to vary with the energy demands of the environment (Nelson, 2004). In seasonal species such as the Siberian hamster, immune functions are tuned to day length information. LPS-induced sickness behavior was found to be less pronounced in Siberian hamsters maintained in short day lengths compared to hamsters in long days (Bilbo et al., 2002). This was associated with a lower expression of IL-1β and TNF-α in the hypothalamus in response to systemic LPS (Pyter et al., 2005).

XI. SENSITIZATION OF THE BRAIN CYTOKINE SYSTEM By analogy with allergy, the term sensitization is used to refer to the development, over time, of an exaggerated response of a biological system to a challenge. The challenge can cause a mild response on the first few exposures, but as the sensitization process develops, the response becomes higher with subsequent exposures. Cross-sensitization occurs when the stimulus at the origin of the sensitization process belongs to a different category from the one which the biological system normally responds to. In neurosciences, sensitization refers to a change in behavior or biological response by an organism that is produced by delivering a strong, generally noxious stimulus. As a typical example, repeated injections of the dopaminergic stimulant cocaine result in sensitization of the locomotor response to this drug (Post and Rose, 1976). Exposure to a stressor such as a single epidose of social defeat produces the same result as the repeated exposures to cocaine (Miczek et al., 1999). In the case of cytokines, a single administration of TNF-α to mice resulted in an enhanced behavioral, neurochemical, and pituitary-adrenal response to the same cytokine when it was re-injected 28 days but not 1 day after the initial injection (Hayley et al.,

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2002b). Sensitization occurred only when TNF-α was administered systemically, but not when it was injected into the lateral ventricle of the brain or when the challenge was carried out into the lateral ventricle of the brain of mice that had been initially injected intraperitoneally. Since this sensitization was abolished by antihistaminergic drugs (Hayley et al., 2002a), the possibility that it was not due to TNF-α per se but to the bovine serum albumin carrier added to the cytokine solution was investigated further but turned out to be negative (Anisman et al., 2003). Repeated administration of IL-1β did not induce behavioral sensitization in rats submitted to a two-way conditioned avoidance learning paradigm (Bonaccorso et al., 2003). In the same manner, repeated injections of IFN-α did not result in an enhanced behavioral effect on locomotor activity and food intake (Segall and Crnic, 1990). However, a single administration of IL-1β was able to sensitize the hypothalamic-pituitary-adrenal axis for a period of up to 22 days post IL-1 (Schmidt et al., 2001). This sensitization was manifest when mice were later exposed to novelty, and it was associated with prolonged activation of CRH and CRH-R1 in the paraventricular nucleus of the hypothalamus (Schmidt et al., 2003). Prior administration of IL-2 or IL-6 was also able to sensitize the locomotor response to amphetamine or to dopaminergic stimulants (Zalcman et al., 1999; Zalcman 2001), making cytokines behave as stressors (Anisman et al., 2002a). A low-grade inflammation condition can also sensitize cytokine-induced sickness behavior. The first test of this possibility took place in cerebellar mutant mice (Bluthe et al., 1997a). These animals develop a cerebellar atrophy that is associated with a chronic inflammatory state both at the periphery and in the brain. Cerebellar mutant mice were found to be more sensitive to the behaviorally depressing effects of intraperitoneal (IP) and intracerebroventricular (ICV) administration of LPS and IL-1β (Bluthe et al., 1997a). A similar increased sensitivity to the behavioral effects of IL-1β was observed in pre-diabetic nonobese diabetic mice (Bluthe et al., 1999b). The rationale for this form of sensitization was elaborated by Perry, in the context of brain inflammation and neurodegeneration (Perry, 2004; Perry et al., 2003). Perry gathered experimental and clinical evidence showing that systemic acute inflammation on a background of ongoing inflammation in the brain can be harmful and contribute to the onset or progression of disease. This can even take place in the context of an antiinflammatory profile such as the one occurring in pre-clinical murine prion disease since under the influence of systemic inflammation it rapidly switches

to a pro-inflammatory profile (Combrinck et al., 2002). The key cellular event that underlies sensitization is the priming of microglia by early or subtle pathological changes. Passage of immune signals from the periphery to the brain during the course of a systemic immune activation further activates microglia and results in aggravated signs of sickness. In contrast, an exaggerated sickness response to a systemic immune challenge or to a direct local stimulation can be used to reveal this priming state of the microglia. This has been demonstrated to be the case in aged mice (Godbout et al., 2005) and in type II diabetic mice (O’Connor et al., 2005).

XII. CYTOKINES, DEPRESSION, AND ANXIETY Uncontrolled inflammation or previous sensitization of the brain cytokine network will lead not only to exaggerated sickness behavior, but also to mood disorders and ultimately neuropathology. In this section, the clinical and experimental evidence showing that activation of the brain cytokine network can induce mood disorders will be presented and the mechanisms discussed. A more detailed discussion of the role of cytokines in mood disorders can be found in Chapter 24, “Psychoneuroimmunology of Depressive Disorder: Mechanisms and Clinical Implications,” by Capuron et al.

A. Depressed Mood Caused by Immune Activation in Human Subjects The first studies documenting the depressive effects of cytokines were carried out clinically (Capuron et al., 2000; Capuron et al., 2001a; Capuron et al., 2001b; Raison et al., 2005). These studies reported that whereas all patients responded to the treatment by manifesting behavioral symptoms of sickness, a significant percentage of them also developed mood disorders. These observations were carried out in patients given cytokine immunotherapy (e.g., IFN-α, IL-2) for the treatment of cancer (kidney cancer and melanoma with metastasis) and viral infections such as hepatitis C. The exact clinical presentation of mood disorders varies according to the immunotherapy treatment and the underlying pathology. Cancer patients display relatively pure depressive disorders, with sadness, decreased selfesteem, and psychomotor retardation, whereas hepatitis C patients develop episodes of depression alternating with episodes of manic symptoms, although not severe or long enough to be catego-


rized as mania. Irritation and anxiety dominate the clinical presentation in these patients (Capuron and Miller, 2004). In all cases, these psychiatric symptoms develop over a background of neurovegetative sickness symptoms, including fatigue, decreased appetite, and sleep disorders. The fact that mood disorders occur only in about one-third of the patients receiving immunotherapy points to the importance of individual vulnerability factors. Patients who have clinically diagnosed depressed mood at baseline, before the initiation of immunotherapy, are more at risk than patients with normal mood scores (Capuron and Ravaud, 1999). The risk of developing a depressive disorder is also higher in patients whose pituitary-adrenal axis response to the first injection of IFNα is more intense (Capuron and Miller, 2004). Whether these features are characteristic of patients who have the usual risk factors for mood disorders or for a differential activation of the innate immune response (e.g., single nucleotide polymorphisms accounting for higher production of pro-inflammatory cytokines or a decreased production of anti-inflammatory cytokines) remains unknown. Similar relationships between immune activation and depressed mood have been observed in conditions of less drastic immune activation. Volunteers injected with Salmonella abortus equi endotoxin developed mild illness symptoms associated with significant increases in anxiety and mood (Reichenberg et al., 2001). Mild inflammatory stimulation caused by typhoid vaccination of student volunteers led to a significant decline in mood without any neurovegetative sign (Strike et al., 2004). This was associated with increased plasma levels of IL-6, and negative changes in mood following vaccination significantly correlated with increases in IL-6 (Wright et al., 2005).

B. Immune Activation and Depressive- and Anxiety-like Behavior in Laboratory Rodents Indirect evidence for an association between inflammation and depression comes from the demonstration that animal models of depression (e.g., olfactory bulbectomized rats or rats submitted to mild chronic stress) display evidence of chronic activation of the innate immune system (Connor et al., 2000; Kubera et al., 1998; Kubera et al., 2001; Mormede et al., 2004). More direct evidence comes from experimental studies showing that peripheral or central administration of pro-inflammatory cytokines induces depressive-like behaviors in laboratory rodents (Avitsur and Yirmiya, 1999; Merali et al., 2003; Yirmiya et al., 1999). These

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behavioral alterations include social withdrawal, increased duration of immobility in the forced swim test, failure to learn to escape from painful electric shocks (learned helplessness), and decreased sensitivity to reward or pleasure (anhedonia). Anhedonia can be evidenced by an increased threshold for a rewarding electrical stimulation in the brain (Anisman et al., 2002b) or a decreased attraction to sweetened solutions (Yirmiya et al., 1999). These behavioral alterations are associated with decreased brain serotoninergic and dopaminergic neurotransmission, as well as some implications for the noradrenergic system. In general, the behavioral and neurochemical alterations that are assumed to model depression in mice and rats are more profound in aged than in young individuals (Onodera et al., 2000; Schulz et al., 2004; Slotkin et al., 1999), which is consistent with the low-grade inflammation status that characterizes aging (see Chapter 17, “Aging, Neuroinflammation, and Behavior,” by Johnson and Godbout, this volume). In the absence of a fully validated animal model of depression, it is important to note that convergent evidence from several behavioral tests together with pharmacological validation (i.e., the observed alterations in behavior must be alleviated by antidepressant treatment) is required before any conclusion on depressive disorders can be reached (Redei et al., 2001; Willner and Mitchell, 2002). Studies on the so-called anhedonic effects of cytokines provide a good illustration of this issue. As pointed out by Merali and colleagues, it is not easy to dissociate the sickness elicited by cytokines, including anorexia, from effects related to anhedonia since both conditions result in a decreased behavioral performance (Merali et al., 2003). In an attempt to reach this goal, they trained rats in a progressive ratio schedule for a sucrose solution reward, in which rats had to progressively increase their rate of responding in order to gain access to the reward. This had already been done in mice by Larson et al. who showed that intraperitoneal administration of IL-1β decreased the breaking point; i.e., IL-1β–treated animals stopped earlier in the sequence than salinetreated mice (Larson et al., 2002). Merali et al. observed the same effect, but in their experiments rats had free access to laboratory chow at the same time they performed on the progressive ratio schedule (Merali et al., 2003). IL-1β decreased both chow consumption and progressive ratio performance. This by itself was not demonstrative of anhedonia, but the observation that chronic fluoxetine treatment attenuated the disturbance of progressive ratio performance but had no effect on reduced chow consumption allowed them to conclude that it is possible to dissociate the anhedonic from the anorexic effects of IL-1β, since the former but

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not the latter one was sensitive to the effects of an antidepressant drug supposed to abrogate anhedonia. Such results do not necessarily imply that reduced preference for a palatable solution in cytokine-treated animals is a measure of anhedonia. In a detailed analysis of the effects of LPS on preference for saccharin and aversion for quinine using a two-bottle test procedure, LPS was found to decrease the preference for saccharin and attenuate the aversion to quinine, on a background of reduced intake of fluid (Aubert and Dantzer, 2005). Taste reactivity, as assessed by scoring of ingestive and aversive taste reaction patterns to separate solutions of sucrose and quinine, was not altered by LPS treatment. However, LPS decreased the ingestive and increased the aversive taste reaction patterns to a mixture of sucrose and quinine. Although the results on taste reaction patterns to sucrose are consistent with those of other studies showing that LPS decreased sucrose intake but did not alter sucrose palatability (CrossMellor et al., 1999), the data on the taste reactivity to the bitter-sweet solution point to an increased finickiness of sick animals that would prevent them from ingesting any potentially toxic compound even when normally tolerated by healthy animals (Aubert and Dantzer, 2005). Most of the studies on the relationships between depressive-like behavior and cytokines have been carried out in rodents injected with cytokines. However, in an effort to model the depression that is associated with coronary artery disease, Grippo et al. assessed the effects of experimental heart failure induced in rats by coronary artery ligation on responses to rewarding electrical brain stimulation in the lateral hypothalamus (Grippo et al., 2003). Heart failure led 7 days later to a rightward shift in the current-response relationship in the brain self-stimulation paradigm, indicative of reduced rewarding properties of the brain stimulation. This effect was prevented by treatment with a TNF-α antagonist administered 24 hours before brain self-stimulation. Another approach to study the possible contribution of cytokines to the pathogenesis of depression is the use of genetic animal models of depression. For instance, the Fawn-Hooded rat is an inbred strain of rats that has been reported to display many of the proposed animal equivalents of depression, including a higher immobility in the forced swim test, and all these features are corrected by antidepressant treatment (Rezvani et al., 2002). Compared to normal Sprague-Dawley rats, Fawn-Hooded rats displayed augmented behavioral and neurochemical responses to IL-1a (Simmons and Broderick, 2005). The possibility that pro-inflammatory cytokines have anxiogenic effects has been investigated using

various tests of anxiety (Anisman and Merali, 1999). In the elevated plus-maze, mice treated with LPS displayed a reduced frequency of open-arm visits, whereas visits to the closed arms were unaffected. Similar effects were obtained in rats treated with ICV IL-1β and TNF-α, but not IL-2 nor IL-6 (Connor et al., 1998). In the light-dark box, LPS increased the tendency to avoid the illuminated region (Anisman and Merali, 1999). These behavioral alterations were not due to motor deficiency since LPS-treated mice escaped very quickly from the open arms and the illuminated compartment when they were directly exposed to them. Acute administration of IL-2 had no obvious anxiogenic effect, whereas chronic IL-2 attenuated exploration. IL-1β had anxiogenic effects at low doses when injected either systemically or centrally, whereas these effects were replaced by a general depression of activity at higher doses. The same effects as those obtained with low doses of IL-1β were observed with TNF-α. Increased levels of anxiety-like behavior in the elevated plus-maze were also observed in mice infected orally with the Gram-negative pathogen Campylobacter jejuni at a dose that induced a subclinical infection but no immune activation, at least based on circulating IL-6 levels (Lyte et al., 1998). In accordance with a positive action of pro-inflammatory cytokines on anxiety, mice overexpressing the IL-1 receptor antagonist behaved as if they are less anxious. Compared to wildtype mice, they displayed higher locomotion and decreased habituation in an open-field test and spent a longer time in the open arms of an elevated plus maze (Oprica et al., 2005). As mentioned in “Behavioral Effects of Cytokines,” administration of bacterial T-cell superantigens was found to suppress food intake, these effects being more apparent when animals were presented with a new palatable solution or when the already familiar food was presented in a novel context (Kusnecov et al., 1999). These effects were therefore interpreted in terms of increased anxiety. Similarly, mice challenged with bacterial superantigens displayed greater reactivity to a novel object introduced into an openfield (Kawashima and Kusnecov, 2002). However, when mice treated with bacterial superantigens were tested in the elevated plus-maze and light-dark box, they showed no sign of anxious-like behavior and actually spent a longer time in the open arms of the plus maze (Rossi-George et al., 2004). An anxiogeniclike effect was observed only when mice tested in the light-dark box had an intervening consumption test prior to exposure to the apparatus. This obviously points to the shortcomings of explanation of the behavioral consequences of immune activation in terms of anxiety.


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C. Activation of Indoleamine 2,3 Dioxygenase As a Likely Intermediate in the Relationship between Cytokines and Depression In cancer patients treated with IFNα and/or IL-2, there is a rapid drop in plasma tryptophan that is accompanied by an increase in plasma kynurenine, a metabolite of tryptophan (Capuron et al., 2002). This increase in the kynurenine-to-tryptophan ratio is characteristic of conditions associated with activation of the indoleamine 2,3 dioxygenase (IDO) enzyme (Widner et al., 2000). This ubiquitous enzyme degrades tryptophan along the kynurenine/quinolinate pathway and is activated by cytokines such as IFNγ and TNF-α (Alberati-Giani et al., 1996; Robinson et al., 2003). Although a causal role of IDO in the pathogenesis of depressive disorders has not yet been demonstrated, the demonstration of a significant positive linear correlation between the drop in circulating tryptophan levels and increasing depressive scores in clinical studies (Capuron et al., 2002) is very suggestive of such a possibility. Indeed, a link between IDO activation and clinical depression has been proposed by various authors (Capuron and Dantzer, 2003; Schiepers et al., 2005; Widner et al., 2000). The hypothesized mechanism for this link is a cytokine-induced increase in IDO activation, thereby reducing tryptophan bioavailability for the synthesis of serotonin, an important neurotransmitter involved in the regulation of mood (Evans et al., 2005; Nemeroff and Vale, 2005; Owens and Nemeroff, 1994). IDO degrades tryptophan to kynurenine, leading to an increase in kynurenine and quinolinic acid and a decrease in tryptophan bioavailability (Figure 11). IDO is expressed systemically in dendritic cells, macrophages, and endothelial cells, but it is also expressed in the central nervous system in endothelial cells, macrophages/microglia, and astrocytes (Burudi et al., 2002; Cross-Mellor et al., 2004; Guillemin et al., 2005; Kwidzinski et al., 2005; Santoso et al., 2002). In the brain, the tryptophan hydroxylase enzyme that adds a hydroxyl radical to tryptophan, converting it into the intermediate product 5-hydroxy-tryptophan, is only 50% saturated with its substrate. Therefore, increasing or decreasing the level of bioavailable tryptophan has an immediate impact on brain serotonin levels (Fernstrom and Wurtman, 1971; Moore et al., 2000; Young and Gauthier, 1981). Acute depletion of tryptophan in normal subjects can be achieved by consumption of a tryptophan-free amino acid drinking solution. When subjects at risk for depression because of a personal or a familial history of depression are submitted to this procedure, they develop larger deteriorations in mood than control

FIGURE 11 Mechanisms of the effects of cytokines on mood. Tryptophan derived from food proteins is transported into the brain for the synthesis of serotonin or 5-hydroxytryptamine. The enzyme indoleamine 2,3 dioxygenase is upregulated in response to pathogenassociated molecular patterns (PAMPs). The resulting degradation of tryptophan along the kynurenine/quinolinic acid pathway makes tryptophan less available for the synthesis of serotonin while generating glutamate receptor ligands.

subjects (Riedel et al., 2002; Sobczak et al., 2002; Van der Does, 2001). These findings are consistent with the serotonin hypothesis of depression (Hirschfeld, 2000a; Hirschfeld, 2000b). Other evidence for serotoninergic abnormalities in depressed patients includes a reduction in cerebrospinal fluid concentrations of the main metabolite of serotonin, 5-hydroxyindolacetic acid (5-HIAA), decreased serotonin uptake and transporter binding sites in platelets, and blunted neuroendocrine responses to serotoninergic drugs (Owens and Nemeroff, 1994). Studies in postmortem tissues and neuroimaging studies in depressed subjects have confirmed that depression is associated with alterations in serotonin receptors and the serotonin transporter (Stockmeier, 2003). The mechanisms of cytokine-induced depression can be studied in acute and chronic animal models of immune activation. Acute administration of LPS or peptidoglycan increased circulating levels of IFNγ and activated peripheral and brain IDO (Figure 12) (Lestage et al., 2002). Inoculation of Bacillus Calmette-Guerin resulted in a sustained elevation of circulating levels of IFNγ and a chronic activation of peripheral and brain IDO, associated with decreases in plasma and brain tryptophan (Figure 13) (Moreau et al., 2005). Both treatments were associated with depressive-like behavior, in the form of increased duration of immobility in the forced swimming test.

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FIGURE 12 Acute peripheral immune stimulation induces IFNγ production and increases IDO activity in the mouse brain. Mice (n = 5 per group) were injected with LPS (0.83 mg/kg) at time 0 and sacrificed at different time intervals to measure IFNγ in the serum and brain IDO. Note that IFNγ peaked at 6 hours post LPS, whereas brain IDO activity peaked at 24 hours post LPS. * p < 0.05, ** p < 0.01, *** p < 0.001. (From Lestage et al., 2002.)

FIGURE 13 Chronic peripheral immune stimulation induces IFNγ production and increases IDO activity in the mouse brain. Mice (n = 6–8 per group) were inoculated intraperitoneally with 107 colony-forming units of Bacillus Calmette-Guerin, an attenuated form of Mycobacterium bovis, before being killed at different time intervals to measure IFNγ in the serum and brain IDO. Note the prolonged increase in peripheral IFNγ levels and brain IDO enzymatic activity. * p < 0.05, ** p < 0.01, *** p < 0.001. (From Moreau et al., 2005.)


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FIGURE 14 Bi-directional interactions between immune events and psychoneuroendocrine states. Various stimuli including pathogen-associated molecular patterns (PAMPs), danger signals, and stress can lead to an activation of the innate immune system, resulting in an increase in peripheral cytokines. This increase is mirrored in the brain. Brain cytokines activate the hypothalamicpituitary-adrenal axis and the neural circuits involved in the regulation of sleep, appetite, metabolism, affect, and cognition. Cortisol downregulates the production and action of cytokines both at the periphery and in the brain. Changes in affect and various physiological functions including metabolism and sleep can alter immunocompetence, which impacts on the microbial load and ultimately the degree of activation of the innate immune system.

XIII. CONCLUSION Pro-inflammatory cytokines are produced at the periphery by activated monocytes and macrophages in response to pathogen-associated molecular patterns. Via their actions in the brain, they are responsible for the occurrence of the non-specific symptoms of sickness, including apathy, social withdrawal, anorexia, cachexia, anhedonia, fatigue, pain, and alterations in cognition and mood (Figure 14). Peripheral immune activation is transmitted to the brain by several communication pathways that include saturable transporters of cytokines across the blood-brain barrier, humoral transmission via the circumventricular organs, and neural transmission via the afferent nerves that innervate the site of the body in which the inflammatory response takes place. This ultimately results in the synthesis and release of pro-inflammatory cytokines in the brain. Centrally produced cytokines act probably by volume transmission on those neural structures that are involved in the control of thermoregulation, metabolism, and behavior, resulting in the development of the brain components of the host response to infection. Glucocorticoids that are released by the adrenal cortex in response to the hypothalamic effects of proinflammatory cytokines regulate the expression and actions of cytokines not only in the periphery, but also

in the brain. When the cytokine system is uncontrolled, its repeated or chronic activation can lead to the development of mood disorders that are dependent on cytokine-induced alterations in the metabolism of tryptophan. These findings open new perspectives on the mechanisms and means of controlling the nonspecific symptoms of sickness such as fatigue, anorexia, and social withdrawal that develop in a wide variety of pathological conditions associated with inflammation and can culminate in pathological alterations in mood and cognition (Dantzer, 2004). Sickness behavior can itself lead to changes in immune competence that regulates the host sensitivity to external and internal pathogens, therefore influencing the degree of activation of the innate immune system and the production of peripheral cytokines (Figure 13). These intricate bidirectional interactions between immune events and psychological states ultimately determine the degree of wellness and therefore provide a potential target for interventions aiming at preserving or restoring well-being.

Acknowledgments Supported by INRA, CNRS, University of Bordeaux 2, and the National Institute of Health (MH 71349 to RD, MH 51569 and AI 50442 to KWK).

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C H A P T E R

15 The Differential Role of Prostaglandin E2 Receptors in the CNS Response to Systemic Immune Challenge MICHAEL LAZARUS AND CLIFFORD B. SAPER

nucleus and release of adrenal and pituitary hormones. EP1 and EP3 receptors on neurons in the preoptic area and in the ventromedial hypothalamus modulate activity of the descending pain inhibitory system and cause hyperalgesia or analgesia. EP3 receptors in the sleep-promoting ventrolateral preoptic area and EP4 receptors in the wake-promoting tuberomammillary nucleus might be involved in alteration of sleep behavior during a bacterial infection.

I. INTRODUCTION 319 II. SIGNALING MECHANISMS BETWEEN THE IMMUNE SYSTEM AND THE CNS 320 III. PATHWAYS ACTIVATED BY THE EP RECEPTORS 322 IV. CONCLUSION AND FUTURE STUDIES 331

ABSTRACT The innate immune system of mammals is able to detect bacteria when they infect local tissue or enter the bloodstream and initiate an immediate immune response, but the mechanism by which the innate immune system can activate CNS responses such as fever and sickness behavior is not well understood. Prostaglandin E2 (PGE2) is considered as the most important link between the peripheral immune system and the brain because drugs that block the synthesis of prostaglandins prevent fever and most components of sickness behavior. PGE2 mediates different or even counterposing components of the acute phase reaction via its action on four PGE2 receptors (EP receptors) and their differential expression in various areas of the hypothalamus and brain stem. A fever model is proposed in which EP1 and EP3 receptors in the preoptic area activate thermogenesis to cause fever, while counterpoised EP4 receptors promote hypothermia. The synergistic action of the EP1 and EP3 receptors is thought to be required for activation of the endocrine component of the paraventricular hypothalamic PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

I. INTRODUCTION It is critical for mammals to be able to detect bacteria immediately when they infect local tissue and enter the bloodstream, and to mount a vigorous immune response. This response is mediated in mammals by an innate immune system, which can recognize components of bacterial cell walls even if it has not been previously exposed to them, and initiate an immediate immune response. This response is mediated, in turn, by a cascade of pro-inflammatory mediators, including the elaboration of a wide range of cytokines and prostaglandins. These mediators initiate an array of physiological responses, termed the acute phase reaction, among which is included a suite of CNS-mediated adaptive responses, sometimes called “sickness behavior” (fever, malaise, increased pain sensitivity, changes in wake-sleep cycles, and feeding) and changes in secretion of hormones such as corticosteroids.

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Although it has been known since the nineteenth century that aspirin could prevent many components of sickness behavior, and this compound was widely used clinically for this purpose, the mechanism of action was not understood until Sir John Vane in 1971 made the fundamental discovery that anti-inflammatory compounds such as aspirin act by blocking the formation of prostaglandins (PGs) (Vane, 1971). Originally, PGs were discovered independently by von Euler and Goldblatt in 1935 as a “vasodilatory substance” in seminal fluid and seminal vesicles from most animals including humans (Goldblatt, 1935; von Euler, 1935). They showed that PGs are involved in a large number of biochemical processes, often in extremely low concentrations. PGs are formed from unsaturated fatty acids, primarily arachidonic acid, which are metabolized to cyclic endoperoxides, which constitute an important branching point from which the stable prostaglandins as well as the more unstable thromboxanes and prostacyclin are formed. The task of these mediators is primarily to protect the integrity of the organism, and they are released when homeostasis is jeopardized by trauma, disease, or various stress factors and, thus, are also called the defense hormones. Prostaglandin E2 is now considered to be the most important link between the peripheral immune system and the brain. Produced at the boundaries between the bloodstream and brain tissue, PGE2 is thought to penetrate the blood-brain barrier and enter the CNS, and is therefore an ideal candidate to translate a peripheral immune signal into an acute phase response by modulating neural activity (Engblom et al., 2002b). The receptors for prostaglandins have now been identified and cloned, and there are four specific PGE receptors (EP receptors 1–4). In this chapter, we will summarize the differential role of prostaglandin E2 receptors in the CNS response to systemic immune challenge with major emphasis on our recent work on the regulation of fever by PGE2.

II. SIGNALING MECHANISMS BETWEEN THE IMMUNE SYSTEM AND THE CNS Although the interactions of the immune and the nervous system have been extensively studied in recent years, the mechanisms by which immune signals are transduced to influence the CNS remain controversial. The endotoxin lipopolysaccharide (LPS), a bacterial cell wall component, has been widely used as a model immune stimulant. LPS binds to the CD14 receptor on leukocytes and macrophages. By interac-

tion with the Toll-like receptor 4 (TLR4), this induces secretion of cytokines, including interleukin-1β (IL1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), as well as prostaglandins (Fitzgerald and O’Neill, 2000; Rivest, 2003). In an effort to use a less complex immune stimulus, many studies have used systemically injected cytokines such as IL-1β and TNF-α to stimulate the immune system. However, circulating levels of IL-1β are not elevated in the bloodstream during systemic infection (Hopkins and Rothwell, 1995), and individual cytokines are rarely if ever elevated in isolation of other components of the pro-inflammatory response. Most cytokines are produced at the site of infection and act in a paracrine manner (Konsman et al., 2002), where they inevitably induce the secretion of the remaining pro-inflammatory cytokines and prostaglandins in any case. Hence, in the discussion that follows, we will concentrate on studies which have used LPS as a model of bacterial invasion and examine how it may act upon the CNS.

A. Stimulation of Peripheral Nerves Low levels of LPS, cytokines, or prostaglandins in the abdominal cavity can activate vagal afferents, mainly from the hepatic nerve, which can signal the brain and produce a CNS response (Blatteis et al., 1998; Romanovsky, 2004; Szekely et al., 2000). The role of the vagus nerves in transmission of immune signals to the brain has been assessed by experiments in which the vagus nerves were sectioned below the diaphragm. These studies suggest that vagal afferents can activate brain stem, hypothalamus, and limbic structures in response to peripherally administered LPS and IL-1β (Dantzer et al., 2000; Ek et al., 1998). Macrophages and dendritic cells of the abdominal cavity including Kupffer cells in the liver can produce IL-1β and PGE2 in response to LPS (Goehler et al., 1999; Lazarus et al., 2002; Sehic et al., 1997) and sensory neurons of the vagus nerves express receptors for IL1β and PGE2 that can activate afferent nerve fibers (Ek et al., 1998). In contrast, the abdominal vagus nerve does not seem to be necessary for causing the CNS response to intravenous or subcutaneous administration of LPS or IL-1β (Bluthe et al., 1996; Ericsson et al., 1997; Wan et al., 1994), nor does vagal transection block the CNS response to intraperitoneal administration of higher doses of LPS (Romanovsky et al., 2000), which may spill over into the systemic circulation. These observations suggest that the vagal sensory route may contribute mainly during low-grade peritonitis.

15. The Differential Role of Prostaglandin E2 Receptors in the CNS Response

B. Entry and Formation of Cytokines in the CNS Many early models of immune-CNS interactions hypothesized that the message might be carried by cytokines that would enter the CNS. However, cytokines are large proteins that cannot easily penetrate the BBB. It has been suggested that they may enter the CNS via carrier-mediated transport (for review, see Conti et al., 2004). However, the transport mechanisms that have been described are slow and saturable and, therefore, insufficient quantities of cytokines enter the brain following i.v. administration to explain the rapid responses of the CNS. During fever, brain IL-6 is largely increased, but this appears to be due to synthesis in the brain and not IL-6 crossing the BBB. IL-1β and TNF-α mRNA expression is not induced in the brain after systemic LPS (Breder et al., 1993; Breder and Saper, 1996). Thus, neither systemic cytokines that cross the BBB nor central synthesis of cytokines seems to play a major role in the brain response to systemic inflammation (Konsman et al., 2002).

C. Circumventricular Organs Another way in which cytokines or LPS might activate neurons in the CNS would be by action on circumventricular organs, such as the organum vasculosum of the lamina terminalis (OVLT), which lack a BBB (Blatteis et al., 1983; Saper and Breder, 1992; Stitt, 1985). Evidence showing IL-1β and TNF-α immunohistochemical staining in the brain suggested that PGE2 might act on cytokine-containing neurons in the preoptic and anterior hypothalamic areas and that these neurons might release cytokines from terminals at distal sites involved in producing the autonomic, endocrine, and behavioral components of the immune response of the CNS (Breder et al., 1988; Breder et al., 1993). Unfortunately, follow-up studies using in situ hybridization demonstrated that there is little if any synthesis of IL-1β and TNF-α by neurons in brain, either at baseline or after LPS stimulation. These studies, instead, showed that mRNA for these cytokines was confined to vascular-related cells along small penetrating blood vessels at the boundaries of the brain, and microglial cells in the circumventricular organs, at least during the first few hours after LPS administration (Breder et al., 1994; Nakamori et al., 1994; Quan et al., 1999). The CVOs are specialized neural regions in contact with the cerebroventricular system that have fenestrated capillaries and therefore lack a BBB. Many circulating hormones, such as angiotensin II, cholecystokinin, and leptin, act on neurons

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in the CVOs (Saper et al., 2002; Saper and Breder, 1994). The results of the immune response in CVOlesioned animals are contradictory (Blatteis, 1992; Ericsson et al., 1994; Ericsson et al., 1997; Katsuura et al., 1990; Takahashi et al., 1997). However, complete lesions inevitably damage structures surrounding the CVOs that might have a greater impact on the sickness behavior than the CVO itself. For example, large lesions that include the OVLT can prevent fever, but they also damage nearby preoptic regions that are crucial for thermoregulation (Blatteis, 1992). Animals with lesions of the OVLT and neighboring structures, such as the median preoptic area (MnPO), have high basal body temperature, which may make it difficult to identify fever (which may depend upon this same mechanism, i.e., inhibiting the cells in the MnPO; Romanovsky et al., 2003). As indicated by c-fos expression, neurons within the CVOs are activated by intravenous LPS or IL-1β, but only with substantially higher doses than those necessary to produce fever or corticotrophin responses (Elmquist et al., 1996; Ericsson et al., 1994). On the other hand, blockers of prostaglandin synthesis inhibit fever response in virtually all models, suggesting that prostaglandins are a necessary component of the fever response. Because prostaglandins may not require CVOs to cross the BBB, research in the last decade has turned from the CVOs as playing a primary role in fever, and instead has focused on the roles played by prostaglandins.

D. Central Production of PGE2 A hallmark of the CNS response to inflammation is that many of its manifestations, including fever and corticotropin activation, can be prevented by blocking the production of prostaglandins (Hashimoto et al., 1988; Johnson and von Borell, 1994; Stitt and Bernheim, 1985; Vane, 1971; Zhang et al., 2003). Both LPS and cytokines activate intracellular signaling cascades in endothelial cells in small blood vessels and in vascular-associated macrophages, termed perivascular cells, in the brain [for detailed review, see ref. (Matsumura and Kobayashi, 2004; Schiltz and Sawchenko, 2003)]. This cascade results in phospholipase A2 degrading phospholipids into arachidonic acid and increased expression of cyclooxygenase type 2 (COX-2) (Ivanov and Romanovsky, 2004; Schiltz and Sawchenko, 2002). COX-2 converts arachidonic acids to PGH2, from which specific prostaglandin synthases synthesize various prostaglandins (O’Banion, 1999; Smith and Dewitt, 1996). In the normal brain, COX-2 is present in certain neurons and meningeal cells but little is found in brain vascular cells (Beuckmann et al., 2000b; Breder

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et al., 1992; Breder et al., 1995; Elmquist et al., 1997; Yamagata et al., 1993). Following administration of LPS or IL-1β, there is little change in neuronal expression of COX-2, but intense induction of COX-2 is seen in vascular cells of the brain parenchyma and subarachnoidal space (Breder and Saper, 1996; Cao et al., 1995; Cao et al., 1996; Elmquist et al., 1997). Van Dam and colleagues (Van Dam et al., 1993) have reported PGE2 immunoreactivity in brain blood vessels of rats injected systemically with LPS, providing evidence that PGE2 is a mediator of the CNS response to immune challenge. Various enzymes have been characterized as prostaglandin E synthases (Beuckmann et al., 2000a; Tanioka et al., 2000), but it was not until 1999 that a membrane-bound prostaglandin E synthase (mPGES-1) was identified (Jakobsson et al., 1999) and its coordinate upregulation with COX-2 was shown (Lazarus et al., 2002; Thoren and Jakobsson, 2000). In the normal brain, mPGES-1 is expressed at very low levels in endothelial cells, but its expression is highly upregulated in response to systemic administration of LPS or IL-1β, and the distribution is very similar to that of COX-2 (Ek et al., 2001; Engblom et al., 2002a; Yamagata et al., 2001). A key role of both COX-2 and mPGES-1 in producing fever has been demonstrated in mouse strains lacking these key enzymes (Engblom et al., 2003; Li et al., 1999; Li et al., 2001). Animals lacking either COX-2 or mPGES-1 fail to produce a fever response when stimulated with LPS or IL-1β. Recent work (Kamei et al., 2004) also showed that mice lacking mPGES-1 have reduced pain hypersensitivity in response to LPS stimulation. Similarly, injections of specific COX-2 inhibitors prevent fever responses (Zhang et al., 2003), although COX-1 may also play a role in signaling the brain via the vagus nerve, as activation of c-fos expression in the nucleus of the solitary tract and other components of the visceral sensory system after LPS is blocked by COX-1 but not COX-2 inhibitors.

III. PATHWAYS ACTIVATED BY THE EP RECEPTORS PGE2 is transported or diffuses across the bloodbrain barrier in the CVOs or penetrating venules around the margins of the brain (Ivanov et al., 2003; Taogoshi et al., 2005), and as a result it can act in the adjacent brain on neurons expressing E type prostaglandin receptors (EPRs) differentially to activate various pathways in the CNS in response to systemic immune challenge. EP receptors are G-protein–coupled receptors and classified into four subtypes: EP1, EP2, EP3, and EP4. These receptors can be grouped into

three categories on the basis of their signal transduction: the EP1 receptor increases intracellular Ca2+. The EP2 and EP4 receptors mediate increases in intracellular cyclic adenosine monophosphate (cAMP). The EP3 receptor expressed in the brain is an inhibitory receptor that mediates decreases in intracellular cAMP, although other splicing variants of the EP3R have different signaling pathways. For a general review on prostanoid receptors, see ref. (Narumiya et al., 1999; Narumiya and FitzGerald, 2001).

A. Febrile Response and Hypothermia Fever is one of the most common symptoms of inflammatory disease and is a CNS-mediated component of the acute phase reaction, an adaptive suite of responses to systemic inflammatory stimuli. Fever represents a temporally complex but stereotyped set of physiological events, resulting in increased body temperature. Other responses are sickness behavior, which includes malaise, increased pain sensitivity, changes in wake-sleep cycles and feeding, and changes in secretion of hormones such as corticosteroids (reviewed below). In rodents, systemic administration of LPS is known to induce monophasic fever, multiphasic fever, or hypothermia depending on the species (and possibly strain of animals used), the dose, the route of administration, and the ambient temperature (Oka et al., 2003a; Romanovsky, 2004). We and others have used c-fos expression in the brain after intravenous or intraperitoneal LPS administration as a measure of the activation of specific brain pathways during fever responses. Of course, not all such activation is related to thermoregulation, and not all activated pathways will necessarily express c-fos protein. On the other hand, this method has given us a way to identify and test the importance of specific neuronal pathways in fever responses and allowed us to identify neurons in ventromedial preoptic nucleus (VMPO), paraventricular hypothalamic nucleus (PVH), parabrachial nucleus (PB), nucleus of the solitary tract (NTS), and ventrolateral medulla (VLM) that reliably show c-fos activation after LPS stimulation (Elmquist et al., 1996; Elmquist et al., 1997; Elmquist and Saper, 1996; Zhang et al., 2000). Cell-specific lesions of the VMPO, using ibotenic acid (Lu et al., 2001), had little if any effect on fever responses, although they did make baseline body temperature unstable. There was a much higher variance in body temperature across the day, as if fine control of thermoregulation had been disrupted. By contrast, lesions of the PVH unmasked a brief hypothermic response in the first hour after intravenous LPS, followed by a fever response that was about 0.5°C less than animals with an intact PVH.


Pittman and colleagues observed similar results in rats that had a reduced febrile response to intracerebroventricular application of PGE2 and intraperitoneal injection of LPS after electrolytic lesion of the PVH, whereas the ablation of the PVH had no effect on maintenance of normal body temperature (Horn et al., 1994). E-type prostaglandins have been considered as principal mediators of fever since Milton and Wendlandt reported in 1970 that injection of prostaglandin E1 into the third ventricle of cats elevated body temperature (Milton and Wendlandt, 1970). LPSinduced c-fos expression can be replicated by injecting threshold doses (1–2 ng) of PGE2 into the preoptic area, but only within a radius of less than 1 mm from the anteroventral tip of the third ventricle (Scammell et al., 1996). Conversely, injecting a COX inhibitor into this same site attenuated fever dramatically after i.v. LPS (Scammell et al., 1998b), indicating that both the site of production of PGE2 and its neuronal targets for causing fever are located at the anterior tip of the third ventricle, a region containing the MnPO, OVLT, and VMPO. Recent work examining c-fos expression in the brains of rats that received intravenous LPS along with cyclooxygenase inhibitors confirmed that c-fos expres-

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sion patterns in rat brain can be dissociated from fever responses (Zhang et al., 2003). In fact, COX-1 inhibition, which did not prevent fever, eliminated LPSinduced c-fos expression in the brain stem and PVH, whereas COX-2 inhibition eliminated fever and c-fos expression in the VMPO and PVH, but left LPS-induced c-fos expression in the brain stem intact (Figure 1). One major clue to identifying the key circuitry involved in the CNS responses to systemic immune challenge is the location of EP receptors in the brain. Oka et al. (2000) mapped the distribution of all four EP receptors by using in situ hybridization (Figure 2). These studies and those by others demonstrated that EP3 receptors are highly localized to the MnPO appearing at rostral levels as an inverted Y-shaped structure, capping the OVLT. Lower levels of expression of the EP3 receptor were found in the medial part of the OVLT, the medial preoptic nucleus (MPO), and the caudal aspects of the VMPO (Ek et al., 2000; Nakamura et al., 1999; Nakamura et al., 2000; Yoshida et al., 2003). The localization of intense expression in the MnPO provides an important clue to the genesis of fever responses, as the EP3 receptor is probably the most critical for producing fever. EP1 receptors are much

FIGURE 1 A series of photomicrographs demonstrating the distribution of c-fos–like immunoreactivity (black nuclei) in the VMPO (A–C) and NTS (D–F) after intravenous LPS administration and intraperitoneal injection of COX-2 inhibitor (SC-236; A and D), or COX-1 inhibitor (SC-560; C and F), or vehicle (DMSO; B and E). Expression of c-fos protein is prevented in the VMPO by the COX-2 inhibitor, but expression of c-fos in the NTS is abolished by the COX-1 inhibitor, indicating that different forms of cyclooxygenase drive different CNS pathways. AVPV, anteroventral periventricular nucleus; dm, dorsomedial subnucleus; m, medial subnucleus; OX, optic chiasm; VMPO, ventral medial preoptic area; 3v, third ventricle; 4v, fourth ventricle; 10, dorsal motor nucleus of the vagus. [From (Zhang et al., 2003) with permission from John Wiley & Sons, Inc.].

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strand OC FIGURE 2 Distribution of EP receptor–bearing neurons within the PGE2-sensitive OVLTMnPO-VMPO region that showed c–fos expression after intravenous LPS administration. Gray areas represent the regions where EP4 (A–D), EP3 (E–H), and EP1 (I–L) receptor mRNA is predominantly expressed with darker areas indicating greater density of EP receptor expression. Circles represent c-fos immunoreactive neurons, and squares show neurons expressing EP4, EP3, and EP1 receptor mRNA as well as c–fos expression. [From (Oka et al., 2000) with permission from John Wiley & Sons, Inc.].

more widely distributed. In fact, EP1 receptors are found in each of the key anteroventral preoptic nuclei, including the MnPO, the VMPO, and the OVLT. By contrast, EP4 receptors are more highly localized. They

are found at highest levels in the VMPO and the lateral OVLT, but are also found at lower levels in the MnPO. LPS induces higher levels of EP4R expression in the PVH, although not in the preoptic area (Oka et al.,


2000; Zhang and Rivest, 2000). Oka et al. (2000) did not identify EP2 receptors in rat hypothalamus, although Zhang and Rivest (1999) reported that EP2 receptor mRNA is expressed within the medial preoptic area (MPO). The combination of in situ hybridization for the EP receptors with c-fos staining during fever response revealed that c-fos expression was highly colocalized with EP4 receptor mRNA, especially in the OVLT and VMPO, whereas EP1 receptor mRNA was found diffusely colocalized with c-fos staining, particularly in the OVLT and VMPO (Oka et al., 2000). There was very little c-fos expression by neurons expressing EP3R mRNA. The EP3 receptor found in the rodent hypothalamus inhibits cells by reducing intracellular cAMP (Narumiya et al., 1999). Thus, patterns of c-fos expression may be misleading, because it is unlikely that EP3 receptor stimulation would cause c-fos expression, and it might suppress such expression. Previous studies that used relatively non-specific agonists for all four EP receptors produced conflicting results, suggesting the involvement of the EP1 and the EP2 receptors in producing fever in rats and pigs, respectively (Oka et al., 1997a; Oka et al., 1998; Oka

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and Hori, 1994; Parrott and Vellucci, 1996). In order to clarify which of the EP receptors might be involved in fever responses, Oka and colleagues (2003b) injected highly specific EP receptor agonists intracerebroventricular (i.c.v.) into awake rats and recorded body temperature telemetrically. In these experiments (Figures 3 and 4), it was found that the EP1 receptor agonist (ONODI-004) caused a brisk fever of about 1.5–2.0°C within 20 minutes of injection. The EP3 agonist (ONOAE-248) caused a slightly smaller fever, but there was a delay of about 40 minutes before it reached a maximum. This response was substantially delayed from PGE2, which caused a brisk fever response in about 20 minutes. The EP4 agonist (ONO-AE1-329) caused a 0.5°C hypothermia, whereas the EP2 receptor agonist (ONO-AE1-250-01) had no effect. The magnitude of these responses is difficult to compare, as all doses were limited by the solubility of the specific compounds to 20 nmol and the drugs may have activated their specific receptors to different degrees. However, these experiments demonstrated that the EP1 and EP3 receptors both are capable of producing hyperthermic responses, whereas the EP4 receptor is more likely to produce hypothermic responses.

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FIGURE 3 Effects of i.c.v. injection of EP receptor agonists on core temperature (T) in rats. Rats were injected with EP receptor agonists at 20 nmol (closed circle), i.e., ONO-DI-004 (A) and ONOAE-248 (B), or their vehicles (open circle) at time zero. The data are expressed as differences from the T at time zero, which is shown in the figure. Each point represents mean ±S.E. (n) = number of animals. Symbols adjacent to points represent level of significance when compared with vehicle-injected control. *P < 0.05; **P < 0.01. [From (Oka et al., 2003b) with permission from Elsevier Science B.V.].

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FIGURE 4 Dose-dependent effects of PGE2 and EP receptor agonists on core temperature (T) in rats. The maximal changes in T during 90 minutes after i.c.v. injection of the following drugs is shown: PGE2 at 0.03 and 0.3 nmol, ONO-DI-004 (EP1 agonist) at 2 and 20 nmol, ONO-AE1259-01 (EP2 agonist) at 20 nmol, ONO-AE-248 (EP3 agonist) at 2 and 20 nmol, and ONO-AE1-329 (EP4 agonist) at 2 and 20 nmol. The number in parentheses in each column represents the number of animals. In the parentheses below the compounds, doses (nmol) of drugs are shown. Each point represents mean ±S.E. Symbols adjacent to points represent level of significance when compared with the values of their corresponding vehicle-injected control rats at the same time point. *P < 0.05; **P < 0.01. [From (Oka et al., 2003b) with permission from Elsevier Science B.V.].

An early study of EP receptor knockout mice showed that only mice with EP3 (Ushikubi et al., 1998), but not EP1, EP2, or EP4 receptor gene deletion lacked a hyperthermic response to intracerebroventricular (i.c.v.) PGE2, suggesting a crucial role for EP3 receptors in producing fever in mice. However, in those experiments, body temperature was measured in restrained animals by using rectal robes, and only for one hour after LPS or PGE2 administration. As restraint causes stress that alters the body temperature, Oka et al. (2003a) telemetrically studied the fever response of the same strains of unrestrained EP1 and EP3 receptor knockout mice for up to 12 hours after intraperitoneal LPS administration at a variety of doses. Animals with EP4 receptor deletion were not studied because they rarely survive very long after birth; the ductus arteriosus remains patent, as it requires EP4 receptor stimulation to close (Nguyen et al., 1997; Segi et al., 1998). In the Oka et al. (2003a) study, different doses

of intraperitoneally injected LPS produced different temporal profiles of fever responses in C57BL/6 wild-type mice. A 1 μg/kg dose caused a brief, early hyperthermia (2–4 hours after LPS injection), which was eliminated in EP3 receptor knockout animals. At 10 μg/kg, the early hyperthermia was greater in wild-type mice. In EP1 receptor knockout animals, this response was reduced in magnitude, and in EP3 receptor null mice, only a brief hypothermia from 1–2 hours after LPS was noted. At 100 μg/kg, wild-type mice showed little early fever, but a 1°C hyperthermia from 4–9 hours after LPS administration (Figure 5). EP1 receptor null mice had no fever response, and EP3R knockout animals had a profound hypothermia of 2°C that lasted for 9 hours. At 1 mg/kg of LPS administration, the wild-type mice had a mild hypothermia of 0.5°C from 1 to 3 hours after the LPS injection, and then a more profound hypothermia of 4°C from 4–8 hours. EP1 receptor knockout animals had a similar response, which lasted at least 12 hours, whereas EP3 receptor knockout animals had an even more profound hypothermia, with body temperature dropping more than 6°C from 4–8 hours after the LPS injection. It appears that the EP3 receptor is necessary not only to produce fever responses, but also to prevent profound hypothermic responses, particularly at higher doses of LPS. The EP1 receptor, by contrast, does appear to play an important role in fever, particularly at intermediate (10–100 μg/kg) doses of LPS, where it is required to produce the hyperthermic responses seen in control animals. Thus, the fever curve at different time points and after different doses of LPS appears to reflect the interaction of both hyperthermic and hypothermic processes. EP3 receptors provide a strong hyperthermic drive; and EP1 receptors a substantial but less intense hyperthermic response. However, without these, the main effect of LPS is hypothermia, which appears from agonist studies (Oka et al., 2003b) to be due to EP4 receptors. Studies by Nakamura and colleagues (2000, 2004) have attempted to examine the neuronal pathways by which the EP3 receptor may cause fever. In agreement with other investigators (Oka et al., 2000), they showed that EP3 receptors are highly expressed by neurons in the MnPO, and went on to show that these cells demonstrate descending projections to the raphe pallidus nucleus (RP) in the brain stem. In addition, neurons in both the RP and the MnPO are retrogradely labeled by injection of the transneural tracer, pseudorabies virus, into the intrascapular brown adipose tissue (IBAT), and labeled neurons at both sites also contained EP3 receptors (Cano et al., 2003; Yoshida et al., 2003). On the other hand, blockade of PGE2 synthesis in the preoptic area prevented fever (Scammell et al., 1998b),


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FIGURE 5 Effects of intraperitoneal injection of LPS at 100 μg/kg on core temperature (T) in WT, EP1 receptor KO, and EP3 receptor KO mice. Mice were injected with LPS (closed circle) or 0.9% saline (open circle) at time zero. The data are expressed as differences from the T at time zero, which is shown in the figure. Each point represents mean ±S.E. In the parentheses, the number of animals is shown. Bar shows dark period. Symbols represent level of significance when compared with 0.9% saline-injected control at each time point. *P < 0.05; **P < 0.01. [From (Oka et al., 2003a) with permission from The Physiological Society, UK].

and injection of PGE2 into the RP did not affect body temperature (Tanaka and McAllen, 2005). The RP has been shown in recent years to be a key site for integrating sympathetic responses important in producing increased body temperature, including both increased thermogenesis through activation of brown adipose tissue and reduced passive heat loss through the skin by tail artery vasoconstriction (Morrison, 2001; Morrison, 2004). Injection of a GABA agonist into the RP prevented febrile response to PGE2 injection i.c.v. (Morrison, 2003; Nakamura et al., 2002). Thus, a model for fever response was suggested in which EP3 receptor–bearing neurons in the MnPO are inhibited by PGE2, thus reducing their inhibition of the RP. This disinhibition of the RP would then lead to thermogenesis and heat conservation, resulting in fever. The model shown in Figure 6 is slightly more complex than the model described above, as it also incorporates the finding of Morrison and colleagues (2004) and our own observations. This model accounts for additional observations, such as the attenuation of fever response by lesion of the PVH or GABAergic inhibition of the dorsomedial hypothalamus (DMH), and that fever is attenuated by blocking excitatory amino acid neurotransmission in the RP, indicating that the RP receives both an augmented excitatory amino acid input and a reduced GABA input during fever responses (Lu et al., 2001; Madden and Morrison, 2003; Madden and Morrison, 2004). In our model, the preoptic area contains the mechanisms for both hyperthermic and hypothermic responses. Hyperthermia is

generated by a system of neurons including the paraventricular and dorsomedial nuclei of the hypothalamus and the RP, which activate thermogenic brown adipose tissue and heat conservation (vasoconstriction) responses. This thermogenic system is normally restrained by inhibitory neurons in the MnPO. Thus, inhibition of these MnPO neurons during fever disinhibits the thermogenic system and results in hyperthermia. Additionally, the MnPO neurons can be activated by excitatory neurons in the OVLT and VMPO, which paralyzes the thermogenic system. At ambient temperatures below thermoneutral, this results in hypothermia. Of course, both of these types of neurons in the MnPO and in the OVLT/VMPO whose activity causes hypothermia can be inhibited by local interneurons, whose activity is therefore hyperthermic. Although some MnPO neurons project to the RP, there are also inhibitory projections to neurons in the PVH and the DMH, which in turn provide excitatory inputs to the RP. Thus, inhibition of the MnPO during fever can disinhibit a wide range of descending thermogenic pathways. In this model, during fever, hyperthermic neurons in the MnPO and OVLT/VMPO are inhibited by EP3 receptors, thus disinhibiting, i.e., releasing, thermogenesis. The same neurons can contain EP4 receptors, which excite them and thereby limit thermogenesis, and in the absence of EP3 receptor action can produce hypothermia. At higher dosages of LPS, the EP4 receptors may predominate, resulting in predominantly

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cAMP↓ FIGURE 6 A model for neuronal interactions with EP receptors in the fever response after systemic immune challenge. The action of PGE2 on EP1, EP3, and EP4 receptors surrounding the anteroventral tip of the third ventricle in the preoptic area, including the MnPO, OVLT, and VMPO, appears to be critical for producing fever. EP1 and EP3 receptors generate hyperthermia, and EP4 responses generate hypothermia. The body temperature of the animal represents the net effect of these counterpoised forces, which may vary at different time-points after immune stimuli start signaling the CNS.

hypothermic responses. We hypothesize that EP1 receptors, which are excitatory, are located on inhibitory interneurons in both the MnPO and the OVLT/ VMPO. These play an accessory role in permitting thermogenesis during fever, but in the absence of EP3 receptors, they cannot overcome the hypothermic effect of the EP4 receptors. On the other hand, in the absence of EP1 receptors, at doses of LPS from 10– 100 μg/kg the effects of the EP3 and EP4 receptors effects are nearly balanced, and little if any fever is seen (Oka et al., 2003a).

B. Alterations in the Secretion of Adrenal and Pituitary Hormones PGE2 also plays a key role in activating the hypothalamic-pituitary-adrenal (HPA) axis in response to inflammation. Activation of neurons that secrete corticotropin-releasing factor (CRF) in the parvocellular PVH is responsible for adrenocorticotropic hormone (ACTH) secretion during a stressful immune challenge. Subsequent increases in circulating ACTH drive synthesis and secretion of glucocorticoids by the adrenal glands. During stress, including systemic inflammation, catecholaminergic inputs from the NTS and VLM to the PVH are an essential component of the neuronal circuits, and their role in regulating the activity of CRF neurons is well recognized (Chan et al.,

1993; Ericsson et al., 1994; Sawchenko et al., 1996). The PVH neurons also receive direct inhibitory input from local hypothalamic circuits, including the preoptic area. These circuits might therefore modulate PVH output through disinhibition (Herman and Cullinan, 1997). The HPA axis response to systemic administration of IL-1β is attenuated by COX inhibitors, and intracerebroventricular or intra-preoptic injection of PGE2 triggers the transcription of CRF (Ericsson et al., 1997; Lacroix et al., 1996; Scammell et al., 1996; Zhang and Rivest, 2000). EP4 mRNA is upregulated in CRF neurons in the parvicellular PVH following intravenous LPS or IL-1β injection. EP4 receptors are also present in activated catecholaminergic neurons of the NTS and VLM, although only the A1 cell group of the VLM showed an increase in EP4 receptor transcription by circulating IL-1β (Zhang and Rivest, 1999). This pattern of EP4 expression suggested that EP4 receptors may play a leading role in the control of the corticotroph axis during acute phase reaction. In addition, EP4 receptors are expressed in the dorsal, ventral, and posterolateral parvicellular subnuclei of the PVH, which project to autonomic nuclei in the brain stem and spinal cord (Oka et al., 2000; Zhang et al., 2000). Although the increase in EP4 receptor expression might suggest that an enhanced effect of PGE2 on EP4 receptors in the PVH, in fact there is no evidence that


PGE2 actually accesses the PVH during an inflammatory response. Thus, the function of the increased expression of EP4 receptor in this model remains a mystery. On the other hand, in situ hybridization studies of the EP1 receptor revealed a diffuse distribution in the PVH, suggesting a possible role for the EP1 receptor, too, in activation of the HPA axis (Batshake et al., 1995; Oka et al., 2000). The EP1 receptor is also expressed on neurons in the supraoptic nucleus (SON) and central nucleus of the amygdala (CeA), areas known to show c-fos expression in response to systemic LPS administration (Batshake et al., 1995; Matsuoka et al., 2003). Neural circuits that include the bed nucleus of the stria terminalis (BST) connect the CeA to the PVH (Herman and Cullinan, 1997) and, hence, might stimulate the secretion of glucocorticoids mediated by the EP1 receptor. The dominant expression of the EP3 receptors in the preoptic area, including the MnPO and MPO (cf. “Febrile Response and Hypothermia”), implicates this receptor in blocking GABAergic inputs to the PVH and, thus, triggering activation of the HPA axis. The MnPO is a major source of input to the PVH and SON to depress CRH, vasopressin, and oxytocin neurons in the PVH and SON. Using mice in which the genes for each individual EP receptor has been knocked out has been a useful approach to testing these hypotheses. Narumiya and colleagues showed that mice in which the EP1 or EP3 receptor had been deleted had defective release of ACTH from the pituitary gland in response to LPS challenge, whereas animals with EP2 or EP4 receptor deletion had a normal ACTH response (Matsuoka et al., 2003). Treatment with a highly selective EP1 antagonist, ONO-8713, suppressed LPS-induced ACTH release in wild-type mice (Matsuoka et al., 2003). A comparison of c-fos expression in brain areas of wildtype mice and EP1R- and EP3R-deficient mice after LPS administration highlighted areas that might be involved in activation of the HPA axis. Although the lack of either the EP1 or EP3 receptor did not reduce c-fos expression in PVH neurons after administration of LPS, the treatment of EP3 receptor knockout mice with ONO-8713 (a selective EP1 antagonist) reduced c-fos expression in CRF-containing neurons of the PVH after LPS administration to an extent comparable to that found in animals treated with the COX inhibitor indomethacin Thus, either the EP1 or the EP3 receptor can drive c–fos expression in the CRH cells, but both are needed to cause sufficient excitation to release excess CRH. The location of the neurons that activate the PVH CRH neurons in response to EP receptors remains to be determined. In EP1 receptor knockout mice, the

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expression of c-fos in response to LPS was reduced in the CeA (Matsuoka et al., 2003). CeA neurons were also found to be immunoreactive for EP1 receptors, suggesting that the EP1 receptors found on CeA neurons may play a major role in activating the HPA axis. On the other hand, Sawchenko and colleagues (Ericsson et al., 1994; Ericsson et al., 1997) have found that cutting the ascending noradrenergic input to the PVH from the VLM prevents CRH responses due to i.v. IL-1β. They can also block CRH responses by blocking PG synthesis locally in the VLM with indomethacin. As EP3 receptors are found in the VLM (Ek et al., 2000), they may play a role in activating local noradrenergic neurons. In summary, it may be necessary to activate both an EP1-dependent pathway involving the CeA and an EP3-dependent pathway involving the VLM to activate fully CRH responses to systemic immune challenge.

C. Global Changes in Nociceptive Thresholds Pain enhancement, also known as hyperalgesia, during a period of infection or illness would benefit the organism by minimizing activity, thus saving energetic resources for fighting infection, and for allowing recovery and healing to occur. Hyperalgesia is produced in animal models of acute inflammation that are induced by systemic administration of LPS or intravenous injection of IL-1β (Maier et al., 1993; Watkins et al., 1994; Wicrtelak et al., 1994). The analgesic effects of COX inhibitors such as indomethacin and aspirin confirm that prostaglandins participate in the processing of nociceptive stimuli (O’Banion, 1999; Vane, 1971; Vane and Botting, 2003). LPS-induced hyperalgesia can be abolished by microinjection of COX inhibitors into the preoptic area prior to LPS administration (Abe et al., 2001). The pain modulatory action of PGE2 during the course of acute systemic infection was previously reviewed in detail by Hori and colleagues (Hori et al., 2000). In brief, centrally administered PGE2 mediates nociception in a dose-dependent manner, causing hyperalgesia at low, non-pyrogenic doses (10 pg/kg– 10 ng/kg) and analgesia at high, pyrogenic ones (1 μg/ kg) (Oka et al., 1994; Oka et al., 1995). In order to clarify which of the EP receptors might mediate pain transmission responses, Hori and colleagues injected EP1, EP2, and EP3 receptor agonists i.c.v. into rats and recorded paw-withdrawal latency on a hot plate 15–60 minutes after injection or firing rate responses of the trigeminal wide dynamic range (WDR) neurons to noxious pinching (Oka et al., 1994; Oka et al., 1997b).

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In these experiments, only the EP3 receptor agonist (M&B28767) decreased the paw withdrawal latency and augmented the nociceptive responses of the trigeminal WDR neurons. In contrast, the EP1 agonist (17-phenyl-ω-trinor PGE2) caused a prolongation of the paw-withdrawal latency and inhibition of the nociceptive responses of the WDR neurons. Moreover, the EP1 receptor antagonist (SC19220) prevented the PGE2-induced analgesic effects on the nociceptive behavior and responses of WDR neurons. These experiments suggested that PGE2 produces hyperalgesia at lower levels through actions on the EP3 receptors and at higher levels causes analgesia through actions on the EP1 receptors. The most sensitive sites to microinjected PGE2 and EP3 receptor agonist for producing thermal hyperalgesia seem to be the preoptic area, including its medial and lateral parts, the MnPO, and the diagonal band of Broca (Hosoi et al., 1997). On the other hand, it was found that the PGE2 and EP1 receptor agonist was most effective for evoking analgesia when microinjected into the ventromedial hypothalamic nucleus (VMH; Hosoi et al., 1999). As described above, although the EP3 receptor is highly expressed on neurons in the preoptic area and the receptors can alter activity of those neurons, the expression of EP1 receptors in the VMH has not been reported and the action of this receptor in this area remained to be elucidated in future studies. In contrast, expression of the EP2 receptor has been described in the VMH (Zhang and Rivest, 1999), but butaprost (an EP2 receptor agonist) failed to modulate pain thresholds (Oka et al., 1994; Oka et al., 1997b). It is poorly understood how alteration of neuronal activity in the preoptic and ventromedial hypothalamus by PGE2 can produce hyperalgesia and analgesia, respectively. One hypothesis is that hypothalamic neurons that respond to PGE2 may send inputs to areas that modulate nociception, such as the periaqueductal gray matter (PAG) and the nucleus raphe magnus (NRM) (Jones, 1992; Vanegas and Schaible, 2004). Projections from hypothalamic structures to the PAG are extensive, and previous anatomic studies indicate that the preoptic area, including the MPO and MnPO, is a major source of afferent input to the PAG and that also the VMH sends a projection to this brain stem region (Canteras et al., 1994; Rizvi et al., 1992; Saper et al., 1976; Saper and Levisohn, 1983; Yoshida et al., 2005). Thus, inhibitory EP3 receptors reduce activity of neurons in the preoptic area that may activate anti-nociceptive systems in the PAG, and PGE2 may increase the activity of VMH neurons that activate the descending pain inhibitory system. It would be of interest to perform similar studies using the newer

generation of highly selective EP agonists, and in mice with EP receptor deletions.

D. Changes in the Sleep-Wake Cycle Sleep and wakefulness are induced by a flip-flop mechanism involving sleep-promoting neurons in the ventrolateral preoptic area (VLPO) and wake-promoting neurons in the brain stem and hypothalamus, including the tuberomammillary nucleus (TMN), locus coeruleus (LC), and dorsal raphe nucleus (DR) (Saper et al., 2001). Aminergic neurons in TMN, LC, and DR promote wakefulness by direct excitatory effects on arousal systems in the thalamus, hypothalamus, basal forebrain, and cerebral cortex, as well as by inhibition of sleep-promoting neurons of the VLPO. During sleep, the VLPO inhibits amine-mediated arousal regions through GABAergic and galaninergic projections. Although endogenous PGE2 levels in the hypothalamus were significantly higher during wakefulness than during non-rapid eye movement (NREM) sleep as determined by microdialysis and the simultaneous recording of electroencephalograms in freely moving rats (Gerozissis et al., 1995), PGE2 is known to have contrasting effects on sleep-wake regulation when infused in different areas of the brain. When PGE2 was infused into the subarachnoid space underlying the ventral surface area of the rostral basal forebrain, the sleeping time of rats was significantly increased (Ram et al., 1997). In a pharmacological study using selective agonists for the EP receptors, the EP4 receptor agonist (ONO-AE1329) exhibited a very potent, dosedependent effect on NREM sleep and showed at lower agonist concentrations (1 and 10 pmol/min) a statistically significant increase of rapid eye movement (REM) sleep during infusion into the subarachnoid space (Yoshida et al., 2000). The infusion site at the ventral surface zone of the rostral basal forebrain is topographically located adjacent to the preoptic area, including the sleep-promoting VLPO, an area known for its high expression of the EP4 receptor (Oka et al., 2000; Zhang et al., 2000). It is also noteworthy that either both NREM and REM sleep or only NREM sleep was increased during infusion of the EP2 (ONO-DI004) and EP3 (ONO-AE257) agonists, respectively, into the subarachnoid space surrounding the preoptic area. In contrast to the wide distribution of the EP3 receptor in the preoptic area, expression of the EP2 receptor in the VLPO has not been reported, and at best, low levels of this receptor can be found in adjacent nuclei of the preoptic area (Zhang and Rivest, 1999). Hayaishi and colleagues have intensively investigated the wake-promoting effect of PGE2 in the


posterior hypothalamus (Huang et al., 2003; Matsumura et al., 1989a; Matsumura et al., 1989b). Recently, this group has shown by in situ hybridization that EP4 receptor mRNA was expressed in histaminergic neurons of the TMN (Huang et al., 2003). Moreover, only the EP4 receptor agonist (ONO-AE1329) mimicked the excitatory effect of PGE2 on histaminergic neurons in the TMN, and only perfusion of the EP4 receptor agonist into the TMN induced wakefulness. Investigations in animal research models and in humans have elucidated the role of cytokines in the physiological and inflammatory regulation of sleep (Krueger et al., 2001; Krueger and Majde, 2003; Mullington et al., 2001). Typically, there is a pronounced enhancement of NREM and an accompanied decrease of REM sleep in animals responding to proinflammatory cytokines, whereas the sleep patterns of humans in response to elevated level of cytokines are more complicated and dose-dependent, leading to increased or decreased NREM sleep. In addition, another PG, PGD2, has also been implicated in regulating sleep (Hayaishi and Urade, 2002). PGD2 is also produced by the action of COX-2, but PGD2 synthase is found only in the meninges surrounding the brain, not in the brain parenchyma (Beuckmann et al., 2000b). Focal infusion of PGD2 into the subarachnoid space ventral to the rostral basal forebrain causes profound enhancement of NREM sleep, and this also activates c-fos expression in the VLPO (Scammell et al., 1998a). Thus, Hayaishi and colleagues have hypothesized counterpoised PGE2 arousal and PGD2 soporific systems that may be activated during inflammation, and which may also play a role in daily regulation of sleep and wakefulness (Hayaishi and Huang, 2004). PGE2 evokes a number of physiological responses during systemic inflammation, though the alterations of sleep-wake patterns by PGE2 is poorly understood. The differential role of DP and EP receptors in sleep regulation during systemic inflammation remains to be clarified in pharmacological and physiological studies using DP and EP receptor agonists or knockout mice. These experiments are crucial to understand how elevated levels of PGE2 in the anterior and posterior hypothalamus in response to a peripheral infection may change sleep-wake cycles of the host, as for example, the excitatory EP4 receptor is expressed on neurons of the sleep-promoting VLPO and the wakepromoting TMN. Moreover, inhibitory EP3 receptors are expressed in the VLPO, thus PGE2 acting on those neurons would most likely decrease activity of the VLPO and induce wakefulness or changes between NREM and REM sleep stages.

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IV. CONCLUSION AND FUTURE STUDIES It is critical for mammals to be able to detect bacteria immediately when they infect local tissue or enter the bloodstream, and to mount a vigorous immune response. During a blood infection (septicemia), bacterial cell wall components such as LPS and cytokines would circulate in the blood, whereas if an infection were more localized, only circulating cytokines, such as IL-1β, TNF-α, and others, would reach the brain. Both LPS and cytokines act on specific receptors present on perivascular cells, and amplify the intracellular response that ultimately leads to elevated levels of PGE2 in the hypothalamus of the brain. Due to the variety of four EP receptors and their differential expression in various areas of the hypothalamus and brain stem, PGE2 mediates different or even counterposing components of the acute phase reaction. In the preoptic area, EP1 and EP3 receptors generate hyperthermia, and EP4 responses generate hypothermia. The body temperature of the animal represents the net effect of these opposing forces, which may vary at different time points after the infection. The synergistic action of the EP1 and EP3 receptor is required for activation of the endocrine component of the PVH in response to systemic LPS administration and release of adrenal and pituitary hormones. EP3 and EP1 receptors have opposing effects on neurons in the preoptic area and on VMH neurons that modulate activity of the descending pain inhibitory system and cause hyperalgesia or analgesia. EP receptors in the sleeppromoting VLPO and wake-promoting TMN might be involved in alteration of sleep behavior during a bacterial infection. Transgenic animals with constitutive gene disruptions of the EP receptors are very valuable for understanding the role played by the specific EP receptors for the mechanisms by which an immune stimulus, such as LPS or IL-1β, can cause a set of acute phase responses organized by the brain. However, the use of existing EP receptor knockout lines has several limitations. It is difficult to use EP4 receptor knockout animals because of the poor viability of the animals. Genetic disruption of the mouse EP4 receptor results in perinatal lethality associated with persistent patent ductus arteriosus. Moreover, because the gene knockout had been present for the entire life of the animals, including during development, there may have been some compensation for the lack of the specific receptors. Most importantly, constitutive knockout animals cannot be used to further localize individual brain areas or neurons involved in the acute phase responses.

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The neuron-specific modulation of EP receptors can be critically tested by using Cre- or Flp-recombinase– mediated DNA recombination in genetically engineered mice with conditional knockouts or knockins of the EP receptors (Lewandoski, 2001). Cre recombinase catalyzes deletion of DNA sequences flanked by 34 base pair LOX sites, while Flp recombinase works similarly on FRT sites. By transgenically directing Cre or Flp to discrete populations of neurons, and by crossing these transgenes with mice bearing LOX- or FRTmodified alleles, it is possible to modulate these genes in a neuron-specific fashion (Balthasar et al., 2004). Similarly, adeno-associated viral vectors (AAVs) expressing either Cre or Flp can be stereotaxically injected into specific nuclei in the brains of mice bearing LOX- or FRT-modified alleles to modulate the gene expression in patterns dictated by the site of AAV injection (Scammell et al., 2003). A mouse line with an EP4R gene amenable to conditional deletion using Cre recombinase was recently created by inserting LOX sites into introns flanking the second exon of the EP4R gene (Schneider et al., 2004). Moreover, our lab is currently producing mice with conditional expression of EP1 and EP3 receptors. The conditional knockout mice for the specific EP receptors will be useful to study the effect of focal gene manipulation in different areas of the hypothalamus, such as preoptic area, PVH, SON, TMN, and VMH, on LPSinduced acute phase responses of the CNS. Moreover, the focal deletion of EP receptors in conditional knockout mice would help to resolve the controversy that the EP receptors expressed in brain stem structures partly modulate autonomic or behavioral changes seen during systemic inflammation or that those changes are due to neural afferent signaling. For instance, the parabrachial nucleus projects to many forebrain structures and autonomic nuclei of the hypothalamus and reliably shows c-fos activation after LPS stimulation (Elmquist et al., 1996; Elmquist and Saper, 1996). Because the EP3 and EP4 receptors are expressed on LPSactivated PB neurons, it was speculated that PGE2 might mediate the signaling of PB neurons rather than the c-fos activation being caused by neural inputs from the NTS or the spinal cord (Engblom et al., 2001; Zhang et al., 2000). Animals with conditional expression of the EP receptor genes will help to test this and a wide range of hypotheses about the roles played by prostaglandins in the CNS response to systemic inflammation.

Acknowledgments We thank Takakazu Oka for his help and advice with this review and for permission to reuse figures from his papers in this review. This work was supported by USPHS grant NS33987.

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C H A P T E R

16 The Role of Pro-inflammatory Cytokines in Memory Processes and Neural Plasticity INBAL GOSHEN AND RAZ YIRMIYA

kines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, which coordinate subsequent immune, physiological, metabolic, and behavioral responses that are collectively termed the acute phase reaction (Dinarello, 2000). In addition to their role in immunoregulation of inflammatory processes, pro-inflammatory cytokines play a major role in modulation of neural, neuroendocrine, and behavioral systems during illness (Dantzer, 2004; Maier and Watkins, 1998; Rothwell et al., 1996; Yirmiya, 1997). For example, medical conditions that are associated with elevated brain levels of proinflammatory cytokines, or direct administration of these compounds into the brains of experimental animals, produce fever, activation of the hypothalamicpituitary-adrenal (HPA) axis, and many behavioral symptoms that are collectively termed sickness behavior (Dantzer, 2004; Kent et al., 1992; Maier, 2003; Yirmiya, 1997). Furthermore, recent studies suggest that brain cytokines may also have some physiological roles in neural, neuroendocrine, and behavioral regulation (Vitkovic et al., 2000). One of the most salient symptoms of the sickness behavior syndrome is alteration in learning and memory processes. The aim of this chapter is to summarize the results that have been obtained to date on the role of pro-inflammatory cytokines, particularly IL-1, IL-6, and TNF-α, in learning and memory, both during illness and in healthy individuals. In addition, we propose several mechanisms that can mediate the effects of cytokine-induced memory alterations.

I. INTRODUCTION 337 II. EFFECTS OF IL-1, IL-6, AND TNF-α ON LEARNING AND MEMORY IN RODENTS 342 III. MECHANISMS UNDERLYING THE EFFECTS OF PRO-INFLAMMATORY CYTOKINES ON LEARNING AND MEMORY 353 IV. PRO-INFLAMMATORY CYTOKINES AND MEMORY FUNCTIONING IN HUMANS 360 V. GENERAL SUMMARY AND CONCLUSIONS 366

I. INTRODUCTION In animals and in humans, infectious diseases pose one of the most serious challenges for survival and reproduction. This challenge constitutes a major selective force for the evolution of defense mechanisms against pathogen invaders. The first line of defense is the non-specific, innate immune response (Hoffmann et al., 1999). This response is triggered by conserved molecular patterns on infectious microorganisms, e.g., lipopolysaccharide (LPS—a major constituent of the cell wall of gram negative bacteria), which bind to specific receptors (e.g., the Toll-like receptors) expressed by cells of the innate immune system (e.g., macrophages) (Janeway and Medzhitov, 2002; Takeda et al., 2003). The resultant activation of these cells leads to the destruction of the pathogenic microorganisms, either by phagocytosis or by secreted compounds such as reactive oxygen species. Activated innate immune cells also secrete various compounds that induce inflammation, particularly the pro-inflammatory cytoPSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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A. Pro-inflammatory Cytokines in the Brain Affect Behavioral Processes In order to modulate learning and memory, proinflammatory cytokines have to be able to reach the brain and to affect neural processes. The following section constitutes a short (and non-comprehensive) introduction to the signaling pathways via which proinflammatory cytokines exert their actions, the ways by which pro-inflammatory cytokines, secreted either by immune cells in the periphery or by glia cells and neurons within the central nervous system (CNS), influence neural processes, and the functional consequences of pro-inflammatory cytokines actions in the brain, i.e., their involvement in behavioral alterations during illness and in health. 1. The Signaling Pathways of Pro-inflammatory Cytokines IL-1 Signaling IL-1 signaling is mediated by a complex system, which includes several ligands and receptors. The cytokines IL-1α and IL-1β activate signaling, whereas IL-1 receptor antagonist (IL-1ra) serves to block the effects of IL-1 by binding to the same receptors without triggering signaling (Dinarello, 1996). Many types of IL-1 receptors have been identified (Loddick et al., 1998). In particular, the IL-1 receptor type I (IL-1rI) appears to mediate all of the known biological functions of IL-1, but in order to transmit a signal into the cell it has to form a complex with the IL-1 receptor accessory protein (IL-1rAcP). On the other hand, IL-1 receptor type II, which can be either soluble or membrane bound, is a decoy receptor, which antagonizes IL-1 signaling (Born et al., 2000; Smith et al., 2000). IL-6 Signaling IL-6 is a part of the IL-6–type cytokines family, which also includes IL-11 and ciliary neurotrophic factor (CNTF). In the first step of its signaling, IL-6 binds to the membrane-bound IL-6 receptor (IL-6R), and together they form a high-affinity complex with a homodimer of the signal transducer gp-130 (Heinrich et al., 2003; Naka et al., 2002). No specific endogenous inhibitor of IL-6 was identified. Rather, its signaling is suppressed by non-specific inhibitors, such as suppressor of cytokine signaling (SOCS) (Naka et al., 2002). Similar to IL-1, IL-6 also has a soluble receptor (sIL-6R). However, in contrast to IL-1RII, sIL-6R is an agonist of IL-6 signaling (Jones et al., 2005). Whereas gp-130 is ubiquitously expressed by all body cells, IL6R is differentially expressed in specific cell types. However, IL-6 can induce signaling in every cell that

expresses gp-130 by binding to sIL-6R, a process named transsignaling (Jones et al., 2005). For example, the influence of IL-6 on neuronal cells depends on sIL-6R (Marz et al., 1998; Marz et al., 1999). TNF Signaling TNF is a part of a large gene superfamily, comprising 18 known ligands, including TNF-α and β (Locksley et al., 2001). TNF is only stable as a homotrimer, either membrane-bound or soluble, and can bind to its receptors at either form. From the various proteins comprising the TNF receptor (TNF-R) family, TNF-R1 and TNF-R2 are the main mediators of its signaling (Tartaglia and Goeddel, 1992; Wajant et al., 2003). 2. Pro-inflammatory Cytokines Influence Neural Processes within the Brain Cytokines are produced by many types of cells, including immune cells in the periphery as well as glia and neurons within the brain, and their gene expression and protein production are elevated during various disease states. Peripheral IL-1, IL-6, and TNF-α orchestrate the inflammatory response to various immune stimuli, causing multiple physiological and behavioral responses. Many immune challenges also induce the production and secretion of cytokines in the brain, which directly produce changes in neurotransmitter and neuroendocrine systems, including monoaminergic systems and the HPA axis (Besedovsky and del Rey, 1996; Dunn and Wang, 1995; Turnbull and Rivier, 1999), as well as behavioral symptoms that will be specified below. To induce these effects, cytokines can directly influence the brain, as suggested by their presence and the expression of their receptors in various brain structures. For example, IL-1 and IL-6 receptors are present throughout the brain, specifically within the hippocampus (Cunningham and De Souza, 1993; Loddick et al., 1998; Parnet et al., 2002; Schobitz et al., 1992, 1993). TNF receptors are also expressed in various brain regions (Kinouchi et al., 1991). The fact that administration of IL-1ra into the brain blocks most of the effects of peripheral IL-1 administration (Kent et al., 1992; Rothwell and Luheshi, 2000) further supports the notion of its central influence. The source of cytokines in the brain can be either local synthesis by glia and neurons, or passage of peripherally produced IL-1, IL-6, and TNF-α through the blood-brain barrier (BBB) (Dantzer et al., 2000; Watkins et al., 1995). Because these cytokines are large proteins, such passage can be accomplished either passively in circumventricular organs, in which the BBB is weak, or via active transport in other areas (Banks, 2005). Alternatively, cytokines can induce the synthe-

16. The Role of Pro-inflammatory Cytokines in Memory Processes and Neural Plasticity

sis and secretion of smaller mediators that can easily cross the BBB, such as prostaglandins (Rivest, 2001). In addition, peripheral IL-1 and TNF-α can influence the brain via the activation of vagal afferent fibers. Indeed, vagotomy blocks many centrally mediated effects of peripheral immune activation (Dantzer, 2001; Goehler et al., 2000). 3. The Involvement of Pro-inflammatory Cytokines in Behavioral Alteration during Sickness and in Health Pro-inflammatory Cytokines Mediate Sickness Behavior During illness, IL-1, IL-6, and TNF-α mediate the collection of neurobehavioral symptoms that constitute the sickness behavior syndrome. It is fairly accepted that this syndrome is adaptive, at least under certain circumstances, helping the organism recuperate from the disease (Dantzer, 2004; Hart, 1988; Kent et al., 1992; Maier and Watkins, 1998; Yirmiya et al., 1999). The symptoms comprising the sickness behavior syndrome include anorexia, body weight loss, altered sleep patterns (characterized by an increase in slow-wave sleep and inhibition of rapid eye movement sleep), psychomotor retardation, fatigue, reduced exploratory behavior, impaired social behavior (e.g., reduction in the time spent by adult animals in social olfactory exploration of juvenile conspecifics), reduced libido (particularly in females), impaired sexual activity, altered pain perception, and anhedonia, defined as the diminished capacity to experience pleasure and gratification from activities that previously brought enjoyment. In animals, this parameter was mainly demonstrated by reduced consumption of and preference for sweet solutions, as well as by decreased responding to rewarding intracranial self-stimulation. Mediation of these symptoms by pro-inflammatory cytokines has been indicated by the findings that: (1) In both humans and animals there are correlations between the elevation in cytokine levels during various medical conditions and the prevalence and severity of the above-mentioned sickness behavior symptoms; (2) pharmacological administration (either peripherally, or directly into the brain) as well as geneticallyengineered transgenic overproduction of proinflammatory cytokines (particularly IL-1, IL-6, or TNF-α) produces sickness behavior symptoms; (3) inhibitors of pro-inflammatory cytokine signaling block the behavioral effects of sickness (Dantzer, 2004; Larson and Dunn, 2001; Maier and Watkins, 1998; Yirmiya et al., 1999). Together with the various behavioral alterations detailed above, cytokine-mediated sickness behavior

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is also accompanied by impairments in cognitive abilities, particularly learning and memory processes. The role of cytokines in this particular symptom will be the focus of this chapter. Pro-inflammatory Cytokines Regulate Behavior in Healthy Animals Recently, cytokines were implicated in the regulation of neurobehavioral processes not only during immune activation, but in healthy animals as well (Vitkovic et al., 2000). Most studies in this area rely on genetic mouse strains lacking normal cytokine signaling. For example, we demonstrated a regulatory role for IL-1 signaling in basal pain sensitivity and neuropathic pain, as well as in morphine-induced analgesia and the development of tolerance following repeated morphine administration, using strains of mice genetically impaired in IL-1 signaling (Shavit et al., 2005; Wolf et al., 2003; Wolf et al., 2006). Similarly, deficiencies in IL-6 and TNF signaling also resulted in behavioral modifications: IL-6 knockout mice differed from their wild-type (WT) controls in their sleep patterns (Morrow and Opp, 2005), and both IL-6 knockout and TNF-α knockout mice demonstrated altered emotional behavior (Armario et al., 1998; Yamada et al., 2000). 4. Interim Summary In conclusion, pro-inflammatory cytokines and their receptors are present in the brain, specifically in the areas that are known to be involved in memory formation, such as the hippocampus. Furthermore, pro-inflammatory cytokines mediate the behavioral alterations that accompany immune activation, and are also involved in the modulation of behavior in healthy animals. The combination of these findings, along with clinical and anecdotal reports about illness-associated cognitive impairments, encouraged many research groups to explore the role of proinflammatory cytokines in memory processes, either during sickness or under normal physiological conditions. Before reviewing the results of these studies in detail, we provide a brief introduction to the basic concepts of memory processes and the brain areas that underlie them. Furthermore, because most studies on pro-inflammatory cytokines and memory utilized rodents, and because proper interpretation of the results of these studies requires understanding of the memory testing paradigms, we describe the most common techniques to evaluate different memory types in rodents.

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B. Memory Processes and Their Assessment in Rodents 1. Memory Processes—Basic Concepts Memory is defined by Webster’s dictionary (1996) as “the mental capacity or faculty of retaining and reviving impressions, or of recalling or recognizing previous experiences.” Memories are acquired by the learning process, during which experiences cause lasting changes in the nervous system. The ability of all organisms to adapt to new situations and to react properly to previously encountered stimuli depends on their ability to remember. Memory can be divided into three stages: encoding, storage (which can last from seconds to years), and retrieval. Failure of any one of these processes will result in impaired memory functioning. One of the first to hypothesize that synaptic changes constitute the basis of memory formation was Donald Hebb, who suggested that the synapse between two neurons it amplified if they are active simultaneously (Hebb, 1949). This hypothesis received important empirical support by the studies of Bliss and Lomo (1973), who showed that brief high-frequency stimulation of hippocampal afferents resulted in persistent augmentation of their synaptic strength within the dentate gyrus (DG), a phenomenon termed long-term potentiation (LTP). Since then, synaptic plasticity was implicated in memory storage, in the hippocampus and other brain areas (e.g., Bliss and Collingridge, 1993; Eichenbaum, 1996). It should be noted, however, that although synaptic plasticity was proved in many studies to be necessary for learning and memory, it is not clear whether it is sufficient for memory formation (see Martin et al., 2000, for review). In addition to the biochemical and structural processes of synaptic plasticity, the brain is capable of plasticity in the whole neuronal level, i.e., via the formation of new neurons, a process termed neurogenesis (Gould et al., 2000; Gross, 2000; Kempermann et al., 2004; Rakic, 2002). In the last decade, the importance of neurogenesis in memory processes was demonstrated by showing that memory induces the generation of new neurons (Gould et al., 1999), and that changes in neurogenesis levels are accompanied by similar changes in memory performance (Gross, 2000). Much more research will be needed to fully understand the processes that underlie plasticity within the brain, and the connection between synaptic plasticity and neurogenesis to learning and memory processes. Various brain structures are involved in the modulation of different memory types, sometimes with a certain amount of redundancy (Zola-Morgan and

Squire, 1993). Among them are the amygdala, which modulates the consolidation of memories of emotionally arousing experiences (McGaugh, 2004), as well as the basal ganglia and the cerebellum (Packard and Knowlton, 2002; Thompson, 2005). This review, however, will focus on memory types that require the hippocampus, a brain region located within the medial temporal lobe. In humans, the hippocampus is responsible for conscience declarative memory—the facts and events we can recollect and report about (Squire et al., 2004). Whereas declarative memory can be easily assessed verbally in humans, it is more difficult to characterize in animals. However, the hippocampus also plays a critical role of spatial navigation and the association between discontinuous events (Eichenbaum, 2000; Squire, 1993; Wallenstein et al., 1998), and these functions can be readily assessed in animals, as will be discussed in the following section. 2. Paradigms for Memory Testing in Rodents During the many decades in which the cognitive behavior of laboratory animals has been investigated, many memory assessment techniques were developed, enabling researchers to examine various memory functions in different species (Cahill et al., 2001). These paradigms span from the simple Pavlovian classical conditioning to the complicated radial arm maze spatial task. Simple, elegant variations within some paradigms (the water maze and fear conditioning tests, for example) enable researchers to distinguish between hippocampal-dependent and hippocampalindependent memory performance. In the most prevalent paradigms, the incentive for learning is the avoidance of an aversive stimulus, whereas fewer paradigms utilize appetitive learning, in which the animal has to learn to perform in order to obtain a positive reward. The four paradigms that have been most frequently utilized in the research on the role of pro-inflammatory cytokines in memory will be described in this section. Less common paradigms will be described in the next section, in the context of the results obtained with these paradigms. The Water Maze The water maze paradigm for memory testing was devised by Morris et al. in 1982. Essentially, rodents are placed in a circular pool and trained to find a platform, located at a particular location within the pool. After reaching the platform and standing on it, the animal is taken out of the pool, dried, and given some rest (which can be considered as rewards) before the next trial. The latency to find the platform serves as an indicator for the learning process; the stronger the


memory, the faster the animal will reach the platform. The experiments are usually conducted using a random protocol, in which the entrance point to the maze is varied randomly between trials, and the platform remains in a permanent position (Morris, 1981). This basic paradigm can be applied in several ways, which enable differentiation between hippocampaldependent spatial memory and hippocampalindependent memory. In the non-spatial memory paradigm, the platform is elevated above the water surface and therefore it is visible; thus, spatial learning is not required. The only thing to be learned is the escape opportunity, i.e., that climbing the platform results in extraction from the maze, and rodents acquire this learning rapidly (Morris, 1981). Hippocampal lesions have no effect on the performance level in this paradigm (Morris et al., 1982). In the spatial memory paradigm, animals are trained to find the location of a hidden platform, submerged below the opaque water surface, using extra maze visual cues. In this case, the animal has to learn both the procedure (climbing on the platform in order to stop swimming and be removed from the maze) and the spatial location of the invisible platform. The latter task depends on normal hippocampal functioning, as evident from the finding that hippocampal lesions abolish the ability of spatial navigation in the maze (Morris et al., 1982). Following acquisition of the spatial task, it is customary to perform a transfer test (also known as probe test), in which the platform is removed from the maze. The animal is placed in the maze for a short trial, in which the time it swims in the quadrant in which the platform was located or the number of times it crosses the former location of the platform is measured as another indicator of spatial memory (Morris, 1984). The basic paradigm described above can be modified in various ways and has been used since its invention to investigate the involvement of various pharmacological, genetic, and environmental factors in memory functioning (Brandeis et al., 1989; D’Hooge and De Deyn, 2001). Fear Conditioning Fear conditioning is the learning that a neutral stimulus predicts the appearance of an aversive event. The combination of a neutral (conditioned) stimulus and an aversive (unconditioned) stimulus renders the formerly neutral stimulus a frightful quality, so that even when it appears by itself, without the aversive stimulus, it will elicit a fearful conditioned response (Maren, 2001). Fear conditioning can be rapidly formed in humans and animals, even following a single conditioning trial, and is usually maintained for long periods (Maren, 2001). In rodents, the dominant behavioral fear response is freezing (complete immobility), and

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the most commonly used aversive stimulus is the delivery of a weak, short electrical shock (Fanselow, 2000). The conditioning process itself (i.e., the association between the neutral and aversive stimuli) is mediated primarily by the amygdala (Maren, 2001; Maren and Quirk, 2004), and by using different types of conditioned stimuli, the fear-conditioning paradigm enables differentiation between hippocampaldependent and -independent functions: When a simple perceptual conditioned stimulus (such as a light or an auditory cue) is used, the hippocampus is not required. However, when the conditioned stimulus is a new environment, a mental representation of this new context has to be created so that the amygdala can associate this representation with the aversive stimulus; this function depends on hippocampal functioning (Fanselow, 2000; Maren and Holt, 2000). In the most commonly used version of the fear-conditioning paradigm, animals are placed in a novel conditioning cage, in which they hear an auditory tone, followed by a brief foot-shock. Thus, the animal can associate the aversive stimulus with the new context as well as with the tone. To test contextual fear conditioning, animals are placed again in the original conditioning cage, and freezing is measured. This task is hippocampal dependent, as it cannot be performed following hippocampal lesions (Fanselow, 2000). To test the hippocampal-independent auditory-cued fear conditioning, freezing is measured when the tone is sounded in a differently shaped context (Fanselow, 2000; Maren and Holt, 2000). In this case, hippocampal lesions have no detrimental effect on performance level (Fanselow, 2000). The fear-conditioning paradigm has been employed extensively in research on the neural basis of memory in various pharmacological, genetic, and environmental studies (Fanselow and Poulos, 2005). Passive and Active Avoidance In the passive avoidance paradigm, animals learn to refrain from contacting a formerly desired stimulus, based on their experience that a previous contact with that stimulus resulted in an aversive incident. The aversive experience is usually a mild electrical shock, and the desired stimuli may be, for example, water for a thirsty animal, food for a hungry animal, or a small dark chamber for an animal placed in a spacious, brightly illuminated one (as rodents prefer narrow dark places). The paradigm is termed “passive” avoidance because the animal does not have to actively perform any action, but rather to refrain from performing an activity it desires (approaching drink, food, or a dark chamber, for example). Thus, the latency it takes the animal to perform the frightful activity serves as a measure for its memory of the aversive consequences

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of that action. Passive avoidance is hippocampal dependent, as hippocampal lesions impair learning in this task, as demonstrated by a diminished latency to enter a dark chamber in which an electric shock was administered in the past (Burwell et al., 2004). Induction of hippocampal lesion at different timings along the passive avoidance paradigm demonstrated a role for both the dorsal and ventral hippocampus in the acquisition, consolidation, and retrieval of passive avoidance (Ambrogi Lorenzini et al., 1996, 1997). Contrary to the passive avoidance paradigm, in active avoidance animals learn to perform a specific action in order to avoid an aversive stimulus. The aversive stimulus is usually a mild electrical shock, and the actions required to stop it can be lever press, entry to a protective area, etc. Thus, the latency to perform the avoidance action or the number of actions serves as an indicator of the learning strength. Active avoidance is also hippocampal dependent, as hippocampal lesions impair performance in this task (Cimadevilla et al., 2000; Munoz and Grossman, 1982).

II. EFFECTS OF IL-1, IL-6, AND TNF-a ON LEARNING AND MEMORY IN RODENTS Exposure to pathogens that stimulate the immune system results in altered memory performance, as part of the general sickness behavior syndrome. For example, viral, bacterial, and parasitic infections, as well as exposure to viral coat proteins or bacterial endotoxin, result in impaired memory performance in animals (e.g., Braithwaite et al., 1998; Gibertini et al., 1995a, b; Kamitani et al., 2003; Lee et al., 2000; Li et al., 2004a; Murray et al., 1992; Pugh et al., 1998; Pugh et al., 2000; Sparkman et al., 2005). Because the immune response to these factors and the sickness behavior that accompanies it are mediated by pro-inflammatory cytokines, many research groups sought to directly assess the involvement of pro-inflammatory cytokines in memory processes. The data collected in these studies, specifically regarding the cytokines IL-1, IL-6, and TNF, will be presented in this section.

A. IL-1 The role of IL-1 in memory processes has been extensively studied in the last decade by many research groups, using various models. Most of these studies showed a detrimental effect of IL-1 on memory formation; however, some studies reported no effect, and recent studies demonstrated an important role for IL-1 in normal memory functioning. These seemingly contradictory results will be presented in the following

sections, together with an attempt to accommodate all findings using a theoretical model. 1. Elevated IL-1 Levels Impair Memory Functioning The first attempt to evaluate the effect of IL-1 on memory was performed by Oitzl et al. in 1993 (Oitzl et al., 1993). In this study, IL-1β (100 ng/rat) injected intracerebroventricularly (i.c.v.) 1 hour before the beginning of spatial water maze training caused a transient memory impairment in the first trial of the following day. When the same dose was injected immediately before training, no effect was found, suggesting that IL-1β does not affect the acquisition of spatial memory but rather the retention of this learning and that the processes triggered by IL-1β require some time to exert their influence on memory (Oitzl et al., 1993). In another extensive set of experiments using the water maze procedure, Gibertini et al. (1995a) injected mice peripherally with IL-1β (100 ng/mouse, intraperitoneal—i.p.) on the first and second day of spatial water maze training and reported a complete blockade of spatial learning (Gibertini et al., 1995a). Gibertini then repeated the experiment, this time training mice in both the spatial (random entry) and non-spatial (fixed entry) versions of the water maze. IL-1β impaired learning in the spatial paradigm but had no effect on non-spatial memory (Gibertini, 1996). IL-1β–injected mice were also found to be less flexible in adapting to a change in the position of the hidden platform. This was demonstrated in an experiment in which after the mice were injected with IL-1β and learned to find the platform using the fixed-entry protocol, the position of the platform was changed. Although IL-1β did not interrupt the initial learning, when the platform position was changed, saline-injected mice quickly adjusted to the change, whereas IL-1β–injected mice continued to look for the platform in its former position (Gibertini, 1996). In a subsequent study, Gibertini (1998) further showed that the memory impairment caused by IL-1 is restricted to learning conditions of relatively low motivation: When mice were trained in the water maze using a water temperature of 23°C, the detrimental effect of IL-1β was observed as before. However, when the water temperature of the maze was reduced to 15°C, thus creating a greater motivation to escape, IL-1β injection had no effect (Gibertini, 1998). A recent study confirmed these early studies on the effects of IL-1β on spatial memory, demonstrating that the detrimental effect of i.c.v.-administered IL-1β on spatial memory could also be achieved using a lower dose (15 ng/rat) (Song and Horrobin, 2004). This study also repeated the finding that in contrast with the effect


of IL-1β in the spatial hippocampal-dependent version of the water maze, no effect of IL-1β was found when the same rats were tested in the hippocampalindependent visually guided version (Song and Horrobin, 2004). In contrast with these findings, other studies, using somewhat different regimens of IL-1 administration and testing procedures, found no effect of IL-1 on spatial memory. Lacosta et al. (1999) injected mice daily with IL-1β (1 μg/mouse, i.p.) for a week before training in the water maze, as well as at the week of training itself, and found no effect on spatial learning (Lacosta et al., 1999). In another study, mice that were trained in the water maze using a spaced-learning protocol displayed normal latency to reach the platform following IL-1 injection (100 ng/mouse) (Gibertini, 1998). However, the IL-1β–injected mice used a different strategy to find the platform, as apparent from the fact that whereas saline-injected mice spent more and more time in the quadrant in which the platform was positioned, IL-1β–injected mice remained at a chance level (Gibertini, 1998). Taken together, the results of all the studies presented so far suggest that IL-1 interferes specifically with spatial learning in the water maze, which depends on normal hippocampal functioning. However, these effects are not demonstrated under all conditions and depend on various experimental parameters. The involvement of IL-1 in memory processes was also extensively examined using the fear-conditioning paradigm. Pugh et al. (1999) demonstrated that i.c.v. injection of IL-1β (either 10 or 20 ng/rat), immediately following conditioning, impaired contextual but not auditory-cued fear conditioning (Pugh et al., 1999; Pugh et al., 2001). These findings indicate that the impairment was restricted to the hippocampaldependent version of the fear-conditioning paradigm, without affecting the hippocampal-independent task. Further evidence for the specific localization of the effect of IL-1 was recently provided by demonstrating that bilateral intrahippocampal injection of IL-1β (10 ng/side) also impaired contextual fear conditioning (Barrientos et al., 2004). In another study, a different version of the contextual fear-conditioning paradigm was used. In that version, rats are preexposed to the context one day before the conditioning session, in which they receive an immediate shock when re-entering this context. The pre-exposure provides an opportunity to generate a mental representation of the context, and without it conditioning will not take place when an immediate shock is applied. This separation between the hippocampal-dependent memory of the context and the conditioning (associative) process itself (which is mediated by the amyg-

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dala) enables researchers to pinpoint the exact component of the learning process in which IL-1 is involved. Using this paradigm, Barrientos et al. (2002) bilaterally injected rats with IL-1β (1 ng/side) directly into the hippocampus after pre-exposure to the context. The animals were immediately shocked on the following day and tested for conditioned fear response one day following the shock. Rats that were injected with IL-1β, immediately or 3 hours following pre-exposure, demonstrated a 50% reduction in contextual fear conditioning. However, when rats were pre-exposed to the context and received the intra-hippocampal injection of IL-1β 24 hours later (at the time of conditioning), they demonstrated only a 20% decrease in fear response compared to controls. Finally, when IL-1β was injected at the time of testing (48 hours after preexposure to the context), no effect on the fear response was observed (Barrientos et al., 2002). These data suggest that the detrimental effect of IL-1 on contextual fear conditioning is caused by interference with the formation of the mental representation of the context by the hippocampus, rather than with the association of this representation with the shock. Another learning and memory task in which IL-1 is critically involved is spatial active avoidance: In a series of experiments, mice learned to avoid an electrical shock by entering a specific arm of a T-maze. IL-1 was injected after the learning phase, and retention was tested 1 week later (Banks et al., 2001). When administered intravenously (i.v.), both human IL-1α (0.3 and 1, but not 0.03 and 0.1 μg/mouse) and human IL-1β (1 μg/mouse) impaired memory; i.e., IL-1 injected mice required more trials in order to learn to perform the avoidance response. Much lower doses were needed to achieve this detrimental effect when human IL-1α (0.1, 0.3 and 1, but not 0.03 ng/side) and IL-1β (1 ng/side) were administered bilaterally into the posterior septum (PDS), connecting the hippocampus and the midbrain. Banks et al. (2001) further demonstrated that the effect of i.v.-injected human IL-1α stems from transport of this cytokine across the BBB as well as from endogenous stores of IL-1α, because the memory impairment was partially blocked by antibodies against either human or murine IL-1α injected into the PDS, and fully blocked by injecting the combination of these antibodies. Human IL-1β, however, did not cross the BBB, but induced the secretion of murine IL-1β, as shown by the findings that its influence on memory was blocked by murine anti–IL-1β injected into the PDS, but was unaffected by human anti–IL-1β (Banks et al., 2001, 2002–2003). This set of studies demonstrates again that IL-1 impairs hippocampal-dependent memory formation when administered either peripherally or centrally.

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IL-1β also impairs performance in several other memory-testing paradigms: three-panel runway, radial arm maze, autoshaping, and passive avoidance. In the three-panel runway task, rodents learn to open the correct doors between three consecutive chambers in order to receive a reward. In this task, a smaller number of errors (attempts to open incorrect doors) reflects better working memory performance (Furuya et al., 1988), which critically depends on normal hippocampal functioning (Kitajima et al., 1992; Ohno et al., 1992). Intrahippocampal IL-1β (32 and 100, but not 10 ng/ side) impaired working memory in the three-panel runway task, as shown by an increased number of errors (Matsumoto et al., 2001, 2004). The win-shift version of the radial arm maze is divided into a training session, in which the locations of the arms that will contain food in the future are marked by blocking them for entry, and a testing session in which the rats have to enter these arms in order to obtain a food reward. A smaller number of errors (entrees to empty arms) reflects better working memory functioning, which depends on normal hippocampal functioning (Floresco et al., 1997). I.c.v. IL1β (10 and 50 ng/rat) impaired working memory in the win-shift radial arm maze, as shown by an increased number of errors in the days in which IL-1 was injected 45 minutes before testing. The effect of the higher dose also remained for another day after the injections were terminated (Song et al., 2004). In the autoshaping task, rodents press a lever in order to hasten the appearance of a delayed food reward. The processing of temporal discontinuities (such as the delay between the lever press and the appearance of the reward) is performed by the hippocampus (Rawlins et al., 1985; Wallenstein et al., 1998), and septal lesions result in longer latencies to press the lever (Sagvolden and Holth, 1986). IL-1β (4 μg/rat, i.p.) delayed the acquisition of autoshaping in rats (Aubert et al., 1995). Furthermore, a positive correlation was found between the dose of IL-1β and the latency to press the lever; the higher the dose, the longer the latency, i.e., the greater the learning deficit (Aubert et al., 1995). A complementary approach to verify the detrimental effect of IL-1 administration on memory processes is to examine the possible beneficial effects of IL-1 blockade. Although most studies using this approach reported that such a blockade impairs rather than facilitates memory functioning (see below), one study demonstrated detrimental effects (Depino et al., 2004). In that study, the step-down passive avoidance test was used, in which rats are placed on an elevated platform in a new context, and an electrical shock is delivered upon stepping down from the platform. The latency to step

down from the platform on subsequent trials serves as a measure to memory (longer latencies indicate better memory). Depino et al. (2004) injected rats with an adenovector containing the rat IL-1ra gene to the hippocampi 1 week before passive avoidance training. The vector was expressed in neurons and glia cells, and blocked the activity of IL-1 within the hippocampus. This resulted in enhanced short- and long-term memory in these rats, as shown by longer latencies to step down from the platform compared to control rats 1.5 and 24 hours following training (Depino et al., 2004). Several studies examined the role of IL-1 in memory impairments caused by various inflammatory agents. Gibertini et al. (1995a, b) showed that inoculation with the bacterium Legionella pneumophila, which increases IL-1β levels in mice (Shinozawa et al., 2002), 24 hours before training in the spatial water maze task impaired learning. However, when mice were injected with anti–IL-1β antibodies 2 hours before training, this bacterial infection had no effect on spatial memory (Gibertini et al., 1995 a, b). Subsequently, Pugh et al. (1998) studied the influence of LPS on fear conditioning. They reported a detrimental effect of LPS, which was demonstrated to increase hippocampal IL-1β levels (Nguyen et al., 1998) on contextual fear conditioning, which requires normal hippocampal functioning. However, no effect on auditory-cued fear conditioning was observed. When the LPS injection was immediately followed by IL-1ra administration, the detrimental effect on contextual memory was abolished (Pugh et al., 1998). In an additional set of experiments, Pugh et al. (2000) demonstrated the potential role of IL-1 in AIDS-related memory impairments: They reported that i.c.v. injection of the HIV coat protein gp-120 increased IL-1β level in the hippocampus and impaired contextual, but not auditory-cued, fear conditioning. Moreover, they showed that IL-1 mediates this impairment, because when IL-1ra was administered i.c.v. immediately following HIV gp-120, the detrimental effect of this pathogen on contextual fear conditioning was abolished (Pugh et al., 2000). IL-1 was also found to mediate memory impairments during chronic brain inflammation. Palin et al. (2004) used a chronic neuroinflammation model based on delayed-type hypersensitivity (DTH) response to the bacterium Bacillus Calmette-Guerin in the hippocampus (Matyszak and Perry, 1995), and tested its influence on memory using the Y maze paradigm. In this paradigm, rats are exposed to two arms of a Y maze, and when they are re-introduced to the maze, a third arm is available as well. A rat that remembers the “old” arms will explore the novel arm more extensively (Dellu et al., 1992). Hippocampal DTH response resulted in memory impairment, as demonstrated by


the finding that whereas control rats preferred to explore the novel arm, infected rats demonstrated no preference on day 16 of infection, suggesting that they did not remember the spatial position of the old arms. Additionally, DTH response produced elevated IL-1β levels in the infected hippocampus, compared to the non-infected hippocampus. Palin et al. (2004) showed that IL-1 mediated this DTH-induced memory impairment, evidenced by the finding that when IL-1ra was chronically administered for 10 days before the memory test, rats with hippocampal DTH response performed the task as well as controls. Interestingly, IL-1 mediates not only the effects of inflammatory agents on memory processes, but also the effect of psychological stress. Maier and Watkins (1995) were the first to demonstrate the role of IL-1 in stress-induced modulation of memory functioning. When rats are subjected to inescapable shock 24 hours before training in an active avoidance task, their performance is impaired, a phenomenon termed learned helplessness (Maier, 1990). I.c.v. administration of IL1ra (100 μg/rat) before inescapable shock administration blocked the learned helplessness induced by inescapable shock (Maier and Watkins, 1995). Pugh et al. (1999) further showed that when rats were isolated for 5 hours following fear conditioning, contextual memory was impaired, whereas auditory-cued memory remained intact. Furthermore, they showed that IL-1 mediates this effect, by demonstrating that hippocampal IL-1β levels were increased following social isolation, and that i.c.v. administration of IL-1ra before isolation blocked the effect of isolation on contextual memory (Pugh et al., 1999). To sum up, the data presented so far (summarized in Table 1) clearly demonstrate a detrimental effect of elevated IL-1 levels on memory processes. This negative influence was found in various studies to be specific to memory tasks that depend on normal hippocampal functioning, whereas the performance of hippocampal-independent tasks is spared. This finding was replicated using various doses (of either IL-1α or IL-1β), administration routes (i.p., i.v., i.c.v., intrahippocampal, and intraseptal), and species (rats and mice), thus strengthening the reliability, validity, and generalizability of these findings. 2. Impaired IL-1 Signaling Disrupts Memory Functioning Although most of the evidence presented so far indicates that IL-1’s effects on learning and memory processes are detrimental, accumulating recent evidence suggests that at least under some circumstances IL-1 may be required for the normal physiological

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regulation of memory processes within the hippocampus. The first example of IL-1–induced memory facilitation was published by Gibertini in 1998. He reported expedited acquisition of spatial memory in the water maze in mice that were injected with IL-1β (1 μg/ mouse, i.p.). In 2002, we further reported data supporting a beneficial role of IL-1 in spatial memory: Rats were trained to find a hidden platform in a water maze, and then injected with either IL-1β (10 ng/rat, i.c.v.) or IL-1ra (100 μg/rat, i.c.v.) at the end of the training day, and tested for retention 24 hours later (Yirmiya et al., 2002). We showed that whereas IL-1β had no effect on either spatial or visually guided memory in the water maze, IL-1ra specifically impaired spatial memory, as demonstrated by a longer latency to locate the hidden platform on the test day in IL1ra–injected rats. Visually guided performance was unaffected (Yirmiya et al., 2002), suggesting that basal IL-1 levels are specifically required for hippocampaldependent memory. We further illustrated the role of IL-1 in spatial memory by testing mice deficient in IL-1 receptor type I (IL-1rKO). In these mice, IL-1 signaling is completely abolished, as reflected by lack of responsiveness to exogenous IL-1α or IL-1β administration (Labow et al., 1997). IL-1rKO mice displayed a slower rate of learning in the spatial memory paradigm; i.e., their latencies to reach the platform were significantly longer (Avital et al., 2003). However, compared to controls, IL-1rKO mice also displayed a slower swimming speed, which may affect the latency to reach the platform. Nevertheless, these impairments cannot account for the slower rate of learning, because the learning deficit was also observed with respect to the path length to reach the platform, which provides a measurement of learning that is not dependent on speed. In contrast with these findings, in the non-spatial memory paradigm (visible platform) IL-1rKO mice showed no differences from controls in either the latency to reach the platform or the path length (Avital et al., 2003). Similar results were also observed in mice that overexpress the human IL-1ra gene specifically within the CNS (IL-1raTG), which demonstrate no response to exogenous IL-1 administration (Lundkvist et al., 1999). IL-1raTG mice displayed a slower rate of learning in the spatial memory paradigm; i.e., their latencies to reach the platform were significantly longer, although no differences in swimming speed were observed. However, in the non-spatial memory paradigm (visible platform) IL-1raTG mice showed no difference from controls (Goshen et al., 2003a). Taken together, the results of these studies suggest that IL-1 is specifically necessary for spatial learning in the water maze, which depends on normal hippocampal functioning.

346


Another task in which IL-1 was found to be critically involved is the fear-conditioning paradigm: IL-1rKO mice exhibited impaired contextual fear conditioning; i.e., they displayed a significantly shorter freezing time than their controls in the original conditioning context, 48 hours after the conditioning trial. In contrast, IL-1rKO mice displayed normal auditorycued fear conditioning (Avital et al., 2003). Similarly, IL-1raTG mice also displayed impaired contextual, but normal auditory-cued, fear conditioning (Goshen et al., 2003a). Again, the involvement of IL-1 in memory processes seems to be specific to hippocampaldependent performance. The involvement of IL-1 in memory processes was also examined using the passive and active avoidance paradigms: We examined the role of IL-1 in passive avoidance conditioning by injecting rats with either IL-1β (10 ng/rat, i.c.v.) or IL-1ra (100 μg/rat, i.c.v.) at the end of passive avoidance training, in which they were electrified when approaching a drinking spout. At different time-points afterwards, the latency to contact the water spout was measured, as an indicator for memory (longer latencies reflect better memory of the aversive nature of the spout). One day after conditioning, neither IL-1β nor IL-1ra had any effect on memory. However, at 5–8 days after conditioning, IL1β–injected rats exhibited facilitated memory, whereas IL-1ra–injected rats demonstrated impaired memory (Yirmiya et al., 2002). Another study replicated our results using the spatial form of the passive avoidance test. In this paradigm, the latency to enter a context in which an electric shock was administered serves as an indicator of memory (longer latencies reflect better memory). Rats that were i.c.v. injected with IL-1β (15 ng/rat) 50 minutes before the acquisition trial and then 50 minutes before the memory tests (24 and 48 hours later) demonstrated an improved memory of the context (Song et al., 2003). In contrast, Bianchi et al. (1998) reported that very low doses of IL-1α (0.125, 0.25, 0.5 and 1 μg/mouse, i.p.) had no effect on performance in a passive avoidance task. However, IL-1α (0.25 and 0.5 μg/mouse, i.p.) attenuated the amnesic effect of scopolamine (an anti-cholinergic drug) on memory in this test (Bianchi et al., 1998), again suggesting that under some circumstances IL-1 can have a beneficial effect on memory. Brennan et al. (2003) recently showed the importance of IL-1 in active avoidance, using the following paradigm: Rats were warned by a tone that a shock will be delivered 60 seconds afterwards and had a chance to prevent the occurrence of the shock by pressing a lever (avoidance response). Additionally, they could terminate the shock after it had begun by pressing the same lever (escape response). Injection of low

doses of IL-1β (1 and 3, but not 6 μg/kg, i.p.) 24 hours before training resulted in an increased number of avoidance responses, but did not affect the number of escape responses. In an additional study, this finding was replicated, using a 3 μg/kg dose of IL-1β, which improved avoidance, but not escape, responses (Brennan et al., 2004). As mentioned before, the processing of temporal discontinuities (such as the 1minute delay between the appearance of the tone and the electric shock) is performed by the hippocampus (Rawlins et al., 1985; Wallenstein et al., 1998). This learning is required in order to perform the avoidance response, but not the escape response, which is a direct reaction to the present appearance of the shock. To sum up, IL-1β administration, either peripherally or centrally, can improve performance in passive and active avoidance tasks, whereas IL-1ra administration impairs passive avoidance. As with the other paradigms presented above, these effects are restricted to hippocampal-dependent tests. In conclusion, the data presented in this section (summarized in Table 1) clearly demonstrate the role of low, “physiological” levels of IL-1 in memory processes. This role is further supported by the finding of increased IL-1α gene expression 4 hours after exposure to a single acquisition trial of a passive avoidance paradigm, compared to a control group that received only a shock, without performing the step-down action (Depino et al., 2004). The beneficial influence of IL-1 on memory processes was reported in various studies to be specific to hippocampal-dependent memory tasks, whereas the performance in hippocampalindependent tasks does not seem to require IL-1 signaling. These findings were replicated using either pharmacological approaches (over various species, doses, and administration routes), or the genetic approach (using different manipulations), thus strengthening the reliability, validity, and generalizability of these findings. 3. The Inverted U-shaped Model for the Influence of IL-1 on Memory Functioning The results of the studies presented above (summarized in Table 1) indicate that on the one hand elevated IL-1 levels have detrimental effects on memory, but on the other hand low IL-1 doses can facilitate memory and blockade of IL-1 signaling is associated with impaired memory functioning. Together, these findings suggest that the influence of IL-1 on memory follows an inverted U-shape pattern. According to this hypothesis, physiological levels of IL-1 are needed for memory formation; however, any deviation from the physiological range, either by

347

16. The Role of Pro-inflammatory Cytokines in Memory Processes and Neural Plasticity TABLE 1 Cytokine

Species

Summary of the Studies on the Effects of Interleukin-1 (IL-1) on Memory in Rodents

Dose

Administration route

Memory testing paradigm

Timing

Effect on memory

Reference

Experiments reporting detrimental effects of IL-1 on hippocampal-dependent memory IL-1β

Rat

100 ng/rat

i.c.v.

IL-1β

Rat

15 ng/rat

i.c.v.

IL-1β

Mouse

100 ng/mouse

i.p.

IL-1β

Mouse

100 ng/mouse

i.p.

IL-1β

Rat

10 ng/rat

i.c.v.

IL-1β

Rat

20 ng/rat

i.c.v.

IL-1β

Rat

10 ng/side

Intrahippocampal

IL-1β

Rat

1 ng/side

Intrahippocampal

IL-1α

Mouse

i.v.

IL-1α

Mouse

IL-1β IL-1β IL-1β

Mouse Mouse Rat

0.3 or 1 μg/mouse 0.1, 0.3, or 1 ng/side 1 μg/mouse 1 ng/side 32 ng/side

IL-1β

Rat

100 ng/side

Intrahippocampal

IL-1β

Rat

4 μg/rat

IL-1ra

Rat

Unknown

IL-1β

Rat

10 or 50 ng/rat

Water maze spatial Water maze spatial Water maze spatial Water maze spatial 23°C Fear-conditioning contextual Fear-conditioning contextual Fear-conditioning contextual Fear-conditioning contextual

1 hour before training

Impaired

Oitzl et al., 1993

During training

Impaired

Song and Horrobin, 2004

During training

Impaired

2 hours before training

Impaired

Gibertini et al., 1995; Gibertini, 1996 Gibertini, 1998

Impaired

Pugh et al., 1999

Impaired

Pugh et al., 1999

Impaired

Barrientos et al., 2004

Impaired


Active avoidance

Immediately following conditioning Immediately following conditioning Immediately following conditioning Immediately, 3 hours and 24 hours following preexposure to context Immediately after training

Impaired

Banks et al., 2001

Intraseptal

Active avoidance

Immediately after training

Impaired

Banks et al., 2001

i.v. Intraseptal Intrahippocampal

Immediately after training Immediately after training 10 minutes before training

Impaired Impaired Impaired

Banks et al., 2001 Banks et al., 2001 Matsumoto et al., 2001

10 minutes before training

Impaired

Matsumoto et al., 2001, 2004

i.p.

Active avoidance Active avoidance Three-panel runway Three-panel runway Autoshaping

Impaired

Aubert et al., 1995

Intrahippocampal adenovector i.c.v.

Passive avoidance, step down Radial arm maze

45 minutes before day 2 of training 1 week before training

Enhanced

Depino et al., 2004

45 minutes before testing (5 minutes after training)

Impaired

Song et al., 2004

No effect

Oitzl et al., 1993

No effect

Gibertini, 1998

No effect

Lacosta et al., 1999

No effect

Yirmiya et al., 2002

48 hours following preexposure to context Immediately after training

No effect


No effect

Banks et al., 2001

Immediately after training 24 hours before training 10 minutes before training

No effect No effect No effect

Banks et al., 2001 Brennan et al., 2003 Matsumoto et al., 2001

15 minutes before acquisition

No effect

Bianchi et al., 1998

2 hours before training

Enhanced

Gibertini, 1998

Immediately after training 50 minutes before acquisition and 50 minutes before test

Enhanced Enhanced

Yirmiya et al., 2002 Song et al., 2003

Experiments reporting no effects of IL-1β administration on hippocampal-dependent memory IL-1β

Rat

100 ng/rat

i.c.v.

IL-1β

Mouse

100 ng/mouse

i.p.

IL-1β

Mouse

1 μg/mouse

i.p.

IL-1β

Rat

10 ng/rat

i.c.v.

IL-1β

Rat

1 ng/side

Intrahippocampal

IL-1α

Mouse

i.v.

IL-1α IL-1β IL-1β

Mouse Rat Rat

0.03 or 0.1 μg/mouse 0.03 ng/side 6 μg/kg 10 ng/side

IL-1α

Mouse

0.125, 0.25, 0.5, or 1 μg/mouse

Intraseptal i.p. Intrahippocampal i.p.

Water maze spatial Water maze spatial 15°C Water maze spatial

Water maze spatial Fear-conditioning contextual Active avoidance Active avoidance Active avoidance Three-panel runway Passive avoidance

Immediately before training 2 hours before training Daily injections at the week preceding training, and at the week of training itself Immediately after training

Experiments reporting beneficial effects of IL-1β administration on hippocampal-dependent memory IL-1β

Mouse

1 μg/mouse

i.p.

IL-1β IL-1β

Rat Rat

10 ng/rat 15 ng/rat

i.c.v. i.c.v.

Water maze spatial Passive avoidance Passive avoidance

(Continues)

348

II. Immune System Effects on Neural and Endocrine Processes and Behavior TABLE 1 (Continued)

Cytokine

Species

IL-1β IL-1β

Rat Rat

Dose 1 μg/kg 3 μg/kg

Administration route i.p. i.p.


Timing

Effect on memory

Active avoidance Active avoidance

24 hours before training 24 hours before training

Enhanced Enhanced

Brennan et al., 2003 Brennan et al., 2003; Brennan et al., 2004

Impaired Impaired Impaired Impaired

Yirmiya et al., 2002 Yirmiya et al., 2002 Avital et al., 2003 Avital et al., 2003

Reference

Experiments reporting detrimental effects of IL-1 signaling blockade on hippocampal-dependent memory IL-1ra IL-1ra IL-1R IL-1R

Rat Rat Mouse Mouse

100 μg/rat 100 μg/rat No IL-1R1 No IL-1R1

i.c.v. i.c.v. Knockout Knockout

Water maze spatial Passive avoidance Water maze spatial Fear-conditioning contextual

Immediately after training Immediately after training Constant knockout Constant knockout

Experiments reporting no effect of IL-1 administration or IL-1 signaling blockade on hippocampal-independent memory IL-1β

Mouse

100 ng/mouse

i.p.

IL-1β

Rat

i.c.v.

IL-1β

Rat

10 or 20 ng/rat 10 ng/rat

i.c.v.

IL-1ra

Rat

100 μg/rat

i.c.v.

IL-1β

Rat

15 ng/rat

i.c.v.

IL-1R

Mouse

No IL-1R1

Knockout

IL-1R

Mouse

No IL-1R1

Knockout

Water maze fixed entry Fear-conditioning auditory-cued Water maze Visually guided Water maze Visually guided Water maze Visually cued Water maze Visually guided Fear-conditioning auditory-cued

During training

No effect

Gibertini, 1996

Immediately following conditioning Immediately after training

No effect

Pugh et al., 1999

No effect


Immediately after training

No effect


During training

No effect

Song and Horrobin, 2004

Constant knockout

No effect

Avital et al., 2003

Constant knockout

No effect

Avital et al., 2003

i.p.—Intraperitoneal; i.v.—Intravenous; i.c.v.—Intracerebroventricular.

excess elevation in IL-1 levels or by blockade of IL-1 signaling, results in impaired memory (Figure 1). This hypothetical model is based on: (A) The beneficial role of low-dose IL-1 administration on hippocampal-memory formation; (B) the detrimental effect of elevated IL-1 levels (induced by either exogenously administered IL-1 or by enhanced endogenous release of IL-1, stimulated by exposure to pathogens or stress) on hippocampal-dependent memory; and (C) the fact that blockade of IL-1 signaling either pharmacologically (by IL-1ra administration) or genetically (by deletion of the IL-1 receptor or overexpression of IL-1ra) impairs hippocampal-dependent memory. An inverted U-shape curve has been demonstrated for the effects of IL-1 on other processes, e.g., on the differentiation of mesencephalic progenitor cells into dopaminergic neurons (Ling et al., 1998), and for the influence of other substances on memory. For example, low doses of corticosterone are essential, whereas high doses are detrimental for spatial memory (Conrad et al., 1999). One weakness of this hypothesis is that, presently, no single study systematically assessed the dual role of IL-1 in memory under identical conditions. Rather, the hypothesis is based on data gathered by many groups, using various testing paradigms, experimental approaches (pharmacological or genetic),

species, doses, and administration routes. Future research should further substantiate the inverted Ushaped curve hypothesis by testing the detrimental effects on memory of both excess and absence of IL-1 signaling under similar experimental conditions.

B. IL-6 The effects of IL-6 on learning and memory have been studied by several approaches, including acute administration of IL-6, manipulations that produce chronic elevation of IL-6 levels, and testing the effects of IL-6 blockade. Many of these studies were conducted in the context of aging, because several studies demonstrated an age-dependent increase in brain IL-6 levels in mice (Prechel et al., 1996; Ye and Johnson, 1999, 2001), and a similar increase was also observed in human plasma (Wei et al., 1992). Additionally, IL-6 expression is higher in senescence-accelerated mice at the age of 10 months compared to controls (Tha et al., 2000). Because aging is accompanied by cognitive deterioration, these findings led to the hypothesis that IL-6 may mediate age-related memory impairments (e.g., Godbout and Johnson, 2004). Based on this hypothesis, the role of IL-6 in memory processes has been extensively investigated with an emphasis on


349

B

0.6 0.4 0.2

t) /r a

t) ng 00 (1 -1 IL

-1

(1 ra

(1

00

0

m

ng

g/

/r a

ra

e ic m O IL

-1

IL

Brain IL-1 Level

t)

0.0

High rK

Basal -normal

0.8

-1

Low

1.0

IL

Latency to reach platform (Control group/Treated group)

Memory / Plasticity Level

A

FIGURE 1 The inverted U-shape model for the effects of IL-1 on memory functioning and neural plasticity. (A) According to this model, normal basal levels of IL-1, particularly within the hippocampus, promote memory consolidation and neural plasticity processes, whereas deviations from the physiological range, either by blockade of IL-1 signaling (induced by pharmacological or genetic manipulations) or excessive elevation in IL-1 levels (induced by infectious, autoimmune, or neurodegenerative diseases, trauma, stroke, or exposure to stress), impair memory and neural plasticity. (B) An example for experimental findings supporting the inverted U-shape model. The graph represents the results of several studies, examining the effects of manipulations in IL-1 signaling on spatial (hippocampal-dependent) learning and memory in the water maze. Each bar represents the latency to reach the platform, computed as the ratio between the latency of the control animals in that experiment to the latency of the manipulated animals. A low ratio means that control animals reached the platform faster; i.e., the manipulated animals displayed impaired memory compared to the controls. Specifically, animals with impaired IL-1 signaling (left two columns), induced by either a genetic manipulation (deletion of the IL-1 receptor type 1 in mice) or pharmacological manipulation (i.c.v. administration of IL-1ra [100 mg/kg] in rats), displayed impaired learning and memory (adopted from Avital et al., 2003 and Yirmiya et al., 2002, respectively). Animals injected with a relatively low dose of IL-1 (10 ng/rat, i.c.v.) displayed no impairment in spatial memory (adopted from Yirmiya et al., 2002), whereas animals injected with a high dose of IL-1 (100 ng/rat, i.c.v.) displayed reduced memory functioning (adopted from Oitzl et al., 1993).

progressive age-related processes. Whereas several studies found no effect of IL-6 on memory, all the studies that did find an involvement of IL-6 in learning and memory demonstrated a detrimental role, either by showing that excessive IL-6 levels impair memory, or by reporting improved memory when IL-6 is absent or when its signaling is blocked. The first study examining the role of IL-6 in memory processes was conducted in 1993 by Oitzl et al. In this study, i.c.v. injection of IL-6 (100 ng/rat), either immediately or 1 hour before the beginning of a 2-day spatial water maze training, had no effect on memory at any time-point, whereas IL-1β produced a memory impairment when it was administered 1 hour before training under similar conditions (Oitzl et al., 1993). Two other studies, using different testing paradigms, also reported that exogenous IL-6 administration did not influence hippocampal-dependent memory processes: Bianchi et al. (1997, 1998) reported that low doses of IL-6 (0.031, 0.125, 0.5, and 2 μg/mouse, i.p.), injected

15 minutes before passive avoidance acquisition had no effect on performance in that task. Furthermore, Brennan et al. (2004) reported no effect of IL-6 (3 μg/ kg, i.p.) when it was injected 24 hours before active avoidance training. The age-dependent involvement of IL-6 in memory processes was then extensively examined in mice that overexpress IL-6 using the active avoidance paradigm. The animals had a chance to prevent the occurrence of a shock by quickly entering a specific arm of a Y maze (avoidance response). Additionally, they could terminate the shock after it had begun by entering this arm (escape response). Heyser et al. (1997) employed this paradigm to test the memory of either homozygous or heterozygous transgenic mice overexpressing IL-6 specifically within the CNS (IL-6TG), at the ages of 3, 6, and 12 months. At 3 months of age, homozygous IL-6TG mice demonstrated impaired learning, as shown by a decreased number of avoidance responses, whereas heterozygous IL-6TG mice were able to learn

350


the avoidance task as well as control mice. At the age of 6 months, the same mice were re-tested, and once again homozygous IL-6TG mice demonstrated impaired learning, which worsened compared to their performance 3 months earlier. However, at that age, heterozygous IL-6TG mice also exhibited impaired memory, intermediate to that of the control and homozygous IL-6TG mice. By 12 months of age, the performance of both homozygous and heterozygous IL-6TG mice had declined further and became indistinguishable (Heyser et al., 1997). A similar progression was also found in the number of errors, first affecting homozygous IL-6TG mice (at 6 months of age), and later heterozygous IL-6TG mice as well (at 12 months of age). No differences in the latency of escape responses were observed at any time-point, suggesting no differences in the motivational properties of the shock (Heyser et al., 1997). Together, these findings imply progressive age-related deficits in spatial avoidance learning. Although the effects of IL-6 overproduction support a role for IL-6 in learning and memory, these findings should be interpreted in the context of other effects of IL-6 overproduction, particularly neurodegeneration and gliosis. However, a detrimental role of IL-6 in memory processes was also demonstrated using another approach, i.e., examination of the effects of IL-6 blockade on memory functioning. Braida et al. (2004) tested the performance of IL-6 knockout mice (IL-6KO), using different memory tests: In a passive avoidance task, in which the mice had to remember to avoid a chamber in which they were previously electrified, the performance of 4-month-old IL-6KO mice was similar to that of controls. However, IL-6KO mice were less susceptible to scopolamineinduced amnesia in this task. Furthermore, when IL6KO mice were tested in a more complex spatial task, the radial arm maze, their performance was found to be improved compared to age-matched WT controls; i.e., the percent of IL-6KO mice that fully acquired the task was higher compared to WT controls at both 4 and 12 months of age. This finding was also reflected in a smaller number of errors performed by IL-6KO mice at both 4 and 12 months of age. This increased precision can be explained by the fact that IL-6KO mice employed a more systematic exploration strategy, as shown by the finding that they demonstrated a higher tendency to visit neighboring arms. This difference in the pattern of entries was especially apparent at 12 months of age (Braida et al., 2004). Furthermore, Balschun et al. (2004) reported that even acute blockade of IL-6 signaling can enhance memory formation, using the forced alternation task. In this paradigm, rats are placed in complete darkness in a Y maze, at a different location in any trial, and have to alternately

choose the right or left arm in relation to the arm they were placed in. The forced alternation task is hippocampal dependent, as hippocampal lesions in rats prevent its acquisition (Aggleton et al., 1986). I.c.v. administration of neutralizing anti–IL-6 antibodies 90 minutes after acquisition resulted in enhanced retention 24 hours later. This was demonstrated by a lower percent of errors performed by anti–IL-6 injected rats, compared to controls (Balschun et al., 2004). The findings of improved performance in different memory tasks following blockade of IL-6 signaling, either by deletion of the IL-6 gene or by IL-6 immunoneutralization, suggest that IL-6 may have a physiological role in the inhibition of memory formation. Although the data on IL-6 overexpression and IL-6 blockade suggest a detrimental influence of IL-6 on memory formation, several studies demonstrated a protective role of IL-6 against memory impairments caused by either scopolamine or ischemia: In contrast to the finding of decreased susceptibility of IL-6KO mice to scopolamine-induced amnesia in the passive avoidance task (Braida et al., 2004), Binachi et al. (1997, 1998) reported that i.p. administration of IL-6 (0.125 and 0.5, but not 0.031 and 2 μg/mouse) partially blocked the effect of scopolamine in the same task. Moreover, chronic administration of IL-6 (450 but not 45 ng/day, via subcutaneous osmotic minipumps for a week), starting 2 hours before ischemia, resulted in improved memory in a passive avoidance task at the end of this week (Matsuda et al., 1996). It may be suggested that when brain homeostasis is maintained, IL6 has a detrimental effect on memory processes. However, when this balance is violated, IL-6 plays a protective role. This role is not necessarily directly connected to memory formation per se, but rather to the effort of recovering homeostasis. To sum up, the data presented above (summarized in Table 2) suggest a detrimental influence of IL-6 on memory processes, which is potentiated as the animals become older. This age-dependent effect is demonstrated by showing progressive memory deterioration in mice that overexpress IL-6, as well as a gradual memory improvement in mice lacking IL-6. However, the role of IL-6 in memory is quite complex, as acute administration of IL-6 did not produce any effect on memory in several studies, and in some situations IL-6 was found to have a protective role.

C. TNF-a The involvement of TNF-α in memory processes has been studied by several research groups, using various models. Most studies reported no involvement of TNF-α in memory functioning. However, a few

351

16. The Role of Pro-inflammatory Cytokines in Memory Processes and Neural Plasticity TABLE 2

Cytokine

Species

Summary of the Studies on the Effects of Interleukin-6 (IL-6) on Memory in Rodents

Dose

Effects of IL-6 administration IL-6 Rat 100 ng/rat

Administration route

i.c.v.


Hippocampal involvement

IL-6

Mouse

Unknown, homozygous

CNS-specific IL-6 transgene

Water maze spatial Active avoidance

Dependent

IL-6

Mouse

Unknown, heterozygous


Active avoidance

Dependent

IL-6

Mouse

Unknown, homozygous


Active avoidance

Dependent

IL-6

Mouse

Unknown, heterozygous


Active avoidance

Dependent

IL-6

Rat

3 μg/kg

i.p.

Dependent

IL-6

Mouse

0.031, 0.125, 0.5 or 2 μg/mouse

i.p.

Active avoidance Passive avoidance

Dependent

Dependent

Effects of IL-6 signaling blockade IL-6 Mouse No IL-6

Knockout

Radial arm maze

Dependent

IL-6

Mouse

No IL-6

Knockout

Passive avoidance

Dependent

Anti-IL-6

Rat

—

i.c.v.

Forced alternation

Dependent

Timing

Effect on memory

Reference

1 hour before training 3 months of constant transgenic overexpression 3 months of constant transgenic overexpression 6 or 12 months of constant transgenic overexpression 6 or 12 months of constant transgenic overexpression 24 hours before training 15 minutes before acquisition

No effect

4 or 12 months of constant knockout 4 months of constant knockout Immediately following acquisition

Enhanced

Braida et al., 2004

No effect

Braida et al., 2004

Enhanced

Balschun et al., 2004

Impaired

Oitzl et al., 1993 Heyser et al., 1997

No effect

Heyser et al., 1997

Impaired

Heyser et al., 1997

Impaired

Heyser et al., 1997

No effect

Brennan et al., 2004 Bianchi et al., 1997; Bianchi et al., 1998

No effect

i.p.—Intraperitoneal; i.c.v.—Intracerebroventricular

studies demonstrated a detrimental effect of TNF-α on memory formation, and one study reported a beneficial role for TNF-α. These contradictory results will be presented, along with possible explanations for the discrepancies among them. The effect of TNF-α on memory was first investigated by Fiore et al. (1996), by testing the performance of adult mice (60 days old) that overexpress TNF-α within the CNS (TNFαTG). These mice were tested in the passive avoidance paradigm, in which they had to remember to avoid a chamber in which they were electrified (thus hesitating for a longer time before re-entering this chamber). TNFαTG mice exhibited

delayed acquisition of this task, reflected by the fact that they required a larger number of trials to reach the learning criterion and demonstrated lower latency in the first five trials. Bjugstad et al. (1998) explored the role of TNF-α in spatial memory by daily injecting rats with TNF-α (50 ng/rat, i.c.v.) for a week before water maze training. On day 8, the injections were terminated and a week-long water maze training started. TNF-α–injected rats demonstrated impaired spatial memory, reflected by longer escape latencies during water maze training, and no preference to the quadrant of the pool in which the platform was formerly positioned, in the probe trial (Bjugstad et al., 1998).

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A negative effect of TNF-α has also been reported following central administration (Matsumoto et al., 2002). In this study, the effect of TNF-α on working memory was examined using the three-panel runway task, a hippocampal-dependent task in which rodents learn to open the correct doors between three consecutive chambers in order to receive a reward (Furuya et al., 1988; Kitajima et al., 1992; Ohno et al., 1992). Intrahippocampal TNF-α (560 but not 10, 32, 100, or 320 ng/side) impaired working memory in this task, reflected by an increased number of errors and longer latencies to perform the task on days 1 and 2 postinjection (Matsumoto et al., 2002). Recently, Golan et al. (2004) further demonstrated the role of TNF-α in spatial memory by testing mice with targeted deletion of the TNF-α gene (TNFαKO) in the water maze. These mice exhibited enhanced spatial memory, reflected by their faster learning of the platform position and their better performance in the probe test compared to WT controls. However, TNFαKO mice demonstrated performance that was identical to WT controls in the hippocampalindependent, visually guided version of the water maze. Similarly, we recently found that mice lacking the TNF-R1 (TNF-R1KO) (Pfeffer et al., 1993) demonstrated enhanced contextual fear conditioning compared to their WT controls, as shown by increased freezing in this hippocampal-dependent task. These mice were similar to WT controls in the performance of auditory-cued fear conditioning, which does not require the hippocampus (Goshen and Yirmiya, unpublished observations). The results presented above suggest a detrimental role for TNF-α in memory processes. This role is independent of the species used or the memory-testing paradigm employed. However, it is restricted to the performance of tasks that depend on normal hippocampal functioning, such as the spatial version of the water maze and contextual fear conditioning, whereas hippocampal-independent tasks are spared. Additionally, the detrimental effect of TNF-α on memory seems to be dose dependent, as demonstrated in the study on intrahippocampal TNF-α administration (Matsumoto et al., 2002). In contrast with these findings, the studies presented in the following paragraphs reported no influence of either excess or absence of TNF-α expression on memory functioning. In two related studies, the influence of elevated TNFα levels on memory was studied using two different transgenic strains that overexpress TNF-α specifically within the CNS: TG6074 mice, with glial overexpression of the murine TNF-α gene, which display inflammatory demyelination and neurological abnormalities (Probert et al., 1995); and TGK3, with neuronal overexpression

of the uncleavable mutant human TNF-α gene, which display no neurological symptoms (Probert et al., 1997). Both of these transgenic strains had longer escape latencies compared to their WT controls at the age of 30 days, suggesting TNF-α–induced memory impairment (Aloe et al., 1999b; Fiore et al., 2000). However, compared to controls, these mice also displayed a slower swimming speed, which may affect the latency to reach the platform. Furthermore, the learning deficit was not observed with respect to the path length to reach the platform, which provides a measurement of learning that is not dependent on speed. Moreover, no difference was found in the preference of the quadrant in which the platform was positioned in the probe test, another memory parameter that is not influenced by swimming speed (Aloe et al., 1999b; Fiore et al., 2000). Together, these findings suggest no effect of excessive brain TNFα on spatial memory. The discrepancy between the results of these studies and the report of impaired spatial memory in TNFαTG6074 mice (Fiore et al., 1996) may be explained by differences in the age of the subjects. Because aging is accompanied by cognitive deterioration, and brain TNF-α expression is increased with age (Casolini et al., 2002; Sharman et al., 2002), it can be postulated that TNF-α may be involved in aging-related memory loss. Indeed, impaired spatial memory was observed in adult (60-day-old) mice, but not in young (30-day-old) mice. Fiore et al. (2000) also reported no difference between TNFαTG6074 and control mice in passive avoidance response in juvenile (15-day-old) mice. Thus, the detrimental effect of TNF-α transgenic overexpression in the brain on memory processes seems to be age dependent. Several studies report no effect of TNF-α deficiency on spatial memory. Both Scherbel et al. (1999) and Gerber et al. (2004), using two different lines of TNFαKO mice, reported normal performance in the water maze paradigm, compared to that of WT controls. We found similar results using TNF-R1KO mice, which demonstrated a similar performance to their WT controls in the water maze (Goshen and Yirmiya, unpublished observation). Nevertheless, TNFαKO mice were less susceptible to the memory impairment caused by brain injury, which was inflicted by controlled cortical impact (Scherbel et al., 1999), suggesting that in WT mice TNFα participates in the processes underlying this damage. Yamada et al. (2000) employed another spatial test, the water finding task, to examine the influence of TNF-α deficiency: In this task, mice are allowed to explore an open field, in which an alcove with a water spout is placed. The mice are then re-introduced to this context when they are thirsty, and the latency to find the water spout serves as an index to their spatial memory.


TNFαKO mice performed this task as well as their WT controls (Yamada et al., 2000). A single study reported a beneficial effect of TNF-α administration on memory, using a passive avoidance task, in which rats could prevent the occurrence of a shock by pressing a lever (avoidance response) or terminate the shock after it had begun by pressing the lever (escape response). Brennan et al. (2004) reported that a single injection of TNF-α (6 μg/kg i.p.) 24 hours before training resulted in increased number of avoidance as well as escape responses. TNF-α also seems to have a positive effect on the recovery of memory functioning following infection: Gerber et al. (2004) reported that after surviving pneumococcal meningitis, TNFαKO mice demonstrated impaired water maze performance compared to surviving WT controls, suggesting a beneficial role for TNF-α in recovery. It may be suggested that, similar to IL-6, when brain homeostasis is maintained, TNF-α has a detrimental effect on memory processes, and when this balance is violated, TNF-α may play a protective role. To sum up, the data gathered so far (summarized in Table 3) do not provide definite conclusions regarding the role of TNF-α in memory processes. However, it can still be suggested that (A) basal levels of TNF-α are not required for memory, as TNF-α–deficient mice demonstrate no memory impairments, and one paper even reports improved memory in these mice; and (B) the negative influence of TNF-α appears to be both dose and age dependent. Further studies should clarify the nature of the involvement of TNF-α in learning and memory processes, both under basal conditions and following various inflammatory and noninflammatory challenges.

III. MECHANISMS UNDERLYING THE EFFECTS OF PRO-INFLAMMATORY CYTOKINES ON LEARNING AND MEMORY A. Neural Plasticity 1. Effects of IL-1 on Neural Plasticity Extensive research was performed to study the involvement of IL-1 in hippocampal plasticity (reviewed in Lynch, 2002; O’Connor and Coogan, 1999). Similar to the findings concerning the role of IL-1 in memory processes, this research also yielded seemingly contradictory results, as many studies demonstrated a detrimental effect of IL-1 on hippocampal plasticity, whereas other studies reported a beneficial role for IL-1 in normal hippocampal plasticity. These results will be presented together with an attempt to

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accommodate them into the inverted U-curve theoretical model, which was proposed for the involvement of IL-1 in memory processes. Detrimental Effects of IL-1 on Neural Plasticity Katsuki et al. (1990) were the first to examine the consequence of IL-1β application on LTP. They reported that IL-1β inhibited LTP in the CA3 region of mouse hippocampal slices in vitro. Additional studies then reported similar findings of IL-1–induced LTP inhibition in the rat CA1 area of the hippocampus (Bellinger et al., 1993) and the DG (Cunningham et al., 1996). This detrimental effect of IL-1 was accomplished by the inhibition of both N-methyl-D-aspartate (NMDA)-mediated and NMDA-independent synaptic potentiation (Coogan and O’Connor, 1997, 1999). Murray and Lynch (1998) reported that LTP maintenance is negatively correlated with IL-1 hippocampal levels; i.e., increased IL-1β levels in the hippocampus (caused by aging, stress, or exogenous application) are accompanied by reduced LTP maintenance. Additional studies shed light on the signal transduction mechanisms that underlie IL-1–induced LTP inhibition. One major mechanism that is affected by IL-1 is LTP-associated increase in glutamate release in the DG (Canevari et al., 1994), evidenced by several studies which demonstrated that IL-1β application resulted in inhibited KCl-induced glutamate release in the hippocampus (Kelly et al., 2003; O’Donnell et al., 2000; Vereker et al., 2000, 2001). A few studies showed that the inhibitory effect of IL-1 on LTP is accompanied by increased activity of stress-activated protein kinases. Concomitantly with LTP inhibition, i.c.v. IL-1β increased p38 mitogen-activated protein (MAP) kinase activity in DG synaptosomes (Kelly et al., 2003; Vereker et al., 2000). Co-administration of IL-1 together with the p38 inhibitor SB203580 attenuated the detrimental effect of IL-1 on LTP both in vivo (Kelly et al., 2003) and in vitro (Coogan et al., 1999), and restored KCl-induced glutamate release in IL-1–treated rats (Kelly at al., 2003). IL1β also increased NFκB activation in the hippocampus, and co-administration of the NFκB inhibitor SN50 attenuated the detrimental effect of IL-1 on LTP and restored KCl-induced glutamate release (Kelly et al., 2003). Similarly, IL-1 increased the activity of c-Jun N-terminal kinase (JNK) (Curran et al., 2003; Vereker et al., 2000), and the JNK inhibitor SP600125 blocked the inhibitory effect of IL-1 on LTP (Curran et al., 2003). Finally, IL-1β also enhanced the production of reactive oxygen species (ROS) (Vereker et al., 2000, 2001), and its inhibitory effect on LTP was blocked by application of the antioxidant phenylarsine oxide (Vereker et al., 2001) and even by a diet enriched in the antioxidant vitamins A and C (Vereker et al. 2000).

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II. Immune System Effects on Neural and Endocrine Processes and Behavior TABLE 3

Cytokine Species

Dose

Effects of TNF-α administration TNF-α Rat 50 ng/rat/ day

TNF-α

Summary of the Studies on the Effects of TNF-α on Memory in Rodents Administration route

Water maze spatial

Dependent

Unknown

Glia-specific overexpression

Water maze spatial

Dependent

Unknown

Neuron-specific overexpression

Water maze spatial

Unknown


Unknown

Effect on memory

Reference

Bjugstad et al., 1998

Dependent

Constant No effect overexpression *

Aloe et al., 1999; Fiore et al., 2000

Passive avoidance

Dependent

Constant Impaired overexpression

Fiore et al., 1996


Passive avoidance

Dependent

Constant No effect overexpression

Fiore et al., 2000

Three-panel runway Three-panel runway Active avoidance

Dependent

24 hours before training 24 hours before training 24 hours before training

Matsumoto et al., 2002 Matsumoto et al., 2002 Brennan et al., 2004

TNF-α

Rat

10, 32, 100, or Intrahippocampal 320 ng/side 560 ng/side Intrahippocampal

TNF-α

Rat

6 μg/kg

TNF-α

Timing

Daily injections Impaired at the week preceding training Constant No effect overexpression *

TNF-α

TNF-α

Hippocampal involvement

i.c.v.

Mouse 30 days old Mouse 30 days old Mouse 60 days old Mouse 15 days old Rat

TNF-α


i.p.

Effects of TNF-α signaling blockade TNF-α Mouse No TNFα Knockout TNF-α

Mouse

No TNFα

Knockout

TNF-α

Mouse

No TNFα

Knockout

TNF-α

Mouse

No TNFα

Knockout

TNF-α

Mouse

No TNFα

Knockout

Water maze spatial Water finding test Water maze spatial Water maze spatial Water maze Visually guided

Dependent Dependent

Dependent

Constant knockout Dependent Constant knockout Dependent Constant knockout Dependent Constant knockout Independent Constant knockout

No effect Impaired Enhanced

Aloe et al., 1999; Fiore et al., 2000

Enhanced

Scherbel et al., 1999 Yamada et al., 2000 Gerber et al., 2004 Golan et al., 2004

No effect

Golan et al., 2004

No effect No effect No effect

i.p.—Intraperitoneal; i.c.v.—Intracerebroventricular; *The transgenic mice had longer escape latencies, suggesting memory impairment. However, compared to controls, these mice also displayed a slower swimming speed, which may affect the latency to reach the platform. Furthermore, the learning deficit was not observed with respect to the path length to reach the platform, which provides a measurement of learning that is not dependent on speed, and no differences in preference for the quadrant in which the platform was positioned was found in the probe test.

In addition to its effects on LTP, IL-1 was also reported to affect basal synaptic activity: Bellinger et al. (1993) reported reduced postsynaptic potentials in the CA1 region of hippocampal slices, and Ikegaya et al. (2003) showed that incubation of hippocampal slices with IL-1β decreased basal CA1 synaptic transmission during that time, an effect that lasted for 30 minutes after IL-1 was washed out. This durable effect was dependent on increased GABA levels, as in the presence of the GABA receptor antagonist bicuculin, IL-1 decreased synaptic

transmission only when it was present in the experimental system, but not after it was washed out (Ikegaya et al., 2003). To sum up, the data presented above clearly demonstrate a detrimental effect of elevated IL-1 levels on hippocampal LTP. This negative influence was replicated in various studies, both in vivo and in vitro, using different doses of exogenously applied IL-1β, as well as endogenous IL-1β elevation caused by aging and stress. These adverse effects are mediated by stressactivated protein kinases and ROS.


Beneficial Effects of IL-1 on Neural Plasticity In contrast to the data presented so far on the detrimental effect of increased IL-1 levels on LTP, recent studies suggest that IL-1 may actually be required for the normal regulation of hippocampal plasticity. The first evidence for the physiological involvement of IL-1 in plasticity was provided by Schneider et al. (1998), who reported that IL-1β gene expression is substantially increased during LTP, starting 15 minutes after stimulation in hippocampal slices and 8 hours after LTP induction in freely moving rats (Balschun et al., 2003; Schneider et al., 1998). Furthermore, the in vivo increase in IL-1β gene expression was long lasting, specific to potentiation (i.e., was observed only in the stimulated, but not contralateral, hippocampus) and could be prevented by blockade of potentiation with the NMDA receptor antagonist, (+/−)-2-amino-5phosphonopentanoic acid (AP-5) (Schneider et al., 1998). The elevation in IL-1β gene expression appears to have a physiological role in the maintenance of LTP, as blocking IL-1 receptors by i.c.v. administration of IL-1ra 90 minutes after the induction of LTP impaired its maintenance, whereas when IL-1ra was applied 30 minutes before or immediately after stimulation, it had no effect (Schneider et al., 1998). However, Loscher et al. (2003) reported that i.c.v. administration of IL-1ra 30 minutes before stimulation resulted in a smaller initial potentiation and no maintenance of LTP. The critical role of IL-1 in LTP was also demonstrated in hippocampal slices in vitro: Application of IL-1ra 30 minutes after the induction of LTP in the rat dentate gyrus in vitro reduced synaptic activity back to baseline levels (Coogan et al., 1999), and when IL-1ra was applied to hippocampal sliced in a physiological temperature for 40 minutes before stimulation, the initial small increase in synaptic activity subsided within 30 minutes (Ross et al., 2003). Loscher et al. (2003) investigated the mechanisms that underlie the detrimental effect of IL-1ra on LTP. They reported decreased glutamate release and increased JNK phosphorylation in hippocampal synaptosomes following exposure to IL-1ra. Interestingly, these effects are not mediated by the IL-1 receptor type I (IL-1RI), as they appear both in the presence of neutralizing antibodies against IL-1RI, and in synaptosomes prepared from IL-1rKO mice (Loscher et al., 2003). Lately, we further demonstrated the necessity of physiological IL-1 levels in hippocampal plasticity by reporting no potentiation following high-frequency stimulation in IL-1rKO mice and a complete absence of LTP in hippocampal slices prepared from these mice (Avital et al., 2003). Moreover, we reported, for

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the first time, an involvement of IL-1 in short-term plasticity as well, by showing enhanced paired-pulse inhibition in the DG of IL-1rKO mice in vivo, and decreased paired-pulse potentiation in vitro in hippocampal slices from IL-1rKO mice (Avital et al., 2003). It should be noted, however, that Ikegaya et al. (2003) reported normal LTP and LTD in IL-1α/βKO mice. To summarize, the data presented in the previous paragraphs demonstrate the requirement of physiological IL-1 levels in hippocampal LTP, both in vivo and in vitro. The importance of IL-1 in LTP is further supported by the findings of increased IL-1 gene expression following LTP. A Theoretical Model for the Effects of IL-1 on Neural Plasticity The data presented above suggest that, similar to the involvement of IL-1 in memory processes, an inverted U-curve pattern exists for its influence on hippocampal plasticity as well (Figure 1). Thus, whereas some studies report a detrimental effect of elevated IL-1 levels on LTP, others report that LTP is impaired when IL-1 signaling is blocked. Whereas the hypothesis of the dual role of IL-1 in memory is based on different results that were obtained under various conditions, Loscher et al. (2003) provide data on the effect of both IL-1 and IL-1ra on LTP under identical conditions. They report that i.c.v. administration of either IL-1β or IL-1ra 30 minutes before tetanic stimulation resulted in a decreased potentiation that did not sustain (Loscher et al., 2003). The combination of the data presented above suggests that physiological levels of IL-1 are needed for synaptic plasticity, and that any deviation from the physiological range, either by elevation in IL-1 levels or by blockade of IL-1 signaling, results in impaired hippocampal plasticity. This is postulated based on (A) the LTP-induced increase in hippocampal IL-1 gene expression; (B) the detrimental effect of elevated IL-1 levels (induced by exogenous application, stress, or aging) on hippocampal LTP both in vivo and in vitro; and (C) the fact that blockade of IL-1 signaling either pharmacologically by IL-1ra or genetically by deletion of the IL-1 receptor impairs synaptic plasticity both in vitro and in vivo. This model predicts that coapplication of IL-1 and IL-1ra, which both impair LTP independently, will not result in accumulated LTP impairment, but rather in a reduced inhibitory effect. Indeed, when IL-1β and IL-1ra are applied concomitantly, their detrimental effect on LTP is attenuated both in vivo (Loscher et al., 2003) and in vitro (Ross et al., 2003).

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Disruption in the balance of hippocampal IL-1 activity may be associated with the findings of agedependent decrease in LTP (Lynch, 1998a, b; O’Donnell et al., 2000). Aging is accompanied by an increase in basal hippocampal IL-1β levels together with reduced LTP (Murray and Lynch, 1998; O’Donnell et al., 2000). Furthermore, aged rats display a similar phenomenon to rats treated with IL-1; i.e., they display decreased KCl-induced glutamate release, increased activation of p38 and JNK, and increased ROS production (Murray and Lynch, 1998; O’Donnell et al., 2000). It should be noted, however, that these findings provide only circumstantial evidence, and no data that directly demonstrate a role for IL-1 in aging are available. Aging is also associated with a diminished increase in LTP-induced IL-1β gene expression. IL-1β expression is increased by 10-fold in young rats during LTP, but only by two-fold in mid-aged rats (Balschun et al., 2003). Together these findings suggest that deviations in either direction from the fine balance of hippocampal IL-1 levels may lead to LTP maintenance impairments in aging. 2. Effects of IL-6 on Neural Plasticity Corresponding to the reports of IL-6–induced memory loss, this cytokine was also found to impair hippocampal plasticity. Bellinger et al. (1995) were the first to demonstrate the detrimental effect of IL-6 on hippocampal plasticity in vitro by reporting reduced LTP in the DG of hippocampal slices prepared from transgenic mice with cerebral overexpression of IL-6. Li et al. (1997) further investigated the dose-dependent effect of IL-6 on the different phases of LTP in vitro by using four different concentrations of IL-6 (50, 200, 500, 2000 U/ml). One hour after tetanic stimulation, LTP in the CA1 region was reduced by all four concentrations. However, 10–15 minutes following stimulation only the three highest concentrations caused a decrease in potentiation, and only the two highest concentrations impaired even the initial post-tetanic potentiation (Li et al., 1997). All of these effects were mediated via the IL-6 receptor, as they were all blocked by antibodies against this receptor (Li et al., 1997). Tancredi et al. (2000) assessed the influence of IL-6 on LTP in the CA1 region in vitro, using three lower IL-6 concentrations (1, 5, 50 U/ml). However, they also reported that LTP was impaired in a dose-dependent manner (not affected by the lowest concentration, mildly repressed by the intermediate one, and strongly inhibited by the highest concentration), and that post-tetanic potentiation was also reduced by the two highest concentrations only (Tancredi et al., 2000). Although different IL-6 concentrations were used in the two studies, both suggest that IL-6 is involved mainly in the process of

the transformation between short- and long-term plasticity, but that at higher concentrations it can interrupt the potentiation process itself. No study reported a significant effect of IL-6 on basal synaptic transmission, and elevated IL-6 levels (either by transgenic overexpression or by external application) had no effect on paired-pulse response in vitro (Bellinger et al., 1995; Tancredi et al., 2000), indicating that this cytokine is not involved in short-term plasticity. Several mechanisms that may underlie the detrimental effect of IL-6 on LTP were investigated: IL-6 reduced glutamate release induced by AP-4 in a dosedependent way (D’Arcangelo et al., 2000). Additionally, IL-6 application to hippocampal slices resulted in increased STAT3 tyrosine phosphorylation and reduced activation of the MAP kinases ERK1 and ERK2 (D’Arcangelo et al., 2000; Tancredi et al., 2000), which play an important role in LTP maintenance (Kelleher et al., 2004). The finding that these effects were blocked by the tyrosine kinase inhibitor lavendustin A (D’Arcangelo et al., 2000; Tancredi et al., 2000) further implicates this signaling pathway in mediating the effects of IL-6 on LTP. All of the studies presented above demonstrate a detrimental effect of elevated IL-6 levels on long-term synaptic plasticity. However, recent studies suggest that endogenous IL-6 may actually have a physiological role in LTP inhibition. Jankowsky et al. (2000) reported a 20-fold increase in IL-6 gene expression 4 hours after LTP induction by high-frequency stimulation in vivo. Balschun et al. (2004) also found increased IL-6 gene expression 8 hours after high-frequency stimulation in freely moving rats. This increase was found only in rats in which LTP was robust and maintained for 8 hours, but not when it subsided within 3 hours following highfrequency stimulation (Balschun et al., 2004). Increased IL-6 gene expression was observed 1–3 hours following LTP induction in hippocampal slices in vitro as well (Balschun et al., 2004). Immunoneutralization of IL-6 by i.c.v. administration of anti–IL-6 antibodies 90 minutes following the induction of LTP resulted in longer maintenance of LTP; i.e., LTP was still preserved in anti–IL-6 injected rats after it subsided in control rats. In contrast, anti–IL-6 antibodies that were injected 30 minutes before or 5 minutes after the induction of LTP had no effect (Balschun et al., 2004), suggesting that IL-6 is selectively involved in a specific phase of LTP consolidation. No other endogenous protein is known to interfere with LTP maintenance in such a late phase, without influencing LTP induction. The findings of LTP-induced increase in IL-6 gene expression, together with longer preservation of LTP following IL-6 neutralization, suggest that IL-6 may have a physiological role in the termination of LTP.


3. Effects of TNF-a on Neural Plasticity As reviewed above, TNF-α seems to have a negative effect on memory functioning, which is both dose- and age-dependent, although this effect is less robust than the effects of IL-1 and IL-6. Furthermore, in contrast to IL-1 and IL-6, basal levels of TNF-α are probably not involved in memory processes. The involvement of TNF-α in hippocampal plasticity was examined only in vitro, and the findings are in line with the behavioral conclusions. Tancredi et al. (1992) were the first to report a dosedependent inhibition of LTP in the CA1 region following TNF-α application in vitro. Many additional studies reported reduced potentiation and inhibited LTP maintenance following TNF-α application in vitro in the DG as well (Butler et al., 2002; Cunningham et al., 1996; Curran and O’Connor, 2001; Curran et al., 2003). However, Albensi and Mattson (2000) reported no change in LTP in the CA1 region of hippocampal slices from mice lacking both TNFR1 and TNFR2 (TNFrKO). This finding is in line with the behavioral reports of normal and even enhanced memory in TNFαKO and TNFR1KO mice. Furthermore, these TNFrKO mice demonstrated impaired LTD (Albensi and Mattson, 2000), suggesting a role for TNF-α in synaptic depression. The mechanisms underlying TNF-induced LTP impairment are quite similar to those that mediate the detrimental effect of IL-1 on LTP. Similar to IL-1β, TNF-α induced p38 MAP kinase activation in the DG (Butler et al., 2004). However, p38 MAP kinase seems to mediate primarily the initial inhibitory effect of TNF-α on LTP, as its inhibitor SB203580 completely blocked TNF-α–induced early-phase LTP impairment (1 hour following tetanic stimulation), but only partially blocked the negative effect of TNF-α 3 hours following tetanic stimulation (Butler et al., 2004; Pickering et al., 2005). Furthermore, like IL-1β, the inhibitory effect of TNF-α on LTP is attenuated by the JNK inhibitor SP600125 (Curran et al., 2003) and by nicotine (Curran and O’Connor, 2003), suggesting a role for JNK and acetylcholine (ACh) in TNF-α–induced LTP inhibition. Finally, TNF-α application was shown to increase surface expression of glutamate AMPA receptors, and TNF blockade resulted in decreased AMPA receptor expression (Beattie et al., 2002). However, these new receptors have abnormal stoichiometry, as they lack the GluR2 subunit; thus, they become Ca2+ permeable and may contribute to neurotoxicity (Stellwagen et al., 2005). To sum up, similar to the behavioral studies that imply a dose-dependent negative effect of TNF-α on memory, the electrophysiological studies presented

357

above also suggest a detrimental effect of TNF-α on hippocampal plasticity, which is mediated by mechanisms similar to those mediating the effects of IL-1β.

B. Stress Responses and Glucocorticoids Secretion A broad range of aversive physiological and psychological stimuli can induce stress responses in both humans and animals. The most prominent physiological stress response is activation of the HPA axis: In response to stressful stimuli, hypothalamic neurons secrete corticotropin-releasing hormone (CRH), the major adrenocorticotropic hormone (ACTH) secretagoge, which induces the secretion of ACTH from the anterior pituitary into the systemic circulation, resulting in secretion of glucocorticoids (GC) from the adrenal cortex (Antoni, 1986). Many researchers examined the effects of stress on hippocampal-dependent memory and plasticity, finding seemingly contradictory results. In some studies, stress exposure or administration of stress hormones was found to produce detrimental effects on hippocampal-dependent memory and plasticity, in both humans and animal models. However, in other studies low levels of stress, accompanied by a mild increase in stress hormones, were found to be necessary for memory formation and LTP (reviewed in de Kloet et al., 1999; Kim and Diamond, 2002; McEwen and Sapolsky, 1995). Thus, similar to the finding on the involvement of IL-1 in hippocampal-dependent memory, corticosteroids also exert an inverted Ushaped influence on memory and plasticity (Conrad et al., 1999; Diamond et al., 1992). IL-1 is expressed throughout the components of the HPA axis (Besedovsky and del Rey, 1996; Turnbull and Rivier, 1999) and influences all of its levels: IL-1 induces the secretion of CRH from the hypothalamus and ACTH from the pituitary (Berkenbosch et al., 1987; Bernton et al., 1987; Sapolsky et al., 1987). Additionally, IL-1 stimulates GCs secretion from the adrenal, although no known IL-1 receptors were found in this gland (Besedovsky and del Rey, 1996; Turnbull and Rivier, 1999). The production, secretion, and influence of IL-1 on the HPA axis have been traditionally considered in the context of illness-associated immune activation (Besedovsky and del Rey, 1996; Turnbull and Rivier, 1999). However, over the last decade it became evident that psychological stressors activate the IL-1 system in the brain (Nguyen et al., 1998, 2000; O’Connor et al., 2003; Suzuki et al., 1997). Furthermore, it was demonstrated, by us and by others, that endogenous IL-1 activates the HPA axis not only during sickness, but also in response to various psy-

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chological stressors (Goshen et al., 2003b; Shintani et al., 1995). The findings that the influence of IL-1 and the influence of stress or corticosterone on memory follow the same inverted U shape and that stress can induce IL-1 production, which in turn activates the HPA axis, may lead to two hypotheses: First, IL-1 may mediate the detrimental effects of stress on memory. This hypothesis was confirmed by two studies (as detailed above in the section titled “IL-1”), which showed that i.c.v. administration of IL-1ra prior to stress exposure blocked its harmful effect on memory (Maier and Watkins, 1995; Pugh et al., 1999). Second, HPA axis activation may be involved in the influence of IL-1 on memory formation (both detrimental and beneficial), which is usually assessed in the context of stressful paradigms (e.g., the Morris water maze or fear conditioning). The studies that addressed the latter hypothesis will be discussed below. The possible involvement of HPA axis activation in IL-1–induced memory impairment was demonstrated by two studies (Song and Horrobin, 2004; Song et al., 2004), reporting a concomitant modulation of the effect of IL-1 on memory and corticosterone secretion. These studies found a detrimental effect of i.c.v. administered IL-1β on spatial memory in the water maze as well as on working memory in the win-shift radial arm maze. Eight weeks of feeding with a diet enriched in the antiinflammatory supplement ethyl-eicosapentaenoic acid (E-EPA, 1%) attenuated the IL-1–induced memory impairment and blocked the IL-1–induced increase in serum corticosterone concentration (Song and Horrobin, 2004; Song et al., 2004), suggesting that this increase mediated the effect of IL-1 on memory. The role of HPA axis activation in IL-1–induced memory improvement was directly assessed using GC receptor blockade: Song et al. (2003) reported improved contextual passive avoidance response concomitantly with increased corticosterone secretion in rats that were injected i.c.v. with IL-1β. However, when IL-1 was co-administered together with the GC receptor antagonist RU486, the beneficial effect on memory was abolished, and so was the increase in corticosterone levels (Song et al., 2003). Additionally, IL-1rKO mice, which demonstrated impaired memory performance (Avital et al., 2003), also showed diminished corticosterone secretion in response to mild stressors (Goshen et al., 2003b), suggesting that impaired HPA axis activation may mediate the poor memory performance of these mice. To conclude, the findings reported to date show that on the one hand IL-1 mediates the detrimental effects of stress on memory, and on the other hand HPA axis activation, which affects memory in an inverted U

pattern similar to IL-1, may be involved in both the detrimental and the beneficial effects of IL-1 on memory formation, which had been assessed using stressful paradigms. Based on all the data presented in this section, the following model may be proposed: Stressful stimuli induce an increase in brain IL-1 levels, which in turn contribute to the activation of the HPA axis. Subsequently, the secretion of corticosterone affects memory processes in an inverted U-shaped way.

C. Prostaglandins An increase in IL-1 levels is usually accompanied by the secretion of prostaglandins (PGs), the proinflammatory derivates of arachidonic acid. The first step of prostaglandin synthesis is performed by the enzyme cyclooxygenase (COX). The primary two COX isoforms are the constitutive COX-1 and the inducible COX-2, which are uniquely involved in different physiological processes (Smith and Langenbach, 2001). COX-1 and COX-2 are encoded by different genes and show only about 60% protein homology. The conformational variations between the two can account for their different sensitivities to specific inhibitors (Kurumbail et al., 1996). IL-1 is a strong inducer of COX-2 in brain glia cells (O’Banion et al., 1996), and both IL-1α and β increased the production of hippocampal prostaglandin E2 (PGE2) in a dose-dependent manner (Weidenfeld et al., 1995). Elevated PGE2 levels were shown in the past to mediate some of the physiological (e.g., fever) (Dinarello et al., 1999) and behavioral (e.g., pain perception and sexual behavior) (Avitsur and Yirmiya, 1999; Hori et al., 1998) effects of IL-1 administration; thus, they may also mediate its influence of memory processes. PGs were demonstrated to affect the cognitive state in many inflammatory and neurodegenerative brain diseases in humans (reviewed in Minghetti, 2004). Furthermore, PGs were repeatedly reported to be involved in memory processes in animals. Interestingly, PGs share the same inverted U-shape pattern of influence on memory as IL-1. On the one hand, high PG levels, e.g., following traumatic brain injury or LPS administration, impaired spatial memory, and COX inhibitors improved memory function (Gopez et al., 2005; Shaw et al., 2005). Moreover, mice with transgenic overexpression of COX-2 demonstrated elevated production of PGE2 in the brain, concomitantly with an agedependent spatial memory deficit in the water maze, as well as impaired passive and active avoidance (Andreasson et al., 2001). On the other hand, basal PGE2 levels may actually have a role in hippocampal memory and plasticity, because inhibition of COX


activity in animals without inflammatory stimulation can negatively affect memory and plasticity. For example, the non-selective COX inhibitors indomethacin and ibuprofen and the COX-2 inhibitor NS-398 impaired spatial but not visually guided water maze performance (Shaw et al., 2003; Teather et al., 2002). COX inhibition by ibuprofen resulted in impaired hippocampal LTP as well (Shaw et al., 2003), and a similar finding was reported for specific COX-2 inhibition by NS398 and nimesulide (Chen et al., 2002), an effect that was reversed by PGE2 application (Chen et al., 2002). To date, PGs were found to mediate only the detrimental effect of IL-1 on memory: Intrahippocampal injection of IL-1, which increased hippocampal PGE2 secretion, impaired working memory performance in the three-panel runway task, similar to intrahippocampal injection of PGE2 itself. Furthermore, subcutaneous injection of the COX inhibitor diclofenac 30 minutes before IL-1 administration totally blocked the detrimental effect of IL-1 on working memory (Matsumoto et al., 2004). Another study showed that 8 weeks of feeding with a diet enriched in the antiinflammatory supplement E-EPA attenuated the spatial memory and working memory impairments induced by i.c.v.-administered IL-1β and blocked the IL-1– induced increase in hippocampal PGE2 concentration (Song and Horrobin, 2004; Song et al., 2004). Finally, chronic treatment with the COX-2 inhibitor cellecoxib for 4 months in aged rats diminished hippocampal protein levels of PGE2 and IL-1β, and blocked the agerelated memory impairment (Casolini et al., 2002). Inhibition of PG synthesis by indomethacin also blocked IL-1–induced LTP inhibition (Coogan et al., 1999). To conclude, PG synthesis, which affects memory and plasticity processes in an inverted U-shaped pattern similar to IL-1, seems to play a role in the detrimental effect of IL-1 on memory processes and plasticity. To date, the role of PGs in the beneficial role of IL-1 in memory processes has not been examined. However, the fact that IL-1 induces PG synthesis, which is necessary for normal memory functioning, suggests that PGs may also be involved in IL-1–induced memory facilitation.

D. Developmental Processes, Neurogenesis, and Neurotrophins Much of the data presented in “Effects of IL-1, IL-6, and TNF-α on Learning and Memory in Rodents,” demonstrating the role of pro-inflammatory cytokines in memory processes and plasticity, was obtained using genetically manipulated mice. Because in these animals the modified signaling is present throughout the life span of the animal, from conception to matu-

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rity, it may influence memory either by affecting brain development in a way that will alter memory functioning in adulthood, and/or by directly influencing ongoing memory processes in the adult brain at the time of learning. The ability of pro-inflammatory cytokines to affect memory processed in these ways will be further discussed in this section. Pro-inflammatory cytokines are expressed in human and mouse pre-implantation embryos and in the embryonic brain (De los-Santos et al., 1996; Gerwin et al., 1995; Kruessel et al., 1997; Mehler and Kessler, 1997; Pousset, 1994; Sharkey et al., 1995). Furthermore, IL-1β, IL-6, and TNF-α levels were reported to increase with time in embryonic human forebrain cells (Mousa et al., 1998), and they have a marked influence on neuronal differentiation and survival (Akaneya et al., 1995; Ling et al., 1998; Marz et al., 1999; Munoz-Fernandez and Fresno, 1998), as well as on neurite outgrowth (Edoff and Jerregard, 2002; Johanson and Stromberg, 2002; Neumann et al., 2002). Thus, pro-inflammatory cytokines may be involved in brain development, and interference with their signaling during the prenatal period may have behavioral effects in adulthood as well. The feasibility of this hypothesis was recently demonstrated in a study on TNFαKO mice. In the first postnatal week, these mice exhibited an accelerated maturation of the DG region of the hippocampus, which may have contributed to the improved memory that these mice displayed in adulthood (Golan et al., 2004). Pro-inflammatory cytokines may also influence ongoing memory processes in the adult brain either by one of the mechanisms that were suggested above (neural plasticity, stress hormones, and secretion of prostaglandins) or by altering neurogenesis, the formation of new neurons, which is considered a part of the biological substrate for memory formation (Gould et al., 2000; Gross, 2000; Kempermann et al., 2004). Recently, immune activation was demonstrated to have a detrimental influence on neurogenesis, suggesting a negative role for pro-inflammatory cytokines on this process. Specifically, microglia, the macrophage-like cells within the brain, were suggested as the mediators of this detrimental effect (e.g., Kempermann and Neumann, 2003). Ekdahl et al. (2003) demonstrated that intracortical LPS administration, which induced the activation of microglia, resulted in decreased neurogenesis. They also reported an inverse correlation between the amount of microglial activation and the generation of new neurons (Ekdahl et al., 2003). Moreover, in order to affect neurogenesis, the inflammatory stimulus does not have to be localized to the brain, as demonstrated by Monje et al. (2003), who reported that a single i.p. LPS

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injection increased the number of activated microglia and reduced hippocampal neurogenesis. Monje et al. (2003) further showed that in vitro, neuronal differentiation is decreased by co-culture with activated but not resting microglia, and that this effect is mediated by soluble factors, as conditioned medium (CM) from activated microglia produced the same effect. One of these factors may be IL-6, as IL-6 inhibited neuronal differentiation, and anti–IL-6 blocked the detrimental effect of IL-6 and of the CM from activated microglia on neuronal differentiation. Interestingly, IL-6 specifically blocked the generation of new neurons, without affecting the development of astrocytes or oligodendrocytes (Monje et al., 2003). Similarly, mice with transgenic overexpression of IL-6 displayed decreased proliferation and survival of neurons, with no effect on the number of glia cells (Vallieres et al., 2002). This finding corroborates the results of a previous study demonstrating that IL-6 promotes differentiation of cerebral cortical precursor cells into astrocytes and inhibits differentiation of cortical precursors along a neuronal lineage in vitro (Bonni et al., 1997). Liu et al. (2005) also showed that CM from LPS-activated microglia reduced neuronal differentiation, and that this effect was at least partly mediated by TNF-α: TNF-α secretion from activated, compared to resting, microglia, was increased, and pentoxifylline, a TNF-α inhibitor, reduced the detrimental effect on neuronal differentiation (Liu et al., 2005). The influence of pro-inflammatory cytokines on either brain development or adult neurogenesis, as well as their acute effects on memory in adulthood, may be mediated by neurotrophic factors. IL-1, IL-6, and TNF-α influence the expression and secretion of various neurotrophic factors, e.g., nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (Frei et al., 1989; Gadient et al., 1990; Ishida et al., 1997; Murphy et al., 2000; Schulte-Herbruggen et al., 2005; Steiner et al., 1991). In addition to their role in the development of the CNS, NGF and BDNF have been implicated in hippocampal-dependent memory (e.g., Chen et al., 1997; Hall et al., 2000). A set of studies conducted by Barrientos et al. (2003, 2004) provides strong evidence that the detrimental effect of IL-1 on memory is mediated by interference with the increase in BDNF expression that accompanies contextual memory formation (Hall et al., 2000). Indeed, IL-1β reduced the increase in hippocampal BDNF mRNA following exposure to fear conditioning (Barrientos et al., 2004). Barrientos et al. (2003) also showed that social isolation stress impaired the acquisition of contextual fear conditioning and decreased BDNF mRNA in the hippocampus. Furthermore, intrahippocampal injection of IL-1ra before isolation prevented both the

memory impairment and the decrease in BDNF expression (Barrientos et al., 2003). Other studies suggest that NGF is involved in TNF-α–induced modulation of memory. Golan et al. (2004) reported that TNFαKO mice, which demonstrated enhanced memory performance, also exhibit increased expression of NGF following performance of the learning task. Moreover, TNFαTG mice, which showed impaired memory performance (Fiore et al., 1996), also demonstrated decreased hippocampal NGF secretion (Aloe et al., 1999a, b). To conclude, the ability of pro-inflammatory cytokines to influence neuronal proliferation and differentiation, as well as the secretion of neurotrophic factors both in the developing and the adult brain, may mediate their effect on memory processes. Future studies should elucidate further the role of specific cytokines in each of these processes.

IV. PRO-INFLAMMATORY CYTOKINES AND MEMORY FUNCTIONING IN HUMANS In humans, the relationships between proinflammatory cytokines and memory functioning were studied using three approaches: (1) examining the correlations between serum levels of these cytokines and cognitive functioning in various conditions that are associated with inflammatory processes (e.g., infectious, autoimmune, or neurodegenerative diseases, as well as aging); (2) directly assessing the effects of cytokine administration on memory; and recently (3) studying the relationships between polymorphisms in inflammatory cytokine genes and the risk and severity of neurodegenerative diseases and dementia.

A. Correlating Pro-inflammatory Cytokine Levels and Memory Functioning Cytokine levels and memory functioning were mainly examined in the context of normal and pathological aging. Ample evidence indicates that in normal aging, and particularly in aging-associated neurodegenerative diseases, the regulatory mechanisms responsible for inflammatory responses are ineffective or damaged, resulting in adverse pathological conditions (Bodles and Barger, 2004; Ferrucci et al., 2004; Krabbe et al., 2004). One of the most consistent findings in gerontological surveys of cytokines is an agedependent increase in IL-6 levels (Ershler et al., 1993; Forsey et al., 2003; Hager et al., 1994; Roubenoff et al., 1998; Wei et al., 1992). Some studies reported that plasma levels of TNF-α are also increased in elderly


populations (Bruunsgaard et al., 1999; Paolisso et al., 1998), although other studies were not able to detect this effect (e.g., Peterson et al., 1994). Although the associations between inflammatory cytokines and aging are quite consistent, it is still not certain whether the increase in inflammatory markers results directly from the aging process per se, or whether it is mediated by indirect processes, particularly sub-clinical disorders like chronic infections and atherosclerosis. More clear and direct associations have been reported between inflammatory cytokines and aging-associated neurodegenerative diseases, particularly Alzheimer’s disease (AD). This condition is accompanied by elevated serum and cerebrospinal fluid (CSF) levels of TNF-α, IL-6, and IL-1 (Blum-Degen et al., 1995; Cacabelos et al., 1991; Licastro et al., 2000a, b; Tarkowski et al., 1999, but see Pirttila et al., 1994), and lower IL-1ra levels in the CSF (Tarkowski et al., 2001). Furthermore, several lines of evidence suggest that within the brain, inflammatory cytokines, particularly IL-1, are involved in the disease process itself (Griffin and Mrak, 2002; McGeer and McGeer, 2003): (1) IL-1 is involved in acute neurodegeneration, particularly by exacerbating various forms of brain injury (Rothwell, 2003). (2) Senile plaques, one of the hallmarks of AD, are surrounded by activated astrocytes and microglia, wherein levels of both IL-1α and IL-6 are elevated (Akiyama et al., 2000; Bauer et al., 1991; Griffin and Mrak, 2002; McGeer and McGeer, 2003; Mrak and Griffin, 2001). (3) IL-1 stimulates the expression of amyloid precursor protein (APP) (Griffin and Mrak, 2002). (4) IL-1 has been implicated in the hyperphosphorylation of tau (Li et al., 2003), another characteristic feature of AD. (5) IL-1 can elevate the levels of acetylcholine esterase (AChE), contributing to the reduction in cholinergic neurotransmission in AD patients (Li et al., 2000). (6) Recently, attention was allocated to the aggravating effect of systemic infections on the symptoms of AD, suggesting that infectioninduced production of peripheral pro-inflammatory cytokines, such as IL-1, is detrimental for AD progression (Perry et al., 2003). (7) Finally, anti-cholinesterases, which offer the only beneficial treatment for AD, were recently reported to reduce the levels of IL-1 within the hippocampus (Pollak et al., 2005). Ample evidence indicates that increases in proinflammatory cytokines, particularly IL-6, are associated with cognitive impairment in elderly people. Several longitudinal population-based studies showed that elderly subjects who had high levels of blood IL-6 (usually defined as being in the highest third for plasma IL-6) were also more likely to exhibit cognitive decline over 2.5–7-year follow-ups (Weaver et al., 2002; Yaffe et al., 2003). Interestingly, IL-6 levels as a risk factor for cognitive decline were particularly evident

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in subjects with apparent cognitive impairment at baseline (Dik et al., 2005). Consistently, in demented subjects with AD, higher levels of IL-6 were correlated with the severity of the dementia (Kalman et al., 1997). Serum levels of IL-1β may also be associated with cognitive decline, as AD subjects who had detectable serum levels of IL-1β at baseline had an increased rate of cognitive decline over a 2-month follow-up, compared with those with no detectable levels of IL-1β (Holmes et al., 2003). Together, these findings suggest that elevated pro-inflammatory cytokine levels, either in normal elderly people or in AD patients, play an important role in promoting cognitive decline. It should be noted that in all the studies mentioned above, cognitive functioning was measured by global neuropsychological rating scales (either the Mini Mental State Examination or a composite global score from various attention, memory, language, and executive function tests), and therefore these reports do not allow us to draw conclusions regarding specific impairments in memory. Contrary to IL-1 and IL-6, the increase in TNF-α seems to play a protective role in AD (Tarkowski et al., 2003b): AD is accompanied by elevated CSF levels of TNF-α (Tarkowski et al., 1999, 2000), and increased CSF TNF-α levels were detected in patients with mild cognitive impairment only if they developed AD 9 months later (Tarkowski et al., 2003a). However, CSF TNF-α levels were negatively correlated with protein levels of Tau, as well as with Fas/APO-1, a protein involved in apoptosis, and positively correlated with bcl-2, which inhibits apoptosis (Tarkowski et al., 1999). These correlations suggest a protective role for TNF-α in AD, although no causal relationship has been demonstrated yet. High levels of pro-inflammatory cytokines are also found in Down syndrome (DS) patients. Griffin et al. (1989) found a larger number of IL-1 immunoreactive cells in the brains of fetuses, neonates, and 3.5-monthold babies with DS compared to age-matched controls. Higher levels of IL-6 were found in the plasma of DS children compared to controls (Corsi et al., 2003; Licastro et al., 2001), and two groups also reported a positive correlation between IL-6 levels in the serum and the degree of mental retardation and dementia in DS patients (Carta et al., 2002; Kalman et al., 1997). Moreover, higher IL-6 levels in DS were already found before the appearance of dementia, suggesting that it may be an early sign of brain alterations leading many years later to cognitive deterioration and dementia (Licastro et al., 2005). Together, these findings suggest that elevated levels of the pro-inflammatory cytokines IL-6 and IL-1 may play an important role in promoting cognitive decline in DS patients.

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Further evidence for the involvement of IL-1 in mental retardation is offered by studies reporting that in humans, deletions in different parts of the encoding region of the IL-1 receptor accessory protein-like gene (IL-1RAPL), which result in premature translation termination, are responsible for a non-specific form of Xlinked mental retardation (Carrie et al., 1999; Jin et al., 2000). Although the function of the IL-1RAPL protein is presently unknown, the homologous mouse IL1RAPL gene is highly expressed in brain areas subserving memory processes, such as the hippocampus and entorhinal cortex (Carrie et al., 1999), further supporting a role for this gene in memory processes. Only a few studies examined the specific relationship between inflammatory markers and memory. In normal elderly subjects, IL-6 was not found to be associated with either immediate or delayed recall (Dik et al., 2005) in the hippocampal-dependent Auditory Verbal Learning Test. However, in another study, higher IL-6 levels were associated with poorer sensory memory, assessed by the intentional memory test (Elwan et al., 2003). It should be noted that in that study, IL-6 levels were not significantly associated with another measure of memory (incidental shortterm memory) and not related to several other cognitive measurements (Elwan et al., 2003), so this single finding should be interpreted cautiously. The importance of co-morbidity for the association between inflammation and memory was demonstrated in another study, in which a significant association was found between TNF-α/IL-10 ratio and memory functioning (including immediate and delayed verbal recall) in subjects above 85 years of age who also had cardiovascular disease. Interestingly, memory functioning did not depend on this inflammatory parameter when cardiovascular disease was absent (van Exel et al., 2003). Inflammatory cytokines are obviously elevated in many medical conditions other than neurodegenerative diseases, including acute and chronic infectious diseases, autoimmune diseases such as lupus and multiple sclerosis, as well as following stroke, surgery, or trauma. Although these conditions are also characterized by transient memory decline and in some conditions even by the development of dementia, the role of specific cytokines in illness-associated memory disturbances has not been elucidated. Interestingly, in one of the only studies in which the relationship between IL-6 levels and autoimmunity-induced memory impairments were investigated, patients with systemic lupus erythematosus, but not with rheumatoid arthritis, exhibited a significant impairment in learning of verbal and non-verbal information, and higher levels of IL-6 in the plasma were associated with higher learning

scores (reflecting better short-term memory functioning). This relationship was substantial, accounting uniquely (i.e., after adjustment for depression score and somatic symptoms) for 17% of the variance in learning scores (Kozora et al., 2001). We have recently found additional support for a possible protective effect of IL-6 in illness-associated memory disturbance (Shapira Lichter et al., 2004). In our study, 33 generally healthy volunteers, who went through moderate surgery, and 18 gender-, age- and education-matched controls completed a neuropsychological test battery at baseline and 24 hours post-surgery. Our preliminary results demonstrate that surgery stress produced impairments in verbal and visual declarative memory, but not in other cognitive parameters. Furthermore, the memory impairments were inversely correlated with the elevation in IL-6 following the surgery (accounting for 35% of the variance in memory decline). This finding suggests that post-surgery increases in IL-6 levels are associated with protection from surgeryinduced memory disturbances (Shapira Lichter et al., 2004). Together, these findings underscore the complexity of the associations between pro-inflammatory cytokines and memory, which is described in the sections on experimental animals, demonstrating that under some situations elevated levels of IL-6 may be associated with protective rather than detrimental effects on memory functioning. In another set of studies, we adapted a different approach for examining the relationship between cytokines and memory, using a double-blind, crossover study, in which healthy male volunteers completed psychological questionnaires and neuropsychological tests following endotoxin (LPS) administration. In one experiment, 20 volunteers were tested 1, 3, and 9 hours after intravenous injection of Salmonella abortus equi endotoxin (0.8 ng/kg) or saline in two experimental sessions (Reichenberg et al., 2001). Blood samples were collected hourly, and rectal temperature and heart rate were monitored continuously. Although endotoxin had no effects on physical sickness symptoms, blood pressure, or heart rate, it induced mild fever and markedly increased the circulating levels of IL-6, TNF-α, soluble TNF receptors, IL-1ra, and cortisol. Endotoxin administration produced a global decrease in memory functions, during all testing periods, reflected by decreased immediate recall of story items, reduced delayed story recall, a deficit in immediate and delayed recall of figure items, and decreased performance in Word List Learning. Furthermore, endotoxin-induced impairments in immediate and delayed story recall were significantly and positively correlated with the secretion of IL-6, TNF-α, and IL-1ra in the first and second testing periods, but not in the last period. Interestingly, using the same


procedure we demonstrated that in contrast with the cytokine-associated deleterious effects of endotoxin on declarative memory, endotoxin administration induced a significant improvement in working memory performance, reflected by an increased score in the Digit Span Backward Test during all testing periods. This improvement was not associated with cytokine secretion (but it did associate with alterations in cholinergic neurotransmission) (Cohen et al., 2003). In another recent study (Krabbe et al., 2005), we also used a double-blind crossover design, in which 12 healthy young males completed neuropsychological tests before as well as 1.5, 6, and 24 hours after an intravenous injection of a very low dose of endotoxin (0.2 ng/kg) or saline in two experimental sessions. Endotoxin administration had no effect on body temperature, cortisol levels, blood pressure, or heart rate, but circulating levels of TNF-α, IL-6, TNFreceptors, and IL-1ra were markedly elevated. In this model, low-dose endotoxemia did not affect cognitive performance significantly, but declarative memory performance was inversely correlated with endotoxininduced increases in circulating IL-6 levels (Krabbe et al., 2005).

B. Assessment of the Direct Effects of Cytokine Administration on Memory Although in our studies on endotoxin-induced memory disturbances (Krabbe et al., 2005; Reichenberg et al., 2001), as well as in most of the literature reviewed above, higher levels of IL-6 were associated with poor memory functioning or memory decline over time; it should be noted that such a correlation does not prove causal relationship. A better approach for proving causality is to directly examine the effects of proinflammatory cytokine administration on memory. The effects of IL-6 on memory functioning were investigated only in one study, in which IL-6 (3 mg/kg) was administered to 19 chronic fatigue syndrome (CFS) patients and 10 control subjects, and memory (assessed by the Sternberg Memory Scanning Test, measuring speed of searching through short-term memory), as well as other neuropsychological functions, were assessed 6.5 hours later (Arnold et al., 2002). Surprisingly, IL-6 did not produce any memory disturbance. In fact, both CFS patients and controls demonstrated improved performance in this test (Arnold et al., 2002). Although the investigators ascribed this improvement to practice effect, it is possible that the improved cognitive functioning was induced by IL-6 (in the absence of a saline-administered control group, it is impossible to distinguish between these two possibilities). This study again demonstrates that IL-6 is not always associated with memory disturbance and that the conse-

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quences of elevated IL-6 levels are determined by many factors, including age and medical condition of the individual, the particular memory function that is assessed, as well as the magnitude and duration of IL-6 elevation.

C. The Influence of Polymorphisms in Inflammatory Cytokine Genes on Neurodegenerative Diseases, Dementia, and Memory In the last 15 years, extensive research efforts were invested in an attempt to link various gene polymorphisms to cognitive functioning in healthy people, as well as to the risk to develop neurodegenerative diseases, which are accompanied by cognitive deterioration. The evidence for the relationships between pro-inflammatory cytokine levels and dementia in AD and other medical conditions, which was reviewed above, suggests that polymorphisms in the genes encoding these cytokines may also play a role. Although the contribution of these gene polymorphisms to memory functioning has not been tested directly yet, recent genetic studies demonstrated a role for polymorphisms in the genes for IL-1, IL-6, and TNF-α in the risk to develop AD, as well as in the age of onset and the course of this disease. These studies are reviewed in the following section. 1. IL-1 Intensive research efforts were dedicated to the possible contribution of different polymorphisms in IL-1 family genes to AD. These studies were performed on thousands of patients from different ages, genders, and ethnic origins. Although the results of these studies are sometimes contradictory, the general conclusion is that several polymorphisms in IL-1 family genes play a role in the risk of developing AD, as well as in the severity of the disease. Most of the research focused on the relationship between IL-1α polymorphisms and AD. One polymorphism that was found to be associated with AD is a C (allele 1) to T (allele 2) transition at position −889 relative to the transcription start point, in the regulatory region of the IL-1α gene. Several studies reported that IL-1A −889 allele 2 (IL-1A −889 T) frequency is higher in AD patients compared to controls (Bosco et al., 2004; Du et al., 2000; Nicoll et al., 2000; Seripa et al., 2005). IL-1A −889 allele 2 serves as a risk factor in a dosedependent manner, as the risk of developing AD with two copies of the IL-1A allele 2 is higher than the risk with only one copy (Combarros et al., 2002; Du et al., 2000; Grimaldi et al., 2000).

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Some studies report that the IL-1A −889 T/T genotype frequency was significantly higher in early onset AD compared to late onset AD and to age-matched controls (Sciacca et al., 2003), and that the presence of the IL-1A −889 T allele was associated with increased risk of early onset AD (Du et al., 2000; Grimaldi et al., 2000; Rebeck, 2000), and with an earlier age of onset in general (Kolsch et al., 2001; Rebeck, 2000). However, a different study reported that the IL-1A −889 T/T genotype was associated with late onset AD but not with early onset AD (Hedley et al., 2002). Many other studies found no association between the presence of IL-1A −889 T allele and AD in different populations (Fidani et al., 2002; Green et al., 2002; Ki et al., 2001; Kolsch et al., 2001; Kuo et al., 2003; Li et al., 2004b; Minster et al., 2000; Pirskanen et al., 2002; Seripa et al., 2005; Tsai et al., 2003). However, two meta-analyses that were performed on this data set confirmed that the IL-1A −889 allele 2 is a risk factor for AD in an age-related pattern: One meta-analysis found that the IL-1A −889 T/T genotype is a risk factor for developing AD in general, and that the frequency of this genotype was increased only in patients with early-onset AD (Rainero et al., 2004). The second metaanalysis found that the IL-1A −889 T/T genotype contributes only to the risk of early-onset but not late-onset AD (Combarros et al., 2003). To conclude, polymorphisms in the regulatory region of the IL-1α gene are involved in the risk of developing AD. The effect of the IL-1A −889 T/T genotype may stem from overproduction of IL-1α in its carriers. Indeed, the IL-1A −889 T/T genotype significantly increased the transcriptional activity of the IL-1α . gene with respect to the IL-1A −889 C/C genotype, and this increase was accompanied by an increase of the IL-1α mRNA and protein levels in the plasma (Dominici et al., 2002a). Polymorphisms at different locations in the IL-1β gene were also found to be associated with AD. One polymorphism is a C to T transition at position −511 in the promoter region of the IL-1β gene. The IL-1B −511 T/T genotype was more frequently found in late onset AD than in healthy patients (Wang et al., 2005). The presence of this genotype was associated with increased risk for AD in general (Bosco et al., 2004), and specifically a risk for late-onset AD and delayed age of onset (Grimaldi et al., 2000). However, the concomitant presence of the IL-1B −511 T/T and alpha1antichymotrypsin T/T genotypes was associated with increased risk of AD and decreased age at disease onset (Licastro et al., 2000b). It should be noted that other studies found no association between the IL-1B −511 T allele and AD (Ehl et al., 2003; Green et al., 2002; Hedley et al., 2002; Li et al., 2004b; Licastro et al., 2004;

Ma et al., 2003; McCulley et al., 2004; Minster et al., 2000; Nishimura et al., 2004). However, in some of these studies, even though IL-1β −511 polymorphism did not influence the risk of AD, it was found to have a pathophysiological influence on the disease process. For example, Ehl et al. (2003) found that Aβ42 levels in the CSF were higher in carriers of the IL-1B −511 T allele, and Green et al. (2002) also reported an impact of IL-1B −511 on Aβ(40) load. The effect of the IL-1B −511 T polymorphism may be mediated by increased IL-1β levels in AD patients, which can contribute to the progression of the disease. Indeed, AD patients with the IL-1B −511 T/T genotype showed higher levels of plasma IL-1β compared to controls (Licastro et al., 2000b). The other polymorphism in the IL-1β gene is a C to T transition, located at position +3953 on exon 5. Hedley et al. (2002) found an increased prevalence of the IL-1B +3954 T/T genotype in late-onset AD patients compared to controls. Furthermore, patients carrying the IL-1B +3953 T allele had a decreased age of onset (Sciacca et al., 2003) and shorter survival (Licastro et al., 2004). These influences may be mediated by increased IL-1β secretion in homozygous carriers of the IL-1B +3953 T allele (Pociot et al., 1992, but see Dominici et al., 2002b). It should be noted, however, that three other studies found no association of the IL-1B +3953 polymorphism with AD (Ma et al., 2003; Rosenmann et al., 2004; Wang et al., 2005). To conclude, the findings presented in the last paragraphs suggest that IL-1β gene polymorphisms in two different loci may influence the age at onset, progression, and survival in AD. The polymorphisms in the IL-1α and IL-1β genes were also found to synergistically influence the risk for AD and the age of onset of the disease. As mentioned above, several studies reported that either IL-1A −889 T or IL-1B +3953 T frequencies were higher in AD patients compared to controls (e.g., Du et al., 2000; Hedley et al., 2002; Nicoll et al., 2000). Moreover, homozygosity for both IL-1A −889 T/T and IL-1B +3953 T/T genotypes conferred the greatest risk to develop AD (Hedley et al., 2002; Nicoll et al., 2000). Furthermore, whereas either IL-1A −889 T/T or IL-1B +3953 T/T genotypes are associated with an earlier onset of the disease (Kolsch et al., 2001; Rebeck, 2000; Sciacca et al., 2003), AD patients carrying both IL-1B +3953 T+ and IL-1A −889 T/T genotypes had the lowest age of onset (Sciacca et al., 2003). These data suggest a cumulative effect of the IL-1α and IL-1β gene polymorphisms in the risk to develop AD and the age of disease onset. The studies that examined the involvement of polymorphisms in the variable number of tandem repeat


(VNTR) at intron 2 of the IL-1ra gene in AD yielded contradictory results. Two studies reported a protective role of IL1-RA allele 2 (2 repeats, instead of 4 in allele 1) on the onset and severity of AD: Grimaldi et al. (2000) reported a later age of onset in homozygous carriers of IL1-RA allele 2, and the presence of this allele was also protective for the severity of dementia, independent of age (Bosco et al., 2004). However, other studies found no association between IL-1-RA intron 2 VNTR and AD (Hedley et al., 2002; Li et al., 2004b; Minster et al., 2000; Wang et al., 2005). More research is needed in order to further clarify the involvement of polymorphisms in the VNTR at IL1RA intron 2 in AD. To conclude, different polymorphisms in the IL-1 gene family seem to be involved in the risk to develop AD, as well as in the progression of the disease. The strongest evidence is for the age-related contribution of the IL-1A −889 T allele to the risk of AD. Transitions in two different loci at the IL-1β gene (+3953 C → T and −511 C → T) also seem to be associated with the risk to develop AD. These polymorphisms in the IL-1α and IL-1β genes also appear to be involved in the age of onset and the progression of the disease. 2. IL-6 The research performed so far on the possible involvement of different IL-6 gene polymorphisms in AD focused on either a G to C transition in position −174 at the promoter region, or the four VNTR alleles (A–D) at the 3′ flanking area, of the IL-6 gene. These studies yielded somewhat ambiguous results, as will be described below. Two studies reported that the frequency of the IL-6 −174 C allele is increased in AD patients, and that carriers of this allele had a greater risk to develop AD (Arosio et al., 2004; Licastro et al., 2003). When the IL-6 −174 C allele appeared concomitantly with the IL-10 −1082 A allele, the risk of AD was even higher (Arosio et al., 2004). Carriers of the IL-6 −174 CC genotype have increased plasma and CSF levels of sIL-6R (Bagli et al., 2003), which can activate IL-6 signaling (Kishimoto et al., 1992). Furthermore, plasma IL-6 levels were higher in AD compared to controls, and the IL-6 −174 CC genotype was accompanied by the highest levels of IL-6 in the plasma and the brain (Licastro et al., 2003). However, other studies reported a lower frequency of the IL-6 −174 C allele in AD patients, and reduced risk of AD in carriers of this allele, suggesting a protective role, specifically for the IL-6 −174 CC genotype (Faltraco et al., 2003; Pola et al., 2002). Accordingly, the IL-6 −174 G allele was higher in AD patients, and the increase in IL-6 plasma levels

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in AD was higher in carriers of this allele (Pola et al., 2002; Shibata et al., 2002). This finding is in line with a report of lower IL-6 levels in healthy humans with the IL-6 −174 CC genotype (Fishman et al., 1998). It should be noted that additional studies found no differences in IL-6 −174 polymorphism distribution in AD patients (Bhojak et al., 2000; Capurso et al., 2004), and no effect on the age of disease onset (Bagli et al., 2000; Depboylu et al., 2004; Zhang et al., 2004). More consistent results were found in studies that examined the role of IL-6 VNTRs in AD. IL-6 VNTR C allele was found to be under-represented in AD patients, and its presence was associated with reduced risk and delayed age of onset of the disease (Licastro et al., 2003; Papassotiropoulos et al., 1999). Moreover, non-carriers of the IL-6 VNTR C allele demonstrated increased plasma levels of sIL-6R (Bagli et al., 2003), which activates IL-6 signaling (Kishimoto et al., 1992). In contrast, the IL-6 VNTR DD genotype is more frequent in AD patients (Licastro et al., 2003; and trend in Shibata et al., 2002). Furthermore, whereas plasma IL-6 levels are higher in all AD patients compared to controls, carriers of the IL-6 VNTR DD genotype had the highest levels (Licastro et al., 2003). It should be noted, however, that other studies found no involvement of IL-6 VNTRs in AD (Bagli et al., 2000). To conclude, although the nature of the role of IL-6 gene polymorphisms in AD is not clear yet, the findings gathered to date suggest that IL-6 gene VNTR polymorphisms play a role in the risk of developing AD, and that IL-6 −174 polymorphisms may also be involved in AD, possibly via interactions with other genes. 3. TNF-a Several polymorphisms in the TNF-α gene were found to be associated with AD, particularly G to A transitions at positions −308 and −238 and a C to T transition at position −850, all in the promoter region of the TNF-α gene, as well as different TNF-α microsatellite alleles. Some studies examined the effects of these specific polymorphisms on AD, whereas others examined the effect of the 2-1-2 TNF haplotype (TNFA −308 A, −238 G, and TNF-a2 microsatellite) on this disease. Polymorphisms in other locations, either in the TNF-α (position −1031) or in the TNF receptor type II genes, were not associated with AD (Nishimura et al., 2004; Shibata et al., 2004). Several studies reported no association between the frequency of different −308, −238, and TNF-α microsatellite alleles (Culpan et al., 2003; Laws et al., 2005; Perry et al., 2001). Two studies examined the involvement of the TNFA −308 A allele in AD age of onset and

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found contradictory results: Perry et al. (2001) reported increased age of onset in carriers of either the TNFA −308 A allele, which is associated with increased TNFα transcription (Wilson et al., 1997), or the TNFa2 microsatellite allele, whereas Alvarez et al. (2002) found an earlier age of onset in carriers of the TNFA −308 A allele. The research on the involvement of the 2-1-2 haplotype in AD also yielded conflicting results: Collins et al. (2000) reported that the presence of both the TNFa2 microsatellite allele and the TNF 2-1-2 haplotype was associated with increased risk of AD. However, Culpan et al. (2003) found that the absence of the 2-1-2 haplotype increased the risk of AD, suggesting that this haplotype plays a protective role, and this conclusion was substantiated by Perry et al. (2001), who reported that this haplotype is also associated with increased age of onset. Whereas one study reported that the TNFA −850 T allele was over-represented in AD patients and that carriers of this allele had an increased risk for AD (Laws et al., 2005), other studies found no association between the TNFA −850 T allele and either early- or late-onset AD (Infante et al., 2002; McCusker et al., 2001; Terreni et al., 2003). However, the TNFA −850 T allele seems to act in synergy with other genes to increase the risk of AD. In particular, the presence of this allele in conjunction with the APOE ε4 allele resulted in doubling the risk for AD (Laws et al., 2005; McCusker et al., 2001, but see Infante et al., 2002). Similarly, whereas individually the involvement of the TNFA −850 T and −308 G alleles in AD is questionable, −850 T/−308 G haplotype frequency is increased in AD patients, and carriers have a greater risk of developing this disease (Laws et al., 2005). To conclude, different TNF-α gene polymorphisms seem to be involved in the risk to develop AD. However, the exact nature of this involvement is not clear, and the data gathered so far suggest that the effects of these polymorphisms are achieved via interactions with polymorphisms in other genes. Further research may be able to resolve these ambiguous results. 4. Interim Summary The recent genetic studies that examined the involvement of the pro-inflammatory cytokines IL-1, IL-6, and TNF-α in AD suggest a role for these cytokines in the risk to develop AD, as well as in the age of onset and the course of this disease. To date, only a single research directly examined the connection between a specific polymorphism (in IL-1RA VNTR) and the degree of cognitive decline, and found an association between the genotype and the extent of demen-

tia (Bosco et al., 2004). Future studies should further elucidate the role of gene polymorphisms of proinflammatory cytokines in AD dementia. Furthermore, because specific polymorphisms result in changes in peripheral and central cytokine transcription levels, they may also be associated with memory performance in healthy people. Indeed, in a recent pilot study (Shapira Lichter et al., 2004), we found a role for IL-1B −511 polymorphism in stressinduced memory impairment in healthy patients going through a moderate surgical procedure: The IL-1B −511 C allele, which is associated with lower plasma IL-1β levels in AD patients (Licastro et al., 2000b), was found to be protective against the memory impairment that accompanied surgery stress (Shapira Lichter et al., 2004). This finding is in line with the reports of an association between the other allele, IL-1B −511 T, and AD (Bosco et al., 2004; Grimaldi et al., 2000; Wang et al., 2005), as described above. Due to the exceptional complexity of both memory processes and the interactions between different genes, it is unlikely that a single gene accounts for differences in normal memory functioning. However, in recent years, following the human genome sequencing many studies have found associations between gene polymorphisms and several cognitive processes, including memory (reviewed in Goldberg and Weinberger, 2004). For example, a BDNF polymorphism was repeatedly found to be associated with medial-temporalcortex–based declarative memory processes (Goldberg and Weinberger, 2004). Presently, the involvement of polymorphisms in pro-inflammatory cytokine genes in variations of normal memory functioning was not examined. Future studies may reveal if polymorphisms in pro-inflammatory cytokine genes may affect learning and memory processes, alone or in synergy with other gene polymorphisms, as found for the risk and severity of AD.

V. GENERAL SUMMARY AND CONCLUSIONS The present review shows clearly that proinflammatory cytokines are critically involved in several aspects of learning and memory. However, cytokine-induced modulation of memory processes is a complex phenomenon, including both detrimental and beneficial effects, depending on the specific pro-inflammatory cytokine, its levels (particularly within the brain), and the particular condition that elicits the cytokine secretion. Overall, several general conclusions can be drawn from the data:


A. In general, elevations in IL-1, IL-6, and TNF-α levels, produced either by exogenous administration in experimental animals or by endogenous secretion during various diseases or exposure to stressful stimuli in both humans and experimental animals, result in impaired learning and memory. B. These impairments are fairly specific to memory tasks that involve the hippocampus, such as declarative memory in humans and spatial or contextual memory in animals. C. The effects of IL-6 and IL-1 seem to be particularly potentiated as humans and animals become older. This age-dependent effect is demonstrated by the high negative correlations between memory functioning and the levels of IL-6 and IL-1 in elderly people, as well as by the progressive memory deterioration in mice that overexpress IL-6, and the gradual memory improvement in mice lacking IL-6. D. Whereas high levels of IL-1 are consistently associated with memory disturbances, the findings on IL-6- or TNF-α–induced memory disturbances are less robust, and in many studies acute elevations in these cytokines, either in experimental animals or in humans, are associated with either no effect or even with protective effects. E. Pro-inflammatory cytokines seem to be involved in dementia, particularly of the Alzheimer type, in which memory loss is usually the first and most disturbing symptom. This involvement is reflected by altered serum levels of pro-inflammatory cytokines in AD patients; associations between polymorphisms of these cytokines and the incidence, age of onset, and severity of dementia; and evidence from studies in animal models for an involvement of cytokines in the mechanisms of AD. This evidence is particularly strong for IL-1 and IL-6, whereas TNF-α may actually play a protective role in AD. F. Pro-inflammatory cytokines, particularly IL-1, have a physiological role in memory processes. In particular, IL-1 within the hippocampus seems to be important for memory consolidation, reflected by the findings that administration of low doses of IL-1 specifically improves hippocampal-dependent memory functioning, whereas genetic or pharmacological impairments in IL-1 signaling produce memory disturbances. IL-6 may also be important physiologically, as a negative modulator of the consolidation process. G. Pro-inflammatory cytokines have marked effects on various neural and neuroendocrine processes

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that underlie learning and memory processes, including induction and maintenance of hippocampal LTP, alterations in intracellular signal transduction mechanisms, secretion of glucocorticoids and prostaglandins, production of neurotrophins, and neurogenesis. H. The influence of IL-1 on memory functioning and on the mechanisms that subserve it follows an inverted U-shape pattern; i.e., physiological levels of IL-1 are needed for optimal memory functioning, neural plasticity, and glucocorticoid secretion, whereas deviations from the physiological range, either by excess elevation in IL-1 levels or by blockade of IL-1 signaling, result in impaired memory and the associated physiological processes.

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17 Aging, Neuroinflammation, and Behavior RODNEY W. JOHNSON AND JONATHAN P. GODBOUT

neuroinflammatory response is accompanied by severe, longer-lasting behavioral deficits and may cause acute cognitive disorders that are often reported in elderly patients with peripheral infections. This chapter will provide a brief background on how the peripheral innate immune system communicates with the brain, discuss evidence that suggests the emergence of a neuroinflammatory state during normal aging, and present important new findings that suggest a peripheral infection induces an exaggerated neuroinflammatory response and severe behavioral deficits in the aged.

I. INTRODUCTION 379 II. BRAIN, MEET IMMUNE SYSTEM—IMMUNE SYSTEM, MEET BRAIN 380 III. IS NORMAL AGING ASSOCIATED WITH NEUROINFLAMMATION? 382 IV. ARE NEUROINFLAMMATION AND SICKNESS EXACERBATED IN THE AGED? 384 V. IF NEUROINFLAMMATION IS INCREASED, WHAT DOES IT MEAN? 385 VI. CODA 386

ABSTRACT Brain microglial cells are ordinarily quiescent but when stimulated can transition to a “primed” or activated state. Both primed and activated microglia are deramified and express markers that suggest activation, but only activated microglia produce appreciable levels of inflammatory cytokines. Primed microglia, however, are hyper-responsive to a secondary stimulus from the peripheral innate immune system and thus can produce an exaggerated cytokine response when provoked. The potential for primed microglia to mount an exaggerated response is important to psychoneuroimmunology because inflammatory cytokines mediate the sickness behavior syndrome and are involved in chronic neurodegenerative diseases. One physiological event that may prime microglial cells for an exaggerated response is aging. Signs of neuroinflammation emerge in healthy aged subjects, and new findings suggest that aged individuals suffer an exaggerated neuroinflammatory response when the peripheral innate immune system is activated. The exaggerated PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

I. INTRODUCTION Aging is a process characterized by deterioration of a mature organism in the absence of detectable disease. The deterioration is time dependent but occurs at different rates in different individuals, so chronological age is not the same as biological age or senescence (Kirkwood et al., 2005). Nonetheless, aging gradually reduces an organism’s ability to cope with intrinsic and extrinsic factors that cause stress, and thus it increases the probability of disease and death. Because worldwide the segment of the population 60 years old and older is rapidly growing and is anticipated to number 2 billion by year 2050, more people than ever will encounter age-related physical and neurobehavioral deficits. Age-related deterioration of the brain produces a variety of behavioral deficits that are unrelated to disease. Cognitive aging, e.g., is a term used to describe

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a pattern of age-related impairments in cognitive functions including fluid reasoning, mental speed, memory, and spatial ability. Other changes such as decreased motivation for food and deficits in motor coordination are also common. Behavioral indications of brain aging are evident by middle age in clinically healthy individuals, so deterioration of the brain precedes the acceleration in mortality rate that is typical in old age. Individual differences in cognitive ability in late life are determined by the baseline of cognitive ability (e.g., intelligence in childhood) and the rate of cognitive aging (Whalley et al., 2004). The latter of these two is of particular interest to the interdisciplinary field of psychoneuroimmunology because the rate of brain aging is substantially influenced by environmental factors (e.g., chronic psychological stress and pathogenic agents) that elicit intrinsic responses that over time impair neural health (Kiecolt-Glaser et al., 2002; Porter and Landfield, 1998; Verkhratsky et al., 2004). One of the very important intrinsic factors that recently emerged as a leading detractor from successful aging is inflammation in the periphery and brain (Finch and Crimmins, 2004; Godbout and Johnson, 2004; Wilson et al., 2002). Thus, interest in the communication pathways between the immune system and brain and how aging influences these pathways has reached unprecedented heights in recent years. Individual differences not withstanding, the immune system deteriorates with age as well, so the elderly are more susceptible to peripheral infection (Armstrong et al., 1999; Castle, 2000; Pinner et al., 1996). This age-associated impairment of immune function is due to weakened defenses by cells involved in innate immunity (Plowden et al., 2004) and a reduction in naïve T-cells, which are critical for mounting both cell-mediated (T-cell) and humoral-mediated (Bcell) adaptive immune responses to novel antigens (Ginaldi et al., 2001; Ginaldi et al., 2005). Ageassociated changes in a number of key cytokines have been observed. For instance, lymphocytes taken from aged individuals secrete less interleukin (IL)-2, but more IL-4 and IL-10 in response to concanavalin A stimulation, indicating an intrinsic polarization of cells from aged individuals towards type-2 cytokine production, at the expense of type-1 cytokine production (Gorczynski et al., 1997). Interleukin-6 is another cytokine whose secretion changes with age, but what makes it unique is the rather dramatic age-related increase in spontaneous secretion. Lymphoid cells isolated from old but otherwise healthy humans and mice spontaneously secrete high levels of IL-6. Consequently, a positive correlation between age and plasma IL-6 concentration has been reported (Daynes et al., 1993; Wei et al., 1992).

In addition to inducing proliferation of activated B-cells, IL-6 is best known for inducing hepatic acute phase protein synthesis and causing metabolic changes that lead to frailty (Ershler and Keller, 2000). Given the cytokines’ metabolic effects and the once-held notion that the brain is an immunoprivileged site free of influence by the immune system, little consideration was given to the idea that an age-related change in a peripheral cytokine might either directly or indirectly hold influence over brain aging. However, dissecting the communication pathways connecting the peripheral immune system to the brain has led to new views on how peripheral inflammatory events influence brain function. Indeed, elevated serum levels of IL-6 were recently determined to be correlated to deficits in cognitive abilities (Weaver et al., 2002). Thus, how IL-6 and other inflammatory cytokines impinge upon the brain to affect brain aging is an important question. The purpose of this chapter is to provide a brief background on how the peripheral innate immune system communicates with the brain, discuss evidence that suggests the emergence of a neuroinflammatory state during normal aging, and present important new findings that suggest a peripheral infection induces an exaggerated neuroinflammatory response and severe behavioral deficits in the aged.

II. BRAIN, MEET IMMUNE SYSTEM— IMMUNE SYSTEM, MEET BRAIN Several working hypotheses on brain aging and inflammation are strongly influenced by research that has dissected the communication pathways that allow the immune system to induce the sickness behavior syndrome (e.g., anorexia, hypersomnia, lethargy, depression) during acute peripheral infection. Some of the first evidence that a cytokine caused sickness behavior came from studies of slow-wave sleep in rabbits (Krueger et al., 1984). Peritoneal macrophages from rabbits were stimulated in vitro with a muramyl peptide that had been shown to stimulate macrophages to secrete endogenous pyrogen—a protein previously shown to cause fever (Atkins, 1960) and activate the hypothalamic-pituitary-adrenal axis (Besedovsky et al., 1981). The cell-free supernatant was collected and later injected into rabbits. Animals injected with supernatant from stimulated macrophages showed an increase in slow-wave sleep compared to those receiving supernatant from unstimulated macrophages. The same behavior could be induced by injecting the supernatant into a lateral cerebral ventricle, indicating it acted at a central site. Thus, a protein (i.e., endogenous pyrogen) secreted by acti-

17. Aging, Neuroinflammation, and Behavior

vated macrophages that acted in the brain was responsible for the change in behavior. Endogenous pyrogen was later determined to be identical to another protein under investigation—IL-1β. It is now clear that mononuclear phagocytic cells secrete several other behaviorally active cytokines, including tumor necrosis factor α (TNF-α) and IL-6. Inflammatory cytokines appear necessary for the immune system to communicate with the brain (Figure 1). As a result of a mutation in a single gene, a certain strain of mice (C3H/HeJ) is resistant to the behavioral effects of lipopolysaccharide (LPS). When injected with LPS, this strain of mice does not become overtly ill like normal mice do (Johnson et al., 1997; Segreti et al., 1997). The mutation is in the Toll-like receptor 4 gene (Hoshino et al., 1999), a gene which encodes an extracellular receptor expressed on the surface of mononuclear phagocytic cells that specifically binds LPS to activate an innate immune response. Therefore, this mutation essentially prevents the animal’s mononuclear phagocytic cells from producing cytokines in response to LPS, so there is a complete breakdown in communication between the immune system and brain. The brain of these mice recognizes cytokines because if injected with recombinant IL-1β, they respond normally by reducing food intake, losing weight, and decreasing social behavior (Johnson et al., 1997).

C3H/HeOuJ

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Plasma IL-1b 1

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How do peripheral inflammatory cytokines instigate communication between the immune system and brain during infection? Although there is clear evidence that inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) can be actively transported from the blood into the brain (Banks and Kastin, 1991; Banks et al., 1991; Banks et al., 1994; Banks et al., 1995; Gutierrez et al., 1993), peripheral cytokines need not enter the brain to elicit behavioral changes. It is now evident that inflammatory stimuli in the periphery (e.g., LPS and inflammatory cytokines) induce IL-1β, IL-6, and TNF-α in discrete brain areas of the mouse (Ban et al., 1992; Laye et al., 1994). For example, inflammatory stimuli in the periphery induce perivascular microglial cells to express cytokines (van Dam et al., 1992; van Dam et al., 1995). Furthermore, cytokines in the periphery can convey a message to the brain via the vagus nerve. After LPS challenge, dendritic cells and macrophages that are closely associated with the abdominal vagus have been shown to express IL-1β protein (Goehler et al., 1999). IL-1 binding sites have been identified in several regions of the vagus as well (Goehler et al., 1997). When activated by peripheral cytokines, the vagus, of course, can activate specific neural pathways that are involved in sickness behavior. However, activation of the vagus also appears to stimulate microglia in the brain to produce cytokines. If the vagus nerve is severed just below the diaphragm in rats, the

The strains are similar but C3H/HeJ mice have a mutated Toll-like receptor 4 (TLR4) gene. TLR4 on the surface of macrophages recognizes LPS and is involved in the innate immune response to Gram-negative bacteria.

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Macrophages of C3H/HeOuJ mice secrete inflammatory cytokines in response to LPS, but due to the mutation in the TLR4 gene macrophages of C3H/HeJ mice do not.

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C3H/HeOuJ mice show typical sickness behavior when challenged with LPS, but C3H/HeJ mice do not. If C3H/HeJ mice are administered recombinant IL-1β, they show typical sickness behavior, indicating inflammatory cytokines are needed for the immune system to convey a message to the brain.

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FIGURE 1 Overview of studies with C3H/HeJ mice, which show the importance of inflammatory cytokines for immune system–to–brain communication. Adapted from Segreti et al. (Segreti et al., 1997) and Johnson et al. (Johnson, 2002; Johnson et al., 1997).

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expression of cytokines in the brain and the sickness behavior that normally occurs after intraperitoneal injection of LPS or IL-1β can be inhibited (Bluthe et al., 1996a, b; Laye et al., 1995). Plasma levels of cytokines are elevated in LPS-injected vagotomized rats, indicating the neural signal is needed for the induction of sickness. The neural pathways activated in the brain by the vagus nerve for rapid immune-to-brain signaling have been described in some detail (Dantzer, 2001). These pathways appear to be responsible for activating the hypothalamic-pituitary-adrenal axis and depressing behavior in response to infection. Other mechanisms that involve induction of prostaglandin synthesis are also important for fever. Based on extensive mapping of the temporal and spatial expression of Fos (a marker for neural activity) and IL-1β, Konsman et al. (1999) proposed a slower immune-to-brain signaling mechanism based on volume transmission. In this method of immune-to-brain communication, production of IL-1β by the brain first occurs in the choroid plexus and circumventricular organs—brain areas devoid of an intact blood-brain barrier. The cytokines then slowly diffuse into the brain by volume transmission, along the way activating neurons and neural pathways relevant for expression of sickness behavior. This pathway may be more important for maintaining sickness behavior than inducing it. Regardless of the pathway(s) at work, it is evident that if inflammatory cytokines are increased in the periphery, they may be increased in the brain too.

III. IS NORMAL AGING ASSOCIATED WITH NEUROINFLAMMATION? Microglial cells are the most important cells of the innate immune system in the brain. Their phenotype is similar to macrophages; and like macrophages in the periphery, microglia in the brain serve as antigenpresenting cells and produce inflammatory cytokines. They are normally quiescent but are activated by factors such as brain trauma, ischemia, neurodegenerative disease, and infection. A critically important but less studied factor that may influence microglia is normal aging (Finch, 2003). In several age-sensitive areas of the hippocampal formation (dentate gyrus and CA1 region) of female mice, the number of microglia and astrocytes is increased about 20% (Mouton et al., 2002). Activated microglia express markers such as MHC class II antigens, which are increased in brains of aged but otherwise healthy humans, non-human primates, and rodents (Perry et al., 1993; Rogers et al., 1988; Sheffield and Berman, 1998; Streit and Sparks, 1997). Recently, morphological changes were charac-

terized in microglia from a healthy, cognitively normal (i.e., non-demented) elderly donor (Streit et al., 2004). Microglia in the aged brain had abnormalities in the cytoplasmic structure and were deramified, indicating a reactive state (i.e., primed but not activated). The morphological changes were distinct from those seen in neurodegenerative disease or brain trauma, where microglia are fully activated. The so-called “microglial dystrophy” has been interpreted to suggest microglial senescence. In aged brains dystrophic microglia were widespread and the number surpassed by nine-fold that seen in the young adult brain (Streit et al., 2004). Surprisingly, the change in microglial cells is reminiscent of the age-associated change in pyramidal neurons that was first reported 30 years ago (Scheibel et al., 1975). Thus, a change in microglial cells may affect neuronal morphology and ultimately function and cause cognitive impairment in the elderly. Very recently microglial senescence was proposed to result in functional defects that trigger an intracerebral inflammatory response that supports the development of age-associated neurodegenerative diseases such as Alzheimer’s disease (AD) (Blasko et al., 2004; Streit, 2004). In addition to signs of activated microglia, there also is good evidence that inflammatory cytokines are increased in brains of aged but otherwise healthy rodents. Much of this work has focused on IL-6, owing to the fact that it was the first inflammatory cytokine shown to be elevated in the periphery of healthy aged animals. For instance, the level of IL-6 in whole brain as well as in the hippocampus, cerebral cortex, and cerebellum was increased in aged mice compared to juvenile and adult mice (Ye and Johnson, 1999). Aging did not appear to increase IL-6 in the hypothalamus. Similarly, 10-month-old senescence-accelerated mice of the P8 strain—a murine model for accelerated aging—had increased IL-6 in the cerebral cortex and hippocampus compared to age-matched controls (Tha et al., 2000). Several studies indicate that the increased IL-6 in the aged brain is centrally produced and not simply a reflection of what is found in the peripheral circulation. Coronal sections of the cerebral cortex taken from aged rodents spontaneously secreted more IL-6 than sections taken from younger controls (Prechel et al., 1996; Ye and Johnson, 2001a). Furthermore, competitive quantitative RT-PCR showed a marked increase in steady-state IL-6 mRNA in whole brain of healthy aged mice compared to adults (Ye and Johnson, 2001b). Studies on glial cells cultured from aged mice also indicate an age-associated increase in IL-6 production. Ye and Johnson (1999) established glial cell cultures from neonate, adult, and aged mice that contained both astrocytes and microglia. The


steady-state level of IL-6 mRNA and spontaneous secretion of IL-6 protein were higher in glial cells cultured from aged mice than in glia cultured from either adult or neonate mice (Ye and Johnson, 1999, 2001b). Two-color flow cytometric analysis of IL-6 and MAC-1 (a marker for microglia) staining showed an agerelated increase in IL-6–positive microglial cells (Ye and Johnson, 1999). Moreover, the IL-6 mean fluorescence intensity increased as a function of age, suggesting more IL-6 expression per cell. Brain endothelial cells, which form the blood-brain barrier, may also contribute IL-6 to the aged brain. Endothelial cells cultured from brains of very old rhesus monkeys spontaneously released more IL-6 than those isolated from adult and fetal/neonatal monkeys (Reyes et al., 1999). Collectively, these studies indicate that the transcription and expression of IL-6 is increased in the brain with age. The dysregulation of IL-6 secretion by microglia in the aged brain has been extended to include a description of how aging influences transcription factors that regulate the IL-6 gene. Interleukin-6 gene expression is regulated by a complex arrangement of several transcription factors including nuclear factor κ B (NFκB) (Libermann and Baltimore, 1990), multiple response element (MRE), C/EBPβ/NF-IL-6, and AP-1 (Li and Karin, 1999). The age-related increase in IL-6 gene expression in the brain, however, appears to be under control of NFκB. Relative to the adult brain, NFκB binding to the IL-6 gene promoter was increased in the brain of aged mice (Ye and Johnson, 2001b). The DNA binding activity of NF-IL-6, AP-1, and MRE was either unchanged or decreased in whole brains of aged mice. Moreover, intracerebroventricular injection of κB decoy—a molecule that competes with the DNAbinding sequences for NFκB—decreased NFκB DNAbinding activity and IL-6 mRNA in the brain of aged mice (Ye and Johnson, 2001b). Parallel studies with mixed glia cultures from aged mice also revealed increased NFκB binding to the IL-6 promoter. Incubating glia from aged mice with κB decoy decreased NFκB activity, IL-6 mRNA, and IL-6 protein secretion to levels similar to that in glia from adults (Ye and Johnson, 2001b). These findings are supported by other studies of age-related or disease-associated increases in NFκB transcriptional activity. For example, NFκB DNA-binding activity is increased in the aged rat forebrain and hippocampus (Korhonen et al., 1997; ToliverKinsky et al., 1997) and in hippocampal and cerebral cortical neurons of AD patients (Terai et al., 1996). Furthermore, in peripheral lymphoid organs of aged mice, NFκB activation is associated with chronic inflammation (Poynter and Daynes, 1999; Spencer et al., 1997).

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It is noteworthy that the concept of age-related neuroinflammation has begun to meld with another wellknown hypothesis on aging—namely, the “free radical hypothesis of aging” (Beckman and Ames, 1998). According to this hypothesis, damage to cell membranes and intracellular proteins increases due to either an increase in oxygen free radicals (i.e., reactive oxygen species, ROS), a decrease in the capacity of the antioxidant defense mechanisms to scavenge ROS, or some combination of these two (Beckman and Ames, 1998). The brain is particularly sensitive to oxidative damage because it is rich in polyunsaturated fatty acids, a principal target for lipid peroxidation. Bolstering the antioxidant defense mechanisms by feeding animals diets supplemented with antioxidant vitamins (e.g., vitamin E) or with fruits and vegetable extracts with high antioxidant activity has been shown to prevent and even reverse several age-related deficits in cognitive and motor function (Joseph et al., 1998). Likewise dietary restriction has been shown to protect neurons from oxidative insults and delay behavioral signs of brain aging (Guo et al., 2000). A recent study indicated that cognitive function in elderly people was related to vitamin E status (Ortega et al., 2002), and several epidemiological studies suggested that consuming an antioxidant-rich diet reduced the risk for age-associated neurodegenerative diseases that involve oxidative damage and impaired cognitive and motor function (Schapira and Olanow, 2004; Zandi et al., 2004). Although many potential mechanisms underlying the deleterious effects of oxidative stress on cognitive and motor behavior have been suggested, precisely how oxidative stress leads to neurobehavioral deficits is not known. The accumulated effects of oxidative stress appear to render neurons more susceptible to apoptosis, perhaps by reducing protein chaperones (e.g., heatshock protein-70; HSP-70) and neurotrophic factors (e.g., brain-derived neurotrophic factor; BDNF). Dietary restriction, which delays brain aging and increases life span, increased both HSP-70 and BDNF and protected neurons from oxidative insults (Mattson et al., 2003). Oxidative stress may also affect cognitive and motor behavior by activating intracellular signaling pathways that ultimately promote the expression of genes whose products cause inflammation and are behaviorally active. The inflammatory cytokines represent one such example. For instance, oxygen-free radicals can activate stress kinase pathways that activate NFκB (Flohe et al., 1997). α-tocopherol—an oxygen free radical scavenging agent—inhibits LPS-induced oxidative stress, NFκB DNA-binding activity, and IL-6 production in microglia and brains. For example, treating primary microglia with α-tocopherol prior to LPS reduced

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intracellular peroxides and IL-6 production (Godbout et al., 2004). Moreover, α-tocopherol was found to reduce NFκB DNA-binding activity, IL-1β, IL-6, and TNF-α in brains of mice challenged peripherally with LPS (Godbout, 2005a). Other studies showed that feeding mice diets high in antioxidants was effective at reducing LPS-induced sickness behavior in both adult and aged mice (Berg et al., 2004; Berg et al., 2005). Further support for melding of the two hypotheses comes from a recent study that investigated the relationship between brain oxidative stress, brain IL-6 production, and psychomotor deficits during aging. Mice reared in SPF conditions and ranging in age from 3–24 months were studied. There was a decline in motor function after 12 months of age and an increase in brain lipid peroxidation and IL-6 production by coronal brain slices ex vivo. Aging mice provided an antioxidant-rich diet for 6 months had improved psychomotor coordination compared to mice fed a diet adequate or low in antioxidants. Furthermore, when mice were tested on successive days, only those fed adequate and high antioxidants exhibited motor learning. Analysis of IL-6 production by coronal brain slices indicated that as dietary antioxidants increased, IL-6 production decreased (Richwine et al., 2005). That oxidative stress and inflammatory cytokines might both contribute to ageassociated deficits in cognition was previously suggested when long-term dietary supplementation with α-tocopherol prevented an age-associated increase in IL-1β and improved long-term potentiation in dentate gyrus of rats [O’Donnell and Lynch, 1998; Murray and Lynch, 1998]. The cytokine IL-1β may cause deficits in LTP by stimulating stress-activated kinases, which in turn inhibit glutamate release (Vereker et al., 2000) Other studies that examined gene expression in the brain on a global scale by microarray analysis also provide compelling evidence for an age-related shift towards a neuroinflammatory state. With an oligonucleotide-based array representing 6,347 genes, Lee et al. (2000) revealed a gene expression profile in the cortex and cerebellum of aged mice indicative of increased inflammation and oxidative stress. The effects of age on gene expression were annulled by caloric restriction—the only treatment known to slow aging and increase maximum life expectancy. Another microarray study that compared gene expression in brains of young adult and aged mice found 38 genes associated with inflammation that were differentially expressed—all but 4 were upregulated in the aged brain (Godbout, 2005b). And another study reported that an inflammatory profile was already evident in the hippocampus of middle-aged rats, and that the emergence of this profile preceded ageassociated cognitive deficits (Blalock et al., 2003).

IV. ARE NEUROINFLAMMATION AND SICKNESS EXACERBATED IN THE AGED? During peripheral infection, the inflammatory cytokines that are produced within the brain target neuronal substrates and elicit a behavioral response that is ordinarily adaptive (Dantzer, 2004; Johnson, 2002). However, excessive or prolonged release of inflammatory cytokines has been shown to produce severe behavioral deficits and promote neurotoxicity (Campbell et al., 1993; Finck and Johnson, 1997; Heyser et al., 1997). A number of studies involving intracerebroventricular administration of inflammatory cytokines or transgenic animal models that overexpress certain cytokines suggest that the magnitude and/or duration of sickness behavior is proportional to the quantity of inflammatory cytokines in the brain (Finck and Johnson, 1997; Heyser et al., 1997; Yao et al., 1999). Thus, circumstances that augment inflammatory cytokine production by microglial cells are likely to lead to pronounced and prolonged behavioral deficits and changes in neuronal circuitry that are counterproductive to recovery and rehabilitation after infection or injury. Several studies showed that coronal brain sections and primary mixed glia from aged rodents produced more IL-1β and IL-6 compared with adults when treated with LPS (Xie et al., 2003; Ye and Johnson, 2001a). The study by Ye et al. (Ye and Johnson, 2001a) also found that sections from aged brains secreted less IL-10 in both the absence and presence of LPS, and a decrease in IL-10 mRNA in the hippocampus of aged rats was recently reported (Frank et al., 2005). Therefore, deficits in important anti-inflammatory cytokines may contribute to the rise in inflammatory ones. Recent studies indicate that normal aging is associated with increased neuroinflammation and that an exaggerated inflammatory response occurs in the brain of healthy aged animals when the peripheral innate immune system is activated. The idea that systemic infection may result in an exaggerated inflammatory response in the aged brain emerged from several studies that suggested neurodegenerative diseases that involve primed or activated microglia are exacerbated by peripheral infection (Perry, 2004; Perry et al., 2003). In this situation, elements unique to the neurological disease provide a primary stimulus for microglial cell activation, and peripheral infection provides a secondary stimulus (Perry et al., 2003). In the ME-7 murine model of prion disease, microglia are activated throughout the limbic system, including the hippocampus where neuron loss occurs (Cunningham et al., 2003). In mice with pre-clinical ME-7–induced prion disease, a peripheral LPS challenge induced an exag-

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gerated depression in locomotor behavior and body temperature, and increased brain IL-1β compared to controls (Combrinck et al., 2002). In a transgenic murine model of amyotrophic lateral sclerosis (ALS), disease progression was exacerbated in presymptomatic mice given repeated i.p. injections of LPS (Nguyen et al., 2004). Toll-like receptor 2 and TNF-α were upregulated in discrete brain and spinal cord regions where degeneration occurred, and the average life span of ALS mice given LPS was decreased by 3 weeks (Nguyen et al., 2004). Finally, inflammatory cytokines produced by microglia are involved in chronic neurodegeneration in MS and AD. Systemic infection is a risk factor that can precipitate relapse in MS patients (Sibley et al., 1985) and trigger delirium in individuals with AD (Holmes et al., 2003). Deposition of β amyloid within senile plaques is a classical feature of AD, and conditions leading to the expression of inflammatory cytokines in the brain are associated with the rapid increase in β amyloid precursor protein (Brugg et al., 1995). In the Tg2576 murine model of AD, intravenous injection of LPS caused age-dependent increases in brain IL-1β and TNF-α levels and increased β amyloid deposition (Sly et al., 2001). This may provoke a positive feedback loop since β amyloid activates microglia (Giulian, 1999). Since activation of the peripheral innate immune system induces brain microglia to produce inflammatory cytokines that are responsible for behavioral deficits, it is reasonable to suspect that aging would exacerbate neuroinflammation and sickness behavior when the peripheral innate immune system is activated. Indeed, this important point has recently been found to be true in studies at the University of Illinois (Godbout, 2005b). Gene expression profiling demonstrated that aged mice had a unique gene expression profile in the brain both before and after an i.p. LPS injection. Moreover, the LPS-induced elevation in brain inflammatory cytokines and oxidative stress was both exaggerated and prolonged compared to adults. Aged mice were anorectic longer and lost more weight than adults following peripheral LPS administration. Remarkably, reductions in both locomotor and social behavior remained 24 hours later, when adults had fully recovered. We recently observed signs of depression in a forced swim test in aged mice 72 hours after LPS injection—a time when other signs of sickness had waned (unpublished data). Another group showed that aged rats suffered a similar exaggerated neuroinflammatory response during a peripheral infection and that the heightened neuroinflammation was associated with deficits in hippocampal-dependent learning and memory (Barrientos et al., 2005). Taken together, these studies show that peripheral infection

leads to exacerbated neuroinflammation in aged rodents. It is important to note that neither Godbout et al. (2005b) nor Barrientos et al. (2005) found peripheral inflammatory cytokines to be a reliable indicator of the exaggerated neuroinflammatory response. Thus, the dysregulated link between the peripheral and central innate immune system is likely to be involved in the severe behavioral deficits that frequently occur in older adults with systemic infections.

V. IF NEUROINFLAMMATION IS INCREASED, WHAT DOES IT MEAN? An exaggerated neuroinflammatory response may underlie the cognitive deficits that are highly prevalent in elderly patients (Figure 2). For example, acute cognitive impairment (i.e., delirium) is the most common psychiatric condition experienced by elderly emergency department patients (Chiovenda et al., 2002; Jackson et al., 2004; Wofford et al., 1996b) and most frequently results from infections that are unrelated to the CNS (Wofford et al., 1996a). In fact, the most striking characteristic of pneumonia (either viral or bacterial) in elderly patients is that it commonly presents clinically as delirium, even in the absence of classical pneumonia symptoms (Janssens and Krause, 2004). Overall, cognitive impairment results in a failure of self-care and is associated with increased hospitalization and delayed recovery (Johnston et al., 1987). The

(+) Infection Cognitive Impairment ↓ Self-Care

•Anorexia •Weight loss •Less likely to visit out-patient clinic

↑ Hospitalization ↑ Mortality Rate FIGURE 2 Elderly patients with a peripheral infection frequently experience cognitive disorders that reduce selfcare behaviors. Thus, cognitive impairment increases the likelihood of developing a new infection and can result in increased hospitalization and mortality.

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severity of dementia has been related to mortality of infectious disease, indicating an insidious relationship between infection and cognition, whereby in the elderly infection induces cognitive impairment and the cognitive impairment exacerbates the infection. In fact, peripheral infection has been found to enhance the risk of dementia in otherwise healthy individuals over the age of 84 (Dunn et al., 2005). Infection also is a risk factor for neurodegenerative diseases including multiple sclerosis (MS) (Sibley et al., 1985) and AD (Holmes et al., 2003). Because microglial cells and inflammatory cytokines are implicated in neurodegenerative diseases, an exaggerated inflammatory response in the aged brain may also underlie this well-documented but poorly understood phenomenon. In support of this hypothesis, a recent microarray study that allowed us to investigate gene expression on a global scale revealed that many inflammatory genes and a number of genes in the β-amyloid family are upregulated in the brains of aged mice but not adults after peripheral LPS challenge (Godbout, 2005b). Most studies concerning interactions between inflammatory cytokines and CNS neurons have focused on neurodegeneration, and more recently, inhibition of neurogenesis in the adult brain (Ekdahl et al., 2003; Monje et al., 2003). However, the transient increase in inflammatory cytokines in the brains of healthy aging subjects and the inflammatory cytokines produced in the brain when the peripheral innate immune system is activated are not likely to be acutely neurotoxic. For instance, although the decline in learning and memory in the aged was originally attributed to hippocampal cell loss (West, 1993), methodologically unbiased studies in rodents and primates, including humans, have now confirmed that in the absence of neurodegenerative disease cell number is preserved in this brain area (Calhoun et al., 1998; Rapp and Gallagher, 1996; Rasmussen et al., 1996). Serge Rivest’s group at Laval University has used in situ hybridization and immunohistochemical staining procedures to carefully examine the effects of peripheral LPS on microglial cell activation in young adult mice (Laflamme et al., 2001; Laflamme and Rivest, 2001). Despite robust activation of microglial cells, no indication of neuron loss under these conditions has been observed (Rivest, 2003). Although the effects of normal aging and systemic infection may conspire and produce an environment that is neurotoxic, it also is reasonable to suspect that changes in function and in existing structure, particularly synapse number and structure, underlie the profound behavioral changes evident in elderly adults with systemic infections. This is important because changes in dendrite morphology are of significant consequence to learning and memory: Treatments that

improve memory and learning, such as environmental enrichment, do so in part by increasing opportunities for synaptic inputs (Moser et al., 1994; Silva-Gomez et al., 2003). Thus, changes in dendrite morphology are likely to underlie the age-associated functional changes (de Brabander et al., 1998; Jacobs et al., 1997; Peters, 2002). A recent study examined the effects of conditioned supernatants from LPS-stimulated microglia on morphology and death of differentiated Neuro2a cells (a neuroblastoma cell line) (Munch et al., 2003). On the one hand, conditioned supernatants from microglia subjected to high concentrations of LPS caused neuronal cell death in a dose-dependent manner. On the other hand, supernatants from microglia exposed to low concentrations of LPS did not induce cell death but inhibited retinoic acid–induced neurite outgrowth and stimulated retraction of already-extended neurites. Another study showed TNF-α prevented neurite outgrowth and arborization in primary murine hippocampal neurons (Neumann et al., 2002). In demented AIDS patients, there is neuronal atrophy but no cell loss in the hippocampal formation (Sa et al., 2000). A recent Golgi impregnation study found diminished dendritic trees in all hippocampus neuron populations examined, including the dentate gyrus and CA1 and CA3 regions (Sa et al., 2004). These results are consistent with what was found in neocortical neurons of AIDS patients (Wiley et al., 1991), and suggest inflammatory cytokines as a common underlying mechanism responsible for the dendritic degeneration. Inflammatory cytokines, especially IL-1β, have been suggested to be involved in the pathogenesis of delirium, which is thought to be due to changes in cerebral metabolism that result in reduced synthesis of acetylcholine (Eikelenboom et al., 2002). IL-1β decreased extracellular acetylcholine in the hippocampus, and LPS administered into the forebrains of rats selectively decreased the number of choline acetyltransferase immunoreactive neurons (Willard et al., 1999). Thus, inflammatory cytokines have potential to influence both neuron structure and function.

VI. CODA Because the elderly are often immunosuppressed and susceptible to infection, the concept that aging may sensitize or prime the brain microglial compartment so that an exaggerated inflammatory response occurs when the peripheral immune system is activated is vitally important. As illustrated in Figure 3, a neuroinflammatory state emerges in the aged, and an exaggerated inflammatory response by primed microg-

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Brain Aging Neuroprotective Growth Factors

Inflammation

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(IL-1b, (IL-1β β, IL-6, TNFa) TNFα α) Innate immune system

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(+++) Sickness Cognitive Impairment Disease Depression Anorexia Weight Loss

FIGURE 3 A heightened inflammatory state emerges in the aged brain, and microglial cells are primed. When a secondary stimulus from the peripheral innate immune system is experienced, primed microglia in the aged brain respond in an exaggerated fashion, producing more inflammatory cytokines than anticipated. The increase in inflammatory cytokines, coupled with age-associated reductions in neuroprotective factors, make neurons more susceptible to injury and exacerbate behavioral deficits. Adapted from Godbout et al. (2005b).

lia may further exacerbate conditions known to be driven in part by inflammatory cytokines. More severe behavioral deficits and delayed recovery are likely to contribute to disease and mortality in elderly patients whose ability to cope with intrinsic and extrinsic factors that cause stress is already diminished by the aging process. Thus, inhibiting neuroinflammation, especially in the presence of a peripheral infection, may be particularly important for enabling successful aging.

Acknowledgments This work was supported by NIH grants AG16710 and MH069148. J.P.G. was supported by a Ruth L. Kirchstein NRSA Postdoctoral Fellowship.

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C H A P T E R

18 Neuroimmune Interactions and Pain: The Role of Immune and Glial Cells LINDA R. WATKINS, JULIE WIESELER-FRANK, MARK R. HUTCHINSON, ANNEMARIE LEDEBOER, LEAH SPATARO, ERIN D. MILLIGAN, EVAN M. SLOANE, AND STEVEN F. MAIER

phine is discussed, including the implications of these findings for clinical pain control.

I. PAIN BASICS 393 II. THE PROBLEM OF CHRONIC PAIN 396 III. HISTORICAL OVERVIEW: DISCOVERIES LEADING TO THE RECOGNITION OF IMMUNE/GLIAL INVOLVEMENT IN CHRONIC PAIN 396 IV. THE CONTRIBUTION OF PERIPHERAL IMMUNE CELLS TO CHRONIC PAIN 398 V. THE CONTRIBUTION OF CNS IMMUNE-LIKE GLIAL CELLS TO CHRONIC PAIN 401 VI. PHYSIOLOGY AND FUNCTION OF GLIA: GENERAL PRINCIPLES 401 VII. PAIN ENHANCEMENT BY ACTIVATED SPINAL CORD GLIA 405 VIII. BEYOND CHRONIC PAIN: GLIAL CELLS OPPOSE THE PAIN-SUPPRESSIVE EFFECTS OF OPIOIDS 408 IX. CONCLUSIONS AND IMPLICATIONS 409

I. PAIN BASICS Pain normally serves adaptive functions (Nash, 2005). First, pain is a sensory warning system. Pain signals relayed by sensory neurons to the spinal cord trigger the activation of protective reflexes. These reflexes rapidly remove endangered body parts from the potentially dangerous stimulus, thereby preventing or minimizing tissue damage. In turn, spinal cord neurons relay the pain message to the brain. Here, adaptive behaviors are organized, such as changing course so as not to continue walking on broken glass. Second, pain serves a recuperative function. After injury, pain motivates attention to the wound, as well as inactivity and behavior related to healing. Thus, normal pain promotes survival. Indeed, the survival value of pain is made all the more apparent by people born without the ability to feel pain (Nash, 2005). Such people lean on hot stoves and realize it only upon smelling their burning flesh, fail to avoid sharp objects, and are unaware of bone breaks or internal injuries which become life threatening as a result. They learn only with great difficulty how to survive in a world full of danger. The pain pathway from body to brain has classically been conceived of as a fairly simple chain of neurons. In skin, joints, muscle, and internal organs, sensory nerve terminals express receptors specialized to detect a wide range of potentially threatening

ABSTRACT This chapter explores how pain can be powerfully amplified by the activation of peripheral immune cells associated with peripheral nerves and by the activation of immune-like glial cells (microglia and astrocytes) within the central nervous system. After review of the basics of pain and pain modulation, the focus will shift to chronic pain, which is poorly if at all controlled by currently available drugs. The thesis will be developed that such pain arises as a consequence of immune and glial activation. The involvement of peripheral immune cells and glia in such pain states is explored. Finally, the very recent finding that glia also oppose the pain-suppressive effects of drugs like morPSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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(A)

(B)

Quiescent glia

Incoming A- /C fiber ‘pain’ signals Normal substance P release, EAA release

NK-1 receptor

PTN

AMPA receptor

Pain message to brain NMDA receptor

Pain stimulus

(C)

(D)

Viruses and bacteria

Non-existent glia

NK-1 receptor

AMPA receptor

PTN cNOS L-arginine

NO

Incoming A- /C fiber ‘pain’ signals Enhanced substance P release, EAA release

Ca2+ NMDA receptor

PTN – NO, PGs, Fractalkine

Primary afferent – Substance P, EAAs, ATP

Activated glia

IL-1, TNF, IL-6, ROS, NO, PGs, EAAs, ATP

Enhance PTN excitability

Enhance primary afferent, substance P and EAA release TRENDS in Neurosciences

stimuli, such as heat, cold, pressure, and various inflammatory mediators (acid, serotonin, proinflammatory cytokines, bradykinin, prostaglandins) (Julius and Basbaum, 2001). Upon activation, these nerve terminals create electrical signals that are relayed to the spinal cord dorsal horns (Figure 1A,B). Pain from the left side of the body is relayed to the left dorsal horn; pain from the right side of the body, to the right dorsal horn. The dorsal horns of the spinal cord are somatotopically organized, so, for example, pain from your abdomen and legs is relayed to the dorsal horns in the lumbar spinal cord. Even here, the neural patterning is exquisite, with a tiny map of the abdomen and legs intricately organized within each spinal cord slice (Clarke and Harris, 2004). At all levels of the spinal cord, the dorsal horns contain neurons that, upon receiving pain messages from sensory neurons, relay this information up to higher brain areas where the pain message is interpreted and acted upon.

However, pain is not simply, passively relayed from the body to the brain. Rather, pain is dynamically modulated. The spinal cord dorsal horns are the major site of pain modulation (Gebhart, 2004). When sensory neurons relay pain information to dorsal horn neurons, the pain message may be suppressed, relayed unaltered, or amplified. For example, if you have surgery and are given morphine for your pain, morphine acts at the spinal cord dorsal horns, preventing pain messages from being relayed to higher brain centers. Thus, you are unaware of the pain. Similarly, life-threatening events activate well-defined brain-to-spinal cord pathways, leading to the release of endogenous morphinelike neurotransmitters (enkephalins) within the spinal cord dorsal horns, again suppressing pain. Analgesia (“without pain”) under such fight/flight situations is adaptive, as being unaware of pain facilitates defense and escape (Gebhart, 2004). On the other hand, hyperalgesia (“exaggerated pain”) can also be adaptive. Hyperalgesia occurs as a

18. Neuroimmune Interactions and Pain: The Role of Immune and Glial Cells

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FIGURE 1 Classical and non-classical views of pain transmission and pain modulation. Panel A: Classical pain transmission pathway. When a noxious (painful) stimulus is encountered, such as stepping on a nail as shown, peripheral “pain”-responsive A-delta and C nerve fibers are excited. These axons relay action potentials to the spinal cord dorsal horn. Here, neurotransmitters are released by the sensory neuron, and these chemicals bind to and activate postsynaptic receptors on pain transmission neurons (PTNs) whose cell bodies reside in the dorsal horn. Axons of the PTNs then ascend to the brain, carrying information about the noxious event to higher centers. The synapse interconnecting the peripheral sensory neuron and the dorsal horn PTN is shown in detail in panels B and C. Panel B: Normal pain. Under basal conditions, pain is not modulated by glia. Under these circumstances, glia are quiescent, and thus not releasing pain-modulatory levels of neuroexcitatory substances. Information about noxious stimuli arrives from the periphery along A-delta and C fibers, causing the release of substance P and excitatory amino acids (EAAs) in amounts appropriate to the intensity and duration of the initiating noxious stimulus. Activation of neurokinin-1 (NK-1) receptors by substance P and activation of AMPA receptors by EAAs cause transient depolarization of the PTNs, thereby generating action potentials that are relayed to the brain. NMDA-linked channels are silent as they are chronically “plugged” by magnesium ions. Panel C: Pathological pain: classical view. In response to intense and/or prolonged barrages of incoming “pain” signals, the PTNs become sensitized and over-respond to subsequent incoming signals The intense and/or prolonged barrage depolarizes the PTNs such that the magnesium ions exit the NMDA-linked channel. The resultant influx of calcium ion activates constitutively expressed nitric oxide synthase (cNOS), causing conversion of L-arginine to nitric oxide (NO). Because it is a gas, NO rapidly diffuses out of the PTNs. This NO acts presynaptically to cause exaggerated release of substance P and EAAs. Postsynaptically, NO causes the PTNs to become hyperexcitable. Glia have not been considered to have a role in creating pain facilitation in this neuronally driven model. Panel D: Pathological pain: new view. Here, glial activation is conceptualized as a driving force for creating and maintaining pathological pain states. The role of glia is superimposed on the NMDA-NO–driven neuronal changes detailed in panel C, so only the aspects added by including glia in the model are described here. Glia are activated (shown as hypertrophied relative to panel [B], as this reflects the remarkable anatomical changes that these cells undergo on activation) by three sources: bacteria and viruses which bind specific activation receptors expressed by microglia and astrocytes; substance P, EAAs, fractalkine, and ATP released by A-delta and/or C fiber presynaptic terminals (shown here) or by brain-to-spinal cord pain enhancement pathways (not shown); and NO, prostaglandins (PGs) and fractalkine released from PTNs. Following activation, microglia and astrocytes cause PTN hyperexcitability and the exaggerated release of substance P and EAAs from presynaptic terminals. These changes are created by the glial release of NO, EAAs, reactive oxygen species (ROS), PGs, pro-inflammatory cytokines (for example, IL-1, IL-6 or TNF), and nerve growth factor. From: Watkins, L. R., Milligan, E. D., and Maier, S. F. (2001). Glial activation: a driving force for pathological pain, Trends in Neuroscience, 24, 450–455. (Copyright permission being requested from TiNS.)

result of enhanced sensory neuron excitability, enhanced release of “pain” neurotransmitters from sensory neurons into the dorsal horn, and/or enhanced excitability of dorsal horn neurons that relay pain messages to the brain (Scholz and Woolf, 2002). Such enhancement of pain signaling in response to tissue damage or infection can be useful. It increases focus on the injury to ensure attention to the injury and can promote learning to avoid such injuries in the future. While many underlying mechanisms have been proposed to account for hyperalgesia (Gebhart, 2004; Scholz and Woolf, 2002), describing the most commonly accepted, classical view will provide the neces-

sary background for understanding sections that follow (Figures 1B,C). In this view, the key event is a change in function of neuronal NMDA receptors in the spinal cord dorsal horns (Petrenko et al., 2003). Pain-responsive neurons in the spinal cord express an array of receptors for transmitters that are released by sensory neurons upon their activation by painful stimuli. These transmitters include substance P and glutamate. Substance P is bound by neurokinin-1 (NK-1) receptors, while glutamate is bound by AMPA and NMDA receptors. Such binding to NK-1 and AMPA receptors always increases the excitability of neurons. In contrast, binding of glutamate to NMDA receptors is normally without effect, as the NMDA-

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associated ion channel is “plugged” by a magnesium ion so no current can pass. However, under conditions of strong and/or persistent pain stimulation, sufficient amounts of substance P and glutamate are released such that sustained depolarization of the spinal cord neurons occur. Under these conditions, the magnesium ions are removed from the NMDA channel, allowing calcium ions to now flow into the cell. This event is key as this influx of calcium causes the production of nitric oxide by calcium-activated neuronal nitric oxide synthase and the production of prostaglandins. These diffuse from the neurons and act to increase both the excitability of spinal cord neurons in response to incoming pain signals, and cause an exaggerated release of “pain” neurotransmitters from spinal cord presynaptic terminals in the spinal cord. Taken together, these downstream effects of NMDA receptor activation result in the amplification of pain messages being relayed to higher brain centers (Petrenko et al., 2003).

II. THE PROBLEM OF CHRONIC PAIN The problem with pain is that pain can become maladaptive, serving no survival advantage. Normally, in the absence of painful stimuli, you feel no pain. Normally, when your injury heals, pain stops. However, that is not the case with chronic pain. In this case, pain occurs spontaneously, in the absence of any identifiable persistent injury or obvious physical reason. In addition, hot, cold, and hard pressure pains are greatly amplified. Indeed, non-painful stimuli such as warm, cool, and light touch are now perceived as pain. Chronic pain occurs in epidemic proportions worldwide. One in four adults suffers from chronic pain that is unresolved by currently available therapies (Louis Harris and Associates, 1999). One or more members of the United States’ 44 million households is in chronic pain (Partners Against Pain, 2000). Such pain affects all aspects of their personal and professional lives. Two-thirds of people with chronic pain have been in pain for more than 5 years and one-third feel that they cannot function as normal people because of their pain (Roper Starch Worldwide, 1999). Many sufferers attempt suicide because of the pain (Partners Against Pain, 2000). A major focus of pain research has been to try to understand why pain goes wrong, and why drugs such as morphine are highly effective for suppressing normal pain yet fail to control chronic pain. Many changes in the functioning of neurons in the pain pathway have been identified in animal models of chronic pain (Woolf and Salter, 2000). For example,

sensory neurons can become spontaneously active, signaling pain when no such stimulus is present. Neurons that normally relay only light touch can change their neurochemistry under conditions of chronic pain. Under such conditions, for the first time, these neurons begin releasing neurotransmitters that signal “pain” in response to light touch. Neurons within the spinal cord dorsal horn have been shown to become spontaneously active and hyperexcitable to incoming pain signals, thus amplifying pain messages sent to the brain (Woolf and Salter, 2000). These, and many other, changes in neuronal functions have been identified, and drugs have been developed to control such neuronal changes. Yet, chronic pain remains unresolved. Indeed, the definition of a good drug for chronic pain is one that fails 80% of the time (McQuay et al., 1995; McQuay et al., 1996). What will become clear, in the sections to follow, is that a reason that chronic pain remains a problem may be the importance of non-neuronal cells in such pain states. Immune cells associated with peripheral nerves and immune-like glial cells within the spinal cord have long been ignored by pain researchers, as they were not thought to influence neuronal function. This view is dramatically changing. Recognition of the key role played by peripheral immune cells and glial cells suggests that development of drugs that target these cell populations may prove efficacious in the control of human chronic pain (Watkins and Maier, 2003).

III. HISTORICAL OVERVIEW: DISCOVERIES LEADING TO THE RECOGNITION OF IMMUNE/GLIAL INVOLVEMENT IN CHRONIC PAIN Three independent and distinct lines of research led to the recognition of the importance of nerve-associated immune cells and spinal cord glial cells in pain enhancement. Interestingly, none originally derived from studies of pain.

A. Sickness Behavior The first line of research, which flourished in the mid-1980s through 1990s, was focused on understanding immune-to-brain communication. These studies sought to understand how viral or bacterial challenge of the immune system could lead to the creation of a wide array of brain-mediated, survival-oriented responses (Hart, 1988). These evolutionarily old, phylogenetically ubiquitous “sickness responses” included physiological changes (e.g., increased sleep, alterations in the composition of blood, fever), endocrine changes


(activation of the sympathetic nervous system and hypothalamo-pituitary response), as well as behavioral changes (e.g., decreased sexual activity, exploration, food/water intake, social dominance, etc.) (Maier and Watkins, 1998). The most recently recognized component of the sickness response was enhanced pain, which has come to be called “sickness-induced hyperalgesia.” This phenomenon was first characterized in the early 1990s (Maier et al., 1992; Watkins and Maier, 2000). The understanding of this enhanced pain state was greatly facilitated by prior studies of mechanisms underlying more classical components of the sickness response. By the early 1990s, it was becoming well accepted that pro-inflammatory cytokines (tumor necrosis factor [TNF], interleukin-1 [IL-1], and interleukin-6 [IL-6]) were key mediators of many sickness responses, both in the periphery and in the brain (Maier and Watkins, 1998; Parnet et al., 2002). In addition, glia were repeatedly implicated as a major source of these pro-inflammatory substances within the brain (Rothwell and Luheshi, 1994). Thus, based on this history, it was logical to explore whether glial pro-inflammatory cytokines within the spinal cord might be critically involved in the generation of sickness-induced hyperalgesia., and indeed, they were (Watkins and Maier, 2000; Watkins et al., 2001). This directly led to the exploration of whether peripheral inflammation/ trauma, more generally, could “tap into” this circuitry. In turn, it was discovered that there are many routes by which spinal cord glia can be activated, causing the release of pro-inflammatory cytokines, resulting in enhanced pain responses (Watkins et al., 2001; Watkins and Maier, 2000; Watkins and Maier, 2003).

B. Peripheral Nerve Damage The second line of research that led to the recognition of a central role of glia in pain regulation also arose independent of an interest in pain, per se. From the early 1970s (Sjostrand, 1971), literature has developed documenting the intriguing enigma that damage to nerve bundles in the periphery mysteriously led to the activation of microglia and astrocytes within the brain and spinal cord. This activation was not random, but rather occurred strictly within central nervous system (CNS) sites where damaged peripheral sensory neurons normally relayed their information to CNS neurons (for example, in the spinal cord dorsal horn). In addition, glial activation was found to occur surrounding CNS neuronal cell bodies (such as motor neurons) whose peripheral axons were damaged. It was not until the early 1990s that the first studies explored whether such glial activation might be poten-

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tially relevant to the understanding of pathological pain. At this time, Garrison and colleagues reported that a classic animal model of neuropathic pain (a form of pathological pain arising as a result of peripheral nerve injury) did indeed lead to marked glial activation within pain-related areas of the spinal cord dorsal horn (Figure 2). Importantly, the administration of a drug known to alleviate neuropathic pain (an NMDA receptor antagonist) also markedly attenuated dorsal horn glial activation (Garrison et al., 1991; Garrison et al., 1994). This finding provided the first evidence that, at minimum, dorsal horn glial activation was strongly correlated with neuropathic pain. As will be reviewed below, studies that followed from other laboratories proved that glial activation was not simply correlated with neuropathic pain, but rather was causal to such pain enhancement.

C. Immune Cells Associated with Peripheral Nerves The third line of research was not focused on spinal cord glia, but rather on immune cells associated with peripheral nerve bundles. It had been known that pain-sensitive peripheral nerves expressed receptors on their terminal endings in skin, for example, that were activated by pro-inflammatory products of nearby activated immune cells. This was not surprising, as it is the job of peripheral nerve endings to be aware of threats to the host. What was surprising was the discovery that immune and immune-like cells including macrophages, endothelial cells, fibroblasts, mast cells, and Schwann cells were resident within nerve bundles (Myers et al., 1999; Watkins and Maier, 2002). In addition, circulating immune cells were recruited to sites of damage within nerve bundles, just as in other bodily tissues (Myers et al., 1996). This led to investigations of the implications of such findings. Initially, these studies focused not on pain, but on nerve degeneration (Powell and Myers, 1986). In the course of the studies, it was documented that peripheral nerve damage led to the activation of resident immune and immune-like cells, leading to the release of chemoattractant factors and pro-inflammatory products. The activation of these resident cells, plus the recruitment and activation of macrophages from the general circulation, was found to be crucially involved in the degeneration of damaged peripheral nerves (Myers et al., 1999). As will be reviewed below, it soon became clear that these same nerve-associated immune and immune-like cells were also crucially involved in the creation of exaggerated pain responses that occur as a result of peripheral nerve damage (Watkins and Maier, 2002).

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IV. THE CONTRIBUTION OF PERIPHERAL IMMUNE CELLS TO CHRONIC PAIN

FIGURE 2 Example of microglial activation in spinal cord dorsal horns in response to a stimulus that creates exaggerated pain states. Peri-spinal administration of the HIV-1 envelope glycoprotein gp120 creates exaggerated responses to both thermal and touch/pressure stimuli (Milligan et al., 2001). Disruption of glial activation abolishes the pain changes. Furthermore, these pain changes are correlated with anatomical evidence of activation of both astrocytes and microglia. An example of such microglial activation is illustrated here. Panel A illustrates the dorsal horn of a rat injected peri-spinally with vehicle. Panel B is identical except that the rat received peri-spinal HIV-1 gp120 at a dose that creates exaggerated pain responses. These photomicrographs are from the tissues collected for analysis reported in Milligan et al. (Milligan et al., 2001). Activation of microglia induces these cells to upregulate their expression of complement type 3 receptors. Thus, enzyme-labeled antibodies directed against complement receptor type 3 (OX-42 monoclonal antibodies) can be used to detect microglial activation by light microscopy. By this method, activated microglia appear darker and more densely stained in accordance with their increased expression of OX-42 antibody-bound receptors. Modified from: Watkins, L. R., Milligan, E. D., and Maier, S. F. (2001). Glial activation: a driving force for pathological pain, Trends in Neuroscience, 24, 450–455. (Copyright permission being requested from TiNS).

Infection, inflammation, and trauma to peripheral nerves can result in pain enhancement perceived as arising from tissues normally innervated by the affected nerve. Thus, for example, insult to the sciatic nerve near the hip leads to the perception of pain radiating throughout the leg. Historically, immune cells were not considered to contribute to such pain phenomena. Rather, pain was thought to be created as a result of changes in neurons. An array of neuronally based mechanisms has been proposed, supported by data from animal studies. As noted above, these include nerve-injury–induced spontaneous electrical activity arising either at the site of injury or from the cell bodies of the injured nerves, and changes in what neurotransmitters are made such that neurons that normally relay touch sensations now, for the first time, begin releasing neurotransmitters that “mean” pain (Woolf and Salter, 2000; Scholz and Woolf, 2002). More recently, the contributions of immune cells to such pain states have been recognized (Marchand et al., 2005). While a thorough review is not possible here, interested readers are referred to one of our recent reviews (Watkins and Maier, 2002), which provides a comprehensive summary of the anatomy and immunology of healthy peripheral nerves, as well as discussion of a variety of ways that immune activation can lead to damage and dysfunction of peripheral nerves from both a basic science and clinical perspective. Here, discussion will focus on the involvement of immune cells to neuropathic pain arising as a result of peripheral nerve trauma, as this is the most common cause of neuropathic pain and the one that has been the focus of the most intense study. Evidence for pain amplification in response to immune activation has accrued through the study of the relatively simple sea slug Aplysia and the rat. From studies of Aplysia, it became clear as early as the mid1980s that sensory nerve damage induces prolonged, exaggerated responses to subsequent mechanical stimuli (Walters, 1994). In studies that followed, it was discovered that immunocytes (macrophage-like cells of Aplysia) migrate to the site of nerve damage (Clatworthy et al., 1994). This was an intriguing finding given that immunocytes, like their mammalian macrophage counterpart, release pro-inflammatory cytokine-like molecules (IL-1–like and TNF-like) upon activation. Indeed, IL-1 and TNF alter ion channels in Aplysia neurons, causing hyperexcitability (Clatworthy and Grose, 1999). Similarly, hyperexcitability of injured nerves is enhanced in the presence of activated immunocytes (Clatworthy and Grose, 1999).


Strikingly parallel findings have been reported in rats. By the mid-1980s it was known that peripheral nerve injury causes enhanced pain states and axonal hyperexcitability, as well as axonal degeneration (called Wallerian degeneration). Both trauma-induced degeneration and enhanced pain are associated with the recruitment and activation of macrophages to the site of injury (Myers et al., 1999). Indeed, simply delaying the recruitment of macrophages to the site of nerve damage delays both the development of neuropathic pain and nerve degeneration (Myers et al., 1996). In contrast, neuropathic pain is increased if activated immune cells are attracted to the injury (Clatworthy et al., 1995). Of the various neuroactive substances released by activated macrophages, the most evidence implicates pro-inflammatory cytokines. These increase at sites of nerve injury, via production by Schwann cells, endothelial cells, and resident and recruited macrophages (Myers et al., 1999). Moreover, the blockade of IL-1, TNF, or IL-6 activity reduces neuropathic pain (Sommer et al., 1998; Sommer et al., 1999). In addition to pro-inflammatory cytokines, activation of the complement cascade and production of reactive oxygen species and nitric oxide have each been implicated in the development of neuropathic pain in animal models (Liu et al., 2000b). More recently, studies of immune involvement in neuropathic pain expanded from studies of traumatic injury of peripheral nerve to the consideration of how neuropathic pain may arise in the absence of frank trauma. Indeed, about half of human neuropathies are inflammatory rather than traumatic in origin (Said and Hontebeyrie-Joskowicz, 1992). How pain arises in such circumstances was a mystery. From early studies of Aplysia, it was clear that sensory neurons become hyperexcitable simply by the presence of immunologically activated immunocytes placed nearby (Clatworthy et al., 1994). In rats, simply exposing otherwise healthy rat peripheral nerves to killed bacteria, algae protein (carrageenan), yeast cell walls (zymosan), or the HIV-1 envelope glycoprotein gp120 increased pain responsivity (Chacur et al., 2001). Such pain changes are mimicked by pro-inflammatory cytokines. TNF and IL-1 injected into peripheral nerves increase pain responsivity, as well as induce nerve inflammation, demyelination, and axonal degeneration (Myers et al., 1999; Zelenka et al., 2005). Importantly, TNF applied to peripheral nerves generates action potentials at that site (Sorkin et al., 1997). Other inflammatory mediators, such as ATP, phospholipase A2, and reactive oxygen species, similarly increase pain responsivity after administration in close proximity to peripheral nerves (Chacur et al., 2004).

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Although it is clear that pro-inflammatory cytokines and other inflammatory mediators induce pain, how they can do this along large nerve bundles is controversial. This is understandable when it is realized that it is heretical to discuss activation of neurons at mid-axon. The classical view of how sensory neurons are activated revolves around the expression of specific receptors on nerve terminals within skin, muscle, or other tissues. It is only at nerve terminals, and certainly not along large nerve bundles, that sensory neurons were thought to be activated by pain-inducing stimuli. Only very recently has the notion been seriously considered that functional receptors may be expressed along the axon (Moalem, 2005), in addition to their expression at their terminations in skin and other tissues. In addition to the possibility of functional receptor expression along axons, inflammatory mediators may have other means for exciting peripheral nerves as well. Of the pro-inflammatory cytokines, TNF has received the most study to date. Studies of the structure of TNF indicate that it may insert into lipid membranes and form a central pore-like region due to its threedimensional configuration. Insertion is facilitated by a physiologically relevant lowering of pH (e.g., acidification), which naturally occurs at sites of inflammation (Kagan et al., 1992). It appears that the inserted TNF molecules form voltage-dependent sodium channels. Additional studies indicate that TNF can interact with sodium and calcium channels naturally expressed along peripheral nerves to increase their inward conductance, an effect that would increase neuronal excitability. Similar to TNF, IL-1 and IL-6 can also produce long-lasting increases in the conductance of voltage-sensitive sodium and calcium channels (Schettini et al., 1988; Wilkinson et al., 1996). In addition to enhancing pain responsivity by altering the function of peripheral nerves, immune activation can also enhance pain responsivity by altering the function of sensory nerve cells within dorsal root ganglia (DRG). There are left and right pairs of DRG in between successive vertebrae of the spinal column. The peripheral projections of these cell bodies innervate specific regions of the body, whereas their central projections innervate adjoining segments of the spinal cord. Each of these DRGs contains the cell bodies of neurons that sense what is occurring to a small “slice” of the body, called a dermatome (“skin slice”). In addition, DRGs contain glially derived satellite cells, dendritic cells, macrophages, and endothelial cells. Each neuronal cell body in the DRG is encapsulated by a layer of satellite cells (Olsson, 1990). Satellite cells are glia-like cells and as such share many of their regulatory and immune-like functions. Like glial cells (as will be described in more detail below), satellite cells regu-

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late the extracellular levels of neuroexcitatory substances such as excitatory amino acids, and can rapidly spread excitability to large regions of the DRG by gap junctional communication across inter-connected satellite cells (Huang and Hanini, 2005). Peripheral nerve injury leads to the activation of satellite cells and immune cells within the DRG, causing the release of pro-inflammatory cytokines and other neuroexcitatory substances (Zhou et al., 1999). Thus, satellite cells are well positioned to regulate neuronal excitability within the DRG. Herniated discs are but one example of the way immune activation can alter pain by actions at the level of the DRG. Herniated discs spontaneously release a variety of pain-producing substances including nitric oxide, pro-inflammatory cytokines, phospholipase A2, cyclooxygenase-2 (COX-2), thromboxanes, leutkotrienes, and prostaglandins (Ahn et al., 2002). These are created both by infiltrating immune cells as well as by histiocytes, fibroblasts, endothelial cells, and chondrocytes of the disc itself. Furthermore, herniated discs become hyperresponsive to inflammatory stimuli. As one example, they release enhanced amounts of nitric oxide, IL-6, and prostaglandins upon stimulation with IL-1 (Kang et al., 1997). Such findings suggest that discassociated pro-inflammatory substances may be a major factor in back pain (Burke et al., 2002). Indeed, there is now a substantial and striking literature accruing that the DRG is vulnerable to immunederived substances. Application of herniated disc tissue to DRG and its adjoining axons causes edema, spontaneous electrical activity in sensory nerves, and enhanced responsivity to pain (Olmarker and Rydevik, 2001). In addition, it causes increased production of pro-inflammatory cytokines, nitric oxide, and phospholipase A2 in DRG (Kawakami et al., 1999). Topical application of TNF to DRG likewise induces spontaneous electrical activity in sensory nerves, edema, and enhanced pain responsivity (Liu et al., 2000a). Other inflammatory mediators, such as bradykinin, chemokines, and phospholipase A2, have also been implicated in enhancing pain responsivity at the level of the DRG (Tang et al., 2004; Zhang et al., 2005). In addition, the meninges offer another means by which the peripheral immune system and the central nervous system (CNS) may interface, thereby potentially influencing pain responsivity. The meninges cover the CNS, creating a barrier between the CNS and the periphery. The meninges are composed of three layers: the pia mater, arachnoid, and dura mater (Patestas and Gartner, 2005). The pia mater lies closest to the parenchyma, which is the unique tissue (neurons and glia) that makes up the central nervous system. The pia mater is a thin, delicate covering that traces

the surface of the parenchyma, and it contains fine blood vessels that penetrate the parenchyma. The arachnoid layer has a spongy consistency and lies between the pia mater and the dura mater. The arachnoid layer does not have a blood supply. The cerebrospinal fluid (CSF) circulates in the subarachnoid space between the arachnoid layer and the pia mater. The dura mater is a tough, outer layer that rests on top of the arachnoid layer. The dura mater is heavily vascularized. At the level of the dura mater, the junctions of the vascular endothelial cells are tight, as this is a primary barrier between CNS vasculature and peripheral blood circulation. The main function of the meninges has previously been presented as one of protection. That is, the meninges, with the circulating CSF, serve to mechanically protect the parenchyma by absorbing shock as the organism moves about, and protects the CNS from bacteria or viruses circulating in the blood via the tight junctions in the dura mater. Of particular interest to those studying communication between the peripheral immune system and the CNS is the cellular composition of the meninges. The meninges are composed, in part, of large numbers of immune cells such as mast cells, dendritic cells, and macrophages (Braun et al., 1993; McMenamin, 1999 #140). Given the proximity of these cells to CSF and the CNS parenchyma, it seems likely that activation of these immune cells could influence neurons and glia in the region. Traditionally, the meninges have been thought of as functionally independent of the CNS parenchyma. However, it has been proposed that the meninges are able to significantly influence the activity of glia and neurons. Mercier and Hatton argue that signals released from the meninges, such as growth factors and cytokines, act on the parenchyma directly, and mediate glial and neuronal activity (Mercier and Hatton, 2004). It should be noted that neither the pia mater nor the basal lamina separating the parenchyma from the pia mater serves as a barrier to large molecules. In other words, any released signal or bacteria or virus able to gain access to the CSF is also able to gain access to the parenchyma as well. In light of the number of immune cells residing in the meninges, it seems possible that inflammation of the spinal cord meninges (meningitis) would lead to pain responses. Indeed, lumbar spinal arachnoiditis (inflammation of the arachnoid layer at the lumbar level of the spinal cord) is associated with back pain (Petty et al., 2000). Further, we have measured elevated IL-1 in the meninges following conditions leading to the exaggerated pain, including peri-spinal (intrathecal) administration of the HIV-1 envelope gp120 and peripheral nerve damage that produces neuropathic pain (Jekich et al., 2006; Wieseler-


Frank et al., 2006). Administration of gp120 into the CSF space induces an inflammatory response by directly acting on immune cells in the meninges in addition to glial cells in the spinal cord. That we see increases in IL-1 following peripheral nerve injury that causes neuropathic pain is interesting, as it suggests that the inflammation of the damaged sciatic nerve at mid-thigh level leads to changes in the meninges as the nerve pierces through the meninges on its way to entering the spinal cord. This could be the result of signals released from the DRG, either inducing an inflammatory response in the meninges or signals from the DRG recruiting inflammatory cells to the meninges. In either case, the peripheral immune system is communicating with the central nervous system and is associated with exaggerated pain. In addition to surrounding the CNS parenchyma, the meninges also project into the brain and spinal cord. These projections are in the form of blood vessel ensheathments. Every blood vessel is ensheathed as it enters and leaves the meninges, and this ensheathment continues into the parenchyma. The cellular composition of these ensheathments differs from the meninges surrounding the parenchyma in that these are composed of fibroblasts and macrophages, which are coupled by gap junctions (Mercier et al., 2002; Mercier et al., 2003; Mercier and Hatton, 2001). The space between the ensheathment and the blood vessel is referred to as the perivascular space. Within the perivascular space are macrophages, termed perivascular macrophages. Perivascular macrophages are unique in that they constitutively express MHCII (Perry, 1998), possibly indicating a heightened state of responsivity. The perivascular macrophages, as well as the ensheathing meningeal macrophages, are well positioned to respond to blood-borne signals, and then release immune mediators that can act on other neurons and glia, and/or can be released back into the blood stream. Given this organization, it is possible that while some substances may not be able to cross the blood-brain barrier, they can still influence glial and neuronal activity by acting on vascular cells, inducing an immune response in the meninges resulting in the release of the neuroexcitatory substances into the CSF. For example, peripheral administration of lipopolysaccharide (LPS) induces cyclooxygenase-2 gene expression in perivascular monocytes/macrophages (Quan et al., 1998). The notion that resident meningeal immune cells may be communicating with glia and neurons in the parenchyma is a newer hypothesis. That it could be another pathway to consider when studying communication between the peripheral immune system and the CNS has not been previously considered.

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V. THE CONTRIBUTION OF CNS IMMUNELIKE GLIAL CELLS TO CHRONIC PAIN Astrocytes and microglia have been the focus of studies exploring the role of CNS immune-like cells in pain dysregulation (Marchand et al., 2005; Watkins et al., 2001; Watkins and Maier, 2003). While, as reviewed below, there are good reasons to implicate these two types of glia in pain facilitation, one must recognize that this focus on astrocytes and microglia is a simple accident of history. That is, astrocytes and microglia caught the attention of pain researchers because, when these cells are activated, they upregulate their expression of cell type-specific markers (e.g., glial fibrillary acidic protein by astrocytes, complement type 3 receptors by microglia). As simple immunohistochemistry can be used to detect increases in these so-called activation markers, microglia and astrocytes were favored for study over other immune and immune-like cells (fibroblasts, endothelial cells, oligodendrocytes, etc.) that do not express such markers. Thus, while it is likely that microglia and astrocytes are not alone in regulating pain, the contributions of other cells types have yet to be investigated. As glia are not yet commonly studied in the field of psychoneuroimmunology, a general overview will be provided prior to discussion of the evidence that these immune-like cells are involved in pain enhancement.

VI. PHYSIOLOGY AND FUNCTION OF GLIA: GENERAL PRINCIPLES A. Microglia Microglia constitute 5–10% of all glia and 5–12% of the total number of cells within the CNS (Cuadros and Navascues, 2001). The embryonic origin of microglia remains controversial, and they likely arise from multiple sources. Most evidence points to a mesodermal origin of these cells, likely of hematopoietic lineage (Cuadros and Navascues, 2001). In addition, peripheral blood monocytes are recruited in early development in response to chemoattractants released within the CNS during programmed cell death (Perry et al., 1985). While these recruited monocytes initially mature into tissue macrophages pursuant to their role in removing cells that die during CNS remodeling, they subsequently become indistinguishable from microglia (Perry et al., 1985). In the adult, resident microglia principally arise from two sources. The first is that resident microglia continue to divide throughout life, and in so doing continuously replenish their numbers. Second, periph-

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eral blood monocytes migrate into the healthy CNS and, once there, mature into resident microglia. These two sources are thought to equally contribute to the number of resident microglia in healthy adult CNS (Lawson et al., 1992). In injured CNS, additional recruitment occurs of circulating monocytes, which again are indistinguishable from microglia when matured (Kaur et al., 2001). Thus, both developmentally and throughout the life span, microglia arise from multiple sources. Regarding their function in healthy CNS, microglia are responsible for immune surveillance and host defense (Nimmerjahn et al., 2005). Logically, given such roles, they express receptors for viruses and bacteria, as well as indicators of cell damage such as heat shock proteins. When not stimulated by such stimuli, microglia actively monitor their microenvironment in their role as sentinels (Nimmerjahn et al., 2005). In this state, microglia express a highly ramified, stellate morphology 30–40 μm in diameter with little-to-no expression of receptors, activation markers, or functions characteristic of activate microglia (Raivich et al., 1999). As microglia are remarkably sensitive to their microenvironment, the shift from their sentinel state to an immune-reactive activated state can occur quite rapidly (Figure 3). Microglia can be activated by CNS trauma, ischemia, tumors, neurodegeneration, bacte-

ria, and viruses. Activation is not all-or-none, but rather is graded, reflected by alterations in morphology (retraction and thickening of processes, hypertrophy, membrane ruffling reflective of exploratory movements), proliferation, upregulated receptor expression (such as complement receptors and scavenger receptors), and changes in function (recruitment to sites of damage, phagocytosis, production of inflammatory mediators such as pro-inflammatory cytokines) (Raivich et al., 1999). Exactly what profile of changes occurs in microglia is defined by the type, intensity, and duration of the initiating stimulus and the prior history of microglial activation. Thus, stating that microglia are activated may not, per se, accurately reflect what form or function has changed. Upon resolution of the acute challenge (infection, damage, etc.), microglia may slowly revert to their basal state. However, microglia may also remain in a “primed,” hypervigilant state for prolonged periods of time (Perry et al., 1985). In such a primed state, microglia are not actively producing pro-inflammatory products but rather respond more rapidly and more vigorously to a new challenge. Thus, upon restimulation, an overproduction of pro-inflammatory products can occur (Perry et al., 1985). There are early indications that re-stimulation of spinal cord glia in such a “primed” state can result in exacerbation of pain (Cahill et al., 2003; Spataro et al., 2005).

FIGURE 3 Microglia under basal and activated states. Microglia in hippocampus were stained with FITC-IB4, which also labels blood vessels (arrowheads, Panels A and C). Microglia under basal conditions exhibit highly branched, ramified morphology (Panel A). A higher magnification of a single microglial cell is shown in Panel B; note the fine, finger-like extensions emanating from the major branches. By 12 hours after activation by isolation from the brain, microglia have lost most of their branches and appear amoeboid in form (Panel C). A comparison of representative microglia under basal versus strongly activated conditions, differences are evident by the reduction in number and length of branches within the first 12 hours after isolation from the brain (Panel D). Modified from: Stence, N., Waite, M., and Dailey, M. E. (2001). Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices. Glia, 33, 256–266. (With permission from Wiley-Liss, Inc.)


B. Astrocytes Astrocytes (also called astroglia) account for 40–50% of all glia and outnumber neurons (Figure 4) (Lee et al., 2000). Embryologically, astrocytes arise from the dorsal neural tube (Goldman and Vaysse, 1991). There are multiple precursor populations, which in turn lead to multiple subpopulations of astrocytes with varying receptor expressions and functions (Goldman and Vaysse, 1991). Mature astrocytes closely ensheath neuronal cell bodies as well as synapses. Thus astrocytes are physically well positioned to intimately detect and respond to neuronal signaling (Aldskogius and Kozlova, 1998). In their basal condition, astrocytes perform an array of functions. They contribute to the formation and structural integrity of the blood-brain barrier, provide neurons with energy sources and precursors for the synthesis of neurotransmitters (Tsacopoulos, 2002), release a variety of growth factors that support the development and maintenance of neurons, regulate extracellular concentrations of ions and neurotransmitters, regulate the formation of synapses, etc. (Perea and Araque, 2002) Beyond these functions, the concept of the “tripartite synapse” has recently been developed. This view redefines synapses as being composed of three, rather than the traditional two, elements: the presynaptic neuronal terminal, the postsynaptic neuronal terminal, and the enveloping astrocyte. Here, astrocytes are conceived of as active contributors to synaptic signaling.

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They receive signals from the neurons and, in turn, they signal back to the neurons and, in so doing, markedly alter neuronal activity and function (Araque et al., 1999; Haydon, 2001). Additionally, astrocytes communicate actively with other astrocytes. While astrocytes do not generate action potentials, as do neurons, they do create calcium waves in which intracellular calcium elevations are propagated in a non-decremental fashion from astrocyte to astrocyte over large distances (Porter and McCarthy, 1997). Such calcium waves, by activating distant astrocytes, lead to the release of glial products at sites distant from the origin of the astrocyte signal (Haydon, 2001). Thus, this can lead to an expansion of glial activation over time (Figure 5).

C. Glia and Glial Products Can Act in Synergy While this issue has just begun to be studied in the pain field, it is important to note that actions of glial pro-inflammatory cytokines can synergize, just as they do in the peripheral immune system. Pro-inflammatory cytokines can also synergize with neurotransmitters and neuromodulators. For example, IL-1 and TNF can synergize with a variety of neurotransmitters/ neuromodulators, resulting in the release of glial products, including pro-inflammatory cytokines (Crusazk et al., 1996). In terms of interactions within the spinal cord, what is known is that nitric oxide (which can be produced

FIGURE 4 Astrocytes under basal and activated states. Astrocytes in the spinal cord were labeled for their expression of glial fibrillary acidic protein (GFAP), an activation marker. Astrocytes in a healthy spinal cord (Panel A: Normal) are morphologically quite distinct from astrocytes strongly activated in response to spinal cord contusion (Panel B: Injured). Modified from: Peters, C. M., Rogers, S. D., Pomonis, J. D., Egnazyck, G. F., Keyser, C. P., Schmidt, J. A., Ghilardi, J. R., Maggio, J. E., and Mantyh, P. W. (2003). Endothelian receptor expression in the normal and injured spinal cord: potential involvement in injury-induced ischemia and gliosis. Experimental Neurology, 180, 1–13. (With permission from Academic Press)

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FIGURE 5 Gap junctions allow activated glia to create widespread excitation. To selectively activate glia in cell culture, one frequently used technique is the application of a mechanical stimulus, which triggers the release of intracellular calcium and, consequently, the stimulation of gap-junctional communication. Such gap-junctional spread of excitability also occurs in response to stimulation of glia with various excitatory substances such as ATP and glutamate. Panel A: A phase-contrast image of a field of cells in a 7-day-old mixed glial culture. A micropipette (P) is positioned over a single cell and is used to stimulate the cell mechanically. Panel B: A fluorescence image of the same field of cells loaded with fura-2 at an excitation wavelength of 380 nm. Panel C: Intracellular calcium ion concentration [Ca2+]in maps of the same field of cells at sequential time-points following mechanical stimulation of a single cell. Time (in seconds) after mechanical stimulation is indicated below each panel. A wave of increased [Ca2+]in is communicated cell by cell in all directions from the stimulated cell. The peak c increase in this example varies from 150–600 nM, as indicated by the pseudocolor scale bar. The wave is initiated at a specific point on each cell and spreads across the cell body and along its processes until it reaches the cell boundary. Scale bars, 100 um. From: Araque, A., Parpura, V., Sanzgiri, R. P., and Haydon, P. G. (1999). Tripartite synapses: glia, the unacknowledged partner. Trends in Neuroscience, 22, 208–215.

by activated glia as well as by activated neurons) potentiates IL-1—induced cyclooxygenase-2 (an inducible enzyme leading to prostaglandin synthesis) (Morioka et al., 2002). Nitric oxide also potentiates IL-1—induced release of substance P from painresponsive sensory neurons that send their presynaptic terminals into the spinal cord so to relay pain information there (Morioka et al., 2002). Also, human

spinal cord astrocytes enhance their release of IL-6 and PGE2 in response to substance P combined with IL-1 (Palma et al., 1997). Regarding microglia-astrocyte interactions, it is clear that astrocytes release substances that stimulate microglial activation, proliferation, and release of a wide array of neuroexcitatory products (Petrova et al., 2000). It is also clear that activation of microglia can


lead, in turn, to the activation of nearby astrocytes (Ledeboer et al., 2005b; Raghavendra et al., 2003). Thus, each of these cell types impacts the functions of the other.

VII. PAIN ENHANCEMENT BY ACTIVATED SPINAL CORD GLIA Normally, glia appear to have little-to-no influence on pain (Marchand et al., 2005; Watkins et al., 2001). That is, if glial activation or their pro-inflammatory products are disrupted, no change in basal pain responsivity is detected. This has been interpreted to mean that, under normal pain states, spinal cord glial cells are simply performing their classical, regulatory functions, quite independently of pain processing. Under such basal conditions, glia are not releasing significant levels of pro-inflammatory products and so do not alter pain responsivity. In contrast, when glia become activated, the proinflammatory products they release do markedly enhance pain. Glial activation occurs under both physiological and pathological conditions. As noted above, peripheral immune challenge is one condition that leads to glial activation within the CNS (Watkins and Maier, 2000). Immune activation in the body can lead in turn to immune-to-brain communication. Such immune-to-brain communication results in sickness responses, including increases in pain. The recognition that many sickness responses are generated as a result of glial activation and consequent pro-inflammatory cytokine production predicted that glia and proinflammatory cytokines would mediate sicknessinduced pain facilitation as well. This did indeed turn out to be the case (Watkins and Maier, 2000). It is now known that glial and pro-inflammatory cytokine regulation of pain extends well beyond sickness responses (Figure 1D). Spinal cord glia are activated in response to a wide array of bodily challenges, including inflammation and damage to peripheral tissues, peripheral nerves, spinal nerves, and spinal cord. Indeed, the involvement of spinal cord glia in enhanced pain is so pervasive that in virtually every animal model of enhanced pain, pain is prevented and/or reversed by disrupting spinal cord glial activation or by disrupting the actions of pro-inflammatory products released by these cells (Marchand et al., 2005; Watkins et al., 2001; Watkins and Maier, 2003). Such disruption of glial effects can be produced by administration of various immunomodulatory drugs, glial metabolic inhibitors, pro-inflammatory cytokine antagonists, or anti-inflammatory cytokines such as interleukin-10 [IL-10] (Milligan et al., 2005a; Watkins

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and Maier, 2003). As one example, chronic exposure of the spinal cord to IL-10 can resolve neuropathic pain arising from peripheral nerve inflammation, traumatic injury, and chemotherapy-induced neurotoxicity (Ledeboer et al., 2005a; Milligan et al., 2005a; Milligan et al., 2005b; Sloane et al., 2005). Such studies are exciting due to the promise they hold for improved treatment of pain, as each model studied is reflective of major pain syndromes that are currently poorly, if at all, treatable in people. The mechanism or mechanisms by which glial proinflammatory products enhance neuronal excitability are still actively being investigated. Neurons within the pain-responsive regions of spinal cord express receptors for pro-inflammatory cytokines (Ohtori et al., 2004) and the excitability of pain-responsive neurons are rapidly increased upon exposure to these glial products, which suggests that a direct effect of these cytokines on neurons may be involved (Reeve et al., 2000). Intriguingly, IL-1 has recently been documented to increase the inward calcium conductance of neuronal NMDA receptors (Viviani et al., 2003), including those of pain-responsive neurons in the spinal cord (Samad et al., 2004). As reviewed in the “Pain Basics” section, such an increase in intracellular calcium would naturally lead to increased production of nitric oxide and prostaglandins, thereby amplifying pain. In addition, TNF has been documented to rapidly increase the number of AMPA receptors on the surface of neurons (Stellwagen et al., 2005), again consistent with increasing the excitability of neurons in a manner that could more easily lead to NMDA activation as a consequence. Lastly, pro-inflammatory cytokines can cause glia to release a variety of neuroexcitatory substances, including nitric oxide, prostaglandins, and a variety of reactive oxygen species (Samad et al., 2001) (Figure 1D). Thus, while pro-inflammatory cytokines can exert multiple effects, all are in the direction of amplifying pain by increasing the excitability of spinal cord neurons. Although, by far, the most focus in the pain literature has been on the role of glial pro-inflammatory cytokines in amplifying pain, it is important to recognize that these are by no means the only neuroexcitatory substances released by activated glia. For example, glia can release a variety of excitatory amino acids, including D-serine that potently enhances NMDA channel function (Snyder, 2004). Furthermore, activated glia can release ATP along with nitric oxide, reactive oxygen species, and prostaglandins, as noted above. Thus, glia are well endowed with releasable products that can enhance pain. Studies of such substances, as relates to how glia regulate pain, are hindered by the fact that neurons as well as glia release

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these. Thus, identifying the relative contribution of glia versus neurons to pain facilitation induced by these mediators is difficult to study in vivo. Also of note here is that glia have recently been implicated in the spread of pain beyond the site of injury. Chronic pain patients often report pain perceived as arising from regions well beyond the actual site of injury, a phenomenon known to be due to a spread of increased excitability within the spinal cord beyond the sensory inputs of the injured body site. Indeed, chronic pain patients may also report that pain is perceived not only in the region of the injury but also in the opposite, so-called mirror-image, body area. Spinal cord glia have been implicated in such spread of pain (Milligan et al., 2003; Spataro et al., 2004). It is intriguing that astrocytes are inter-connected into widespread networks via cell-to-cell connections, called gap junctions (Figure 5). These gap junctions allow for the spread of excitation from astrocyte to astrocyte over larger regions (Araque et al., 1999). As the newly excited, distant astrocytes can then begin releasing neuroexcitatory substances (Araque et al., 1999), this suggests that such spread of excitation by glia might be involved in the spread of pain as well. Indeed, disruption of spinal cord glial gap junctions has been found to abolish both mirror-image pain and pathological pain mediated by spinal immune activation in rats, an effect which may be due to attenuation

of the spread of glial activation and thus the absence of pro-inflammatory cytokine production by these distant glia (Spataro et al., 2004) (Figure 6). One last point that bears discussion here regards the relative role of microglia and astrocytes in pain amplification. As noted above, both astrocytes and microglia are thought to play key roles in pain enhancement. Recent studies have begun to examine the parameters that define which glial cell type or types) become activated. Evidence to date points to microglia as highly reactive, vigilant cells that rapidly respond to disruption of normal homeostasis (Nimmerjahn et al., 2005). While microglia, unlike astrocytes, do not enwrap synapses so have no direct “knowledge” of neuronal synaptic activity, they are rapidly responsive to alterations in the extracellular microenvironment. Microglia appear to be the first glial cell type activated under conditions leading to exaggerated pain responses. Blocking microglial activation with minocycline prevents the development of pathological pain in response to nerve injury (Ledeboer et al., 2005b; Raghavendra et al., 2003). Intriguingly, administration of minocycline after pathological pain has developed now fails to control such pain (Ledeboer et al., 2005b; Raghavendra et al., 2003). Data such as these have led to the current view that microglia are the first glial cell to respond, and they, in turn, lead to the recruitment of astrocytes. With astrocyte recruitment, microglial

FIGURE 6 Blocking spinal cord gap junctions with carbenoxolone blocks both astrocyte activation and interleukin-1 production. The gap junction decoupler carbenoxolone inhibits intrathecal HIV-1 gp120-induced spinal IL-1 production and glial activation (Panels A and B, respectively). Panel A: IL-1 protein in dorsal spinal cord 310 minutes after intrathecal (I.T.) injection of gp120 or vehicle (240 minutes after I.T. carbenoxolone or vehicle). Carbenoxolone (carbenoxolone + gp120) blocks increases in IL-1 typically seen following gp120 (vehicle + gp120). Panel B: Mean integrated density of GFAP immunoreactivity in dorsal spinal cord. Eight hours following I.T. gp120 (7 hours after I.T. carbenoxolone) rats were transcardially perfused with 4% paraformaldehyde, and lumbosacral spinal cords were collected for analysis of glial activation (GFAP) by immunohistochemistry. There is a strong trend even with small sample sizes for carbenoxolone to block gp120-induced GFAP increases.


involvement decreases, and astrocytes maintain pain enhancement over time. The question of which glial cell type is activated first leads naturally to a consideration of the critical stimulus for such glial activation. Immunologically, glia (as noted above) become activated in response to bacteria and viruses. But here, the question is what substance or substances are released within the spinal cord as a consequence of peripheral nerve damage, such that glia become activated? Presumably, something has to be released in the spinal cord by incoming sensory neurons whose peripheral axons have been injured. Or, something could be released by spinal cord neurons upon receiving injury signals from those sensory neurons. In either case, it is presumed that neuron-toglia signals must exist. A number of neuron-to-glia signals have now been posited. Fractalkine is one such signal. Fractalkine is a unique protein (a chemokine) that, in the spinal cord, is expressed exclusively on the outside surface of neurons (Chapman et al., 2000). Fractalkine is released upon strong neuronal activation, thereby forming a diffusable signal. Our studies have shown that, in the spinal cord, only microglia express receptors for fractalkine under either basal or neuropathic pain states (Verge et al., 2004). In support of this conclusion, very strong fractalkine receptor gene expression is observed in enriched microglia cultures derived from rat dorsal spinal cord, while negligible expression is observed in comparable enriched astroglia cultures. Exposure of acutely isolated dorsal spinal cord to fractalkine causes the release of IL-1 (Johnston et al., 2004). As would be expected from this finding, intrathecal microinjection of fractalkine produces hyperalgesia (Milligan et al., 2004). This production of hyperalgesia occurs via activation of microglia, as these pain-enhancing effects are prevented by minocycline, a selective microglial inhibitor (Milligan et al., 2005c). Fractalkine-induced pain enhancement indeed results from the release of nitric oxide and pro-inflammatory cytokines, as these effects are prevented by a nitric oxide synthase inhibitor, an IL-1 receptor antagonist, and IL-6— neutralizing antibodies (Milligan et al., 2005c). Blockade of fractalkine effects, using a neutralizing antibody to the fractalkine receptor, delays the onset of neuropathic pain and also reverses established neuropathic pain for as long as the neutralizing antibody is administered (Milligan et al., 2004). This suggests that peripheral nerve injury causes the sustained release of fractalkine from damaged sensory afferents and/or intrinsic spinal cord neurons, leading to microglial activation and the release of painenhancing substances.

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Heat shock protein is a second putative neuron-toglia signal. This is a novel candidate that is only just beginning to be explored for its pain regulatory effects and effects on dorsal spinal cord glia. Heat shock proteins are best known as intracellular chaperones that protect cells in response to cellular stress. As intracellular signals, they are survival signals. However, when cells are damaged, dying, or dead, heat shock proteins are released and become extracellular signaling molecules to nearby glia. In this role, they can activate glia in their role as immune-like cells. Notably, remarkable upregulation of one member of the heat shock protein family, heat shock protein 27 (Hsp27), has been reported in damaged sensory fibers within spinal cord dorsal horn and surrounding white matter for months after peripheral nerve injury (Costigan et al., 1998). Thus, heat shock proteins may directly activate microglia and/or may synergize with fractalkine. Although speculative, synergy would be a logical mechanism for making glial activation “fail safe”; that is, a combination of “warning signals” may far more potently activate glial cells than any single signal. Synergy would be a logical solution for the regulation of glial activation that leads to pathological pain, as it would constrain the conditions under which such activation would be expected to occur. Notably, preliminary studies indicate that incubation of cultured dorsal spinal cord glia with fractalkine plus Hsp27 potentiates IL-1 gene expression (Wieseler-Frank et al., 2004). Adenosine triphosphate (ATP) is a third putative neuron-to-glia signal. ATP is considered a classical “pain” neurotransmitter, with many purinergic receptor subtypes found on neurons, astrocytes, and microglia. As regards the present proposal, Tsuda et al. (2003) recently demonstrated a key role of microglial P2X4 purinergic receptors in inducing pain enhancement. First, they documented that P2X4 receptors dramatically upregulate in spinal cord ipsilateral to peripheral nerve damage. Critically, these P2X4 receptors are expressed by microglia, but not neurons or astrocytes. Second, they administered a variety of purinergic receptor antagonists to reverse neuropathyinduced allodynia and found that the one specific to microglial P2X4 receptors was effective. Third, they stimulated microglia in vitro with ATP to activate them. Intrathecal microinjection of these in vitro— stimulated microglia was sufficient, in and of itself, to produce mechanical allodynia. While Tsuda et al. did not examine what neuron-to-glia signal so potently upregulates microglial P2X4 purinergic receptors in response to peripheral nerve injury, it may well be that the signal is, at minimum, fractalkine. Preliminary studies indicate that fractalkine upregulates P2X4 gene expression in cultured dorsal spinal cord

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microglia (Wieseler-Frank et al., 2003). Whether heat shock protein, alone or in combination with fractalkine, has similar effects is unknown.

VIII. BEYOND CHRONIC PAIN: GLIAL CELLS OPPOSE THE PAIN-SUPPRESSIVE EFFECTS OF OPIOIDS Up until this point, the discussion has focused on immune-derived and glia-derived enhancement of pain that occurs in response to inflammation, infection and/or injury. Here, the discussion extends to glial activation in response to opioids administered with the aim of relieving such pain (Watkins et al., 2005). The discussion below will develop the concept that spinal cord glia become progressively more activated over time when morphine is repeatedly administered, as would occur when delivered for relief of chronic pain (Watkins et al., 2005). Repeated administration of opioids such as morphine leads to the development of tolerance to the pain-relieving effects of such drugs. This, in turn, leads to progressive increases in drug dosage in an attempt to gain control over pain. Increases in drug dosage leads, in turn, to increases in side effects, such as constipation and respiratory depression (Raith and Hochhaus, 2004). In addition, prolonged opioid administration leads to pain amplification upon discontinuation of the drugs. Such abstinence-associated pain facilitation, like tolerance, is a clinically relevant problem (White, 2004). While tolerance is often thought to occur due to downregulation of opioid receptors, resulting in signaling failure, tolerance can also occur by a quite different process; that is, by the activation of endogenous pain facilitation systems such as those already discussed. The existence of both pain inhibitory and facilitatory systems means that the pain experienced by an individual reflects the summation of these two types of systems. Thus, tolerance could be produced by either a weakening of the pain suppressive system (the typical assumption) by repeated opioid administration, or by a progressive strengthening of pain facilitatory systems. In the latter scenario, the strengthening of the pain facilitatory system would counteract the pain-reducing effects of morphine. Spinal cord glia and the pro-inflammatory cytokine products they produce can be considered such a counteractive system (Watkins et al., 2005). Opioids such as morphine may directly, or indirectly, activate spinal cord glia. Glia elsewhere in the CNS express the full range of opioid receptor subtypes (Chao et al., 1996; Persson et al., 2000), so it is likely (but as yet unproven) that spinal cord glia do as well.

Opioids may also exert indirect effects. Dynorphin is one potential candidate for such indirect effects, as chronic morphine increases spinal dynorphin levels and dynorphin enhances pain, at least in part, via the release of pro-inflammatory cytokines, such as IL-1 (Laughlin et al., 2000; Rady and Fujimoto, 2001). There is also evidence that morphine can release soluble signals from neurons such as fractalkine that serve to induce pro-inflammatory responses from nearby glia (Johnston et al., 2004). Whatever the proximate cause of morphine-induced glial activation and pro-inflammatory cytokine release, it is clear that glial pro-inflammatory cytokines alter the analgesic effects of opioids, such as morphine. Spinal delivery of IL-1 receptor antagonist or the antiinflammatory cytokine IL-10 along with morphine enhances the magnitude and duration of analgesia (Johnston et al., 2004). Similarly, injecting IL-1 into the cerebrospinal fluid of mice, at a dose having no behavioral effect on its own, blocks the analgesic effect of systemic morphine (Shavit et al., 2005). Indeed, if morphine analgesia is allowed to be expressed and then dissipate, potent analgesia can be rapidly reinstated by injecting IL-1 receptor antagonist. This suggests that analgesia only appeared to disappear as a result of the release of pain-enhancing pro-inflammatory cytokines that drive pain-facilitatory effects that counterbalanced the pain-suppressive effects of morphine (Shavit et al., 2005). Such a conclusion is supported by studies of transgenic mice. For example, morphine analgesia is dramatically enhanced in mice with impairments of IL-1 signaling (Shavit et al., 2005). It is clear that chronic morphine activates spinal cord glia, as indicated by the upregulation of gliaspecific activation markers (Raghavendra et al., 2002; Song and Zhao, 2001). That such glial activation contributes to morphine tolerance is supported by the finding that co-administering glial inhibitors along with morphine disrupts the development of morphine tolerance (Song and Zhao, 2001). How glial activation disrupts the effects of morphine is still under investigation. At this point, all evidence points to pro-inflammatory cytokines. Morphine exposure increases spinal cord levels of mRNA, protein and protein release of pro-inflammatory cytokines (Johnston et al., 2004; Raghavendra et al., 2002). These morphine-induced increases in glial pro-inflammatory cytokines are causal to morphine tolerance as blocking pro-inflammatory cytokine function blocks, in turn, both tolerance- and abstinence-induced pain enhancement (Johnston et al., 2004; Raghavendra et al., 2004). Paralleling such findings, transgenic mice with impaired IL-1 signaling demonstrate dramatically reduced morphine tolerance (Shavit et al., 2005).


Interestingly, spinal pro-inflammatory cytokine activation in neuropathic pain pathologies has been linked to the reduced morphine efficacy under neuropathic conditions (Raghavendra et al., 2003), producing a state of naive morphine tolerance. That is, ongoing glial activation in response to neuropathy may “prime” the glia so that they over-respond to morphine, compared to sham controls, rendering morphine ineffective in such pain pathologies. Therefore, spinal cord glia oppose the analgesic effects of morphine via the release of pro-inflammatory cytokines. In response to acute and chronic morphine, this is observed as a decrease in the magnitude and duration of analgesia. In response to cessation of chronic morphine, this is observed as abstinence-induced pain facilitation. Notably, it now appears that morphine will not be alone in having its efficacy undercut by glial activation. Preliminary studies now document that spinal cord glia and pro-inflammatory cytokines similarly compromise the analgesic effects of methadone, at least in part via non-classical opioid receptors (Hutchinson et al., 2005). These data also expand the clinical implications of spinal glial activation, as crosstolerance between opioids may be explained by the activation of the glial pain facilitatory system rendering all attempts to treat chronic pain ineffective. Once it is clear which clinically relevant opioids are affected by glial activation and whether classical opioid receptors or atypical receptors are involved, new therapies can be intelligently designed to separate the painrelieving effects of opioids from their glial-activating effects. Such an outcome would predict far-reaching improvements in the ability of opioids to resolve currently uncontrolled pathological pain states. The clinical implications of glial activation following opioid exposure have now extended to opioid addiction and dependence. Recent data suggest that systemic administration of the blood-brain barrier permeable glial inhibitor, AV-411, can blunt naloxoneprecipitated opioid withdrawal behaviors following chronic morphine administration (Figure 7). These data are supported by the work of Dafny et al. (Dafny et al., 1983; Dougherty and Dafny, 1990), who nearly 30 years ago demonstrated that non-selective immune suppression inhibited morphine-withdrawal behaviors. In light of these recent data, it would now appear Dafny et al. had non-selectively inhibited glial activity as well.

IX. CONCLUSIONS AND IMPLICATIONS Although the present chapter has focused on immune and glial modulation of pain, it should be clear that there is nothing a priori unique about the

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FIGURE 7 Total withdrawal behaviors recorded by a blinded observer, 41–50 minutes following naloxoneprecipitated withdrawal, in rats that had received an escalating dependence regimen of subcutaneous morphine for 5 days, and an intraperitoneal co-administration of vehicle or AV-411, a blood-brain permeable drug with glia suppressive actions. These data indicate that morphine activates glia within the brain, which create a hyperexcitable state expressed as withdrawal behaviors. Data modified from: Shumilla, J. A., Ledeboer, A., Liu, T., Hutchinson, M. R., Skyba, D., Pater, C., Watkins, L. R., and Johnson, K. W. (2005, In press). AV-411, a novel attenuator of neuropathic pain. In Proceedings of the 8th International Conference on the Mechanisms and Treatment of Neuropathic Pain.

nerves or neurons involved. All peripheral nerves, regardless of function, would be expected to be affected to some degree by immune activation in the ways described for pain. Similarly, all CNS neurons, regardless of function, would be expected to be affected by glial activation in the ways described for pain. Indeed, there is growing recognition that glia powerfully influence neuronal functions at all levels of the CNS. This chapter has surveyed immune interactions with pain systems at multiple non-classical sites. Whereas immune regulation of pain has long been recognized at sites in inflammation and damage in the skin, where peripheral nerve terminals have evolved explicitly to accurately sense what is happening to the body, immune regulation of pain along peripheral nerve bundles and within the spinal cord has not. There are several major points that bear reemphasis here. First and foremost is that immune activation and immune-like glial activation are important for pathological pain states, such as neuropathic pain. Classical immune cells (e.g., macrophages, dendritic cells), immunocompetent cells that can release factors thought of as immune cell products (e.g., endothelial cells, fibroblasts, Schwann cells, etc.), and immune-like

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spinal cord glial cells (astrocytes and microglia) can each contribute to pain enhancement. This has profound implications for the understanding of how such pain states occur and how such pain states can be effectively controlled via the development of drug therapies aimed at controlling these recently recognized sources of pain enhancement. The second major point is the pervasiveness of proinflammatory cytokine involvement in pain facilitation, including neuropathic pain. The pain-enhancing effects of pro-inflammatory cytokines at sites of skin or muscle injury have long been recognized. In contrast, pro-inflammatory cytokine enhancement of pain via actions along peripheral nerves, at the DRG, and in spinal cord has only recently been uncovered. These are striking in their implications as novel targets for pain control. The third major point is the breadth of impact that glia have on pain regulation. While most of this chapter reviewed direct effects of glial activation on pain, this topic has recently been expanded by the discovery that opioids such as morphine activate glia as well. The efficacy of opioids for pain relief is hampered by glial release of pro-inflammatory cytokines. Again, the implications of these findings are great. They predict that an understanding of how glia are activated by clinically relevant analgesic drugs will lead, in turn, to improvements in pain control by the development of therapeutics that avoid this unanticipated side effect. The last major point is that investigation of immune and glial involvement in pain is in its infancy. Many more immune cells and immune-derived substances are implicated in the etiology of pathological pain syndromes than have yet been studied for their potential involvement in the pain syndromes that follow, and lastly, very little is understood about the response repertoire and receptor expression of immune-like glial cells within the pain modulatory regions of the spinal cord. The reason is that it has only recently become recognized that glia residing in various sites within the CNS are not all the same. Rather, there can be striking regional heterogeneity in how these cells respond and to what they respond under basal or activated states. Glia are the product of their microenvironment and must be understood in this context. Given that brain glia have almost exclusively been the subject of study, much remains to be learned about the dynamics of dorsal spinal cord glial function under both basal and pathological pain conditions.

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C H A P T E R

19 Cytokines and Non-immune Brain Injury BARRY W. MCCOLL, CHRIS J. STOCK, AND NANCY J. ROTHWELL

effects of inflammation in the injured or diseased brain. This chapter will endeavor to provide an overview of the studies that have been integral to elucidating the putative involvement of cytokines in non-immune brain injury and disease.

I. INTRODUCTION 415 II. EVIDENCE OF CYTOKINE INVOLVEMENT IN CNS INJURY AND DISEASE 416 III. CYTOKINES IN REPAIR AND RECOVERY AFTER CNS INJURY 421 IV. MECHANISMS OF CYTOKINE ACTIONS 422 V. CONCLUSIONS AND THERAPEUTIC POTENTIAL 422

I. INTRODUCTION The importance of cytokine actions in the brain in regulating the host defense response is now well recognized, and described in detail elsewhere in this volume. Cytokines are classically recognized as local mediators of tissue responses to injury, infection, ischemia, and immune activation. Yet this local function was, for many years, largely ignored in the central nervous system. Infections of the brain, such as malaria, measles, and HIV have long been known to elicit local cytokine activation in the CNS and immune diseases, most notably multiple sclerosis has been linked to cytokines for well over a decade (Allan and Rothwell, 2003). However, the potential contribution of cytokines—both beneficial and detrimental to acute and chronic CNS diseases and insults is now well established. The most extensively studied cytokines in brain injury are the pro-inflammatory molecules IL-1, TNFα, and IL-6, but evidence, albeit often circumstantial, also implicates a range of other cytokines including IL-4, IL-10, IL-12, IL-18, interferons, and chemokines, as well as newer members of the IL-1 and TNF families. In many cases evidence is largely indirect and

ABSTRACT Emerging evidence, mainly derived from experimental studies, suggests that cytokines may play key roles in influencing the severity and evolution of acute tissue damage in response to ischemic and traumatic brain injury. Most convincing evidence has accumulated for IL-1, implicating the actions of this cytokine as detrimental to outcome after injury. In addition to their role in acute damage, there is growing interest regarding the involvement of cytokines in the postacute phase, notably in the resolution of acute inflammation and their impact on brain tissue repair and long-term functional recovery. The involvement of cytokines in chronic neurodegenerative disorders is also an area of expanding investigation in light of the discovery of cytokine gene polymorphisms which confer increased risk of developing Alzheimer’s disease. Further studies will be required to clarify the mechanisms by which cytokines can modulate the response to acute brain injury. It is conceivable that such studies may reveal novel targets for intervention that may prove useful in ameliorating the damaging PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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based predominantly on expression patterns. In others a clear role has been established, at least in experimental studies, as is the case for IL-1 in acute experimental brain injury. Some cytokines appear to have complex or even seemingly opposing actions, particularly during the acute phases of developing injury and later repair and recovery. There is less direct evidence to implicate cytokines in chronic CNS diseases associated with injury, but supportive data are now available or emerging for their likely contribution to Alzheimer’s disease, movement disorders, amyotrophic lateral sclerosis, injury resulting from epilepsy or tumors, and the transmissible spongiphorm encephalopathies.

II. EVIDENCE OF CYTOKINE INVOLVEMENT IN CNS INJURY AND DISEASE A. Evidence of Cytokine Overexpression after CNS Injury There exists substantial clinical and experimental evidence of increased local cytokine expression within the CNS in response to insults such as ischemic stroke and traumatic brain injury (TBI), although the spatiotemporal modulation of this expression is incompletely understood. Various anomalous observations, in combination with ample evidence of complex regulatory interactions between cytokines, suggest that the expres-

sion profile of CNS cytokines after injury is highly dynamic. While almost all CNS cell types are capable of expressing the majority of cytokines and their receptors, acute-phase transcription and immunoreactivity (ir) of “pro-inflammatory” cytokines such as IL-1β and TNF-α are localized to cells exhibiting monocytic or microglial morphologies and degenerating neurones (Buttini et al., 1994; Davies et al., 1999; Knoblach et al., 1999; Logan et al., 1994; Ohtaki et al., 2004; Sairanen et al., 2001; Wiessner et al., 1993). In contrast, postacute (days to weeks post-insult) immunoreactivity of “anti-inflammatory” cytokines (e.g., TGF-β and IL-10) is associated predominantly with astrocytes and large phagocytes (Lehrmann et al., 1995; Li et al., 2001; Logan et al., 1994; Wiessner et al., 1993) (Figure 1). There is also evidence of a significant delay (and in some cases a discrimination) between peak transcription and peak protein levels of some cytokines in response to injury, and a spatial transition of expression from pathologic foci to peri-infarct regions over the first 24–48 hours after injury. The majority of these data have been acquired from experimental models of cerebral ischemia in rodents, and it is primarily such studies that will be discussed here. 1. Pro-inflammatory Cytokines: IL-1b, TNF-a, and IL-6 IL-1β transcription is rapidly (1–3 hours) and transiently (up to 3 days, peak at 6–24 hours) upreg-

FIGURE 1 CNS cytokine expression after injury. Expression of several cytokines, including pro-inflammatory IL-1β, TNF-α, IL-6, and anti-inflammatory IL-10, is rapidly upregulated within injured brain tissue (in red). The primary source of this expression is microglia and infiltrating leukocytes, although TNF-α is strongly associated with neural processes. From 3 days onwards, resolution of energy deficits and reparative processes around the injury core (core in red, recovering tissue in green) coincide with significant production of both IL-1β and its endogenous antagonist IL-1RA, in parallel with elevated TGF-β expression within reactive astrocytes and large phagocytes.

19. Cytokines and Non-immune Brain Injury

ulated within the ischemic rodent brain, accompanied by significant, but modest, increases in protein levels (Berti et al., 2002; Buttini et al., 1994; Legos et al., 2000; Liu et al., 1993; Zhai et al., 1997). Both in situ hybridization (ISH) and immunohistochemical (IHC) analyses have identified microglia and cells exhibiting a monocytic morphology as the source of this acute-phase expression (Buttini et al., 1994; Davies et al., 1999). Although IL-1 is believed to signal only through its type I receptor (IL-1RI), it is transcription of the “decoy” type II receptor that is acutely upregulated in response to injury, primarily in peripheral monocytes and endothelia (Docagne et al., 2005; Parker et al., 2000; Wang et al., 1997). In contrast, peak transcription of IL-1RI is not observed until 5 days after injury, which coincides with peaks in gross protein levels of both IL-1β (3–5 days) and its endogenous antagonist, IL-1RA (5 days), which is present in concentrations 20-fold higher than IL1β at these times (Legos et al., 2000; Wang et al., 1997). There is little clinical evidence of IL-1β upregulation, although elevated plasma concentrations of IL-1β are observed at 3 hours post-admission in stroke patients (Mazzotta et al., 2004). Both IL-6 and TNF-α exhibit similar upregulation to IL-1β after cerebral ischemia in the rat; mRNA levels are increased by 2–3 hours, peak at 6–12 hours, and decline to baseline by 3 days (Berti et al., 2002). Protein levels of IL-6 in the ischemic rat brain lag behind transcription, peaking at 24 hours, before declining by 3 days (Legos et al., 2000). Clinical data from stroke, TBI, and subarachnoid hemorrhage (SAH) patients demonstrate rapid (3 hours) increases in plasma and intrathecal IL-6 levels that peak at 2–4 days before declining to baseline, excepting stroke, after which CSF upregulation persists for up to 3 months (Hayakata et al., 2004; Mathiesen et al., 1993; Tarkowski et al., 1995; Vila et al., 2000). Higher cerebrospinal fluid (CSF) IL-6 concentrations cf plasma after such injuries and microdialysis assays of parenchymal IL-6 levels support the contention of significant local CNS production (Mathiesen et al., 1993; Tarkowski et al., 1995; Winter et al., 2004). IHC analysis of IL-6 immunoreactivity after focal ischemia in rodents suggests that it is primarily produced by microglia-like cells and neurones within the peri-infarct zone up to 14 days post-injury (Block et al., 2000). There are few reports of IL-6 receptor expression, although diffuse upregulation of IL-6R mRNA and immunoreactivity in neurones is observed up to 24 hours after striatal stab lesion in parallel with similar increases in IL-6, IL-1β, and IL-1R expression (Yan et al., 1992). Although comparable data are not available for TNF-α, immunohistochemical studies of acute isch-

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emic injury in the rat indicate strong TNF-α protein expression from 6 hours through to 24 hours (Liu et al., 1994; Ohtaki et al., 2004). Such studies demonstrate that this expression is associated with neuronal processes and oligodendrocytes, initially within the infarct zone and at a later time points in the peri-infarct zone. Similar investigations of human brain tissue, taken from severe ischemic stroke patients, indicate a biphasic expression profile of TNF-α with peaks at 1 and 3 days, localized to neuronal processes and microglia at 1 day and astrocytes from 3–8 days (Sairanen et al., 2001). This expression pattern is also exhibited by TNF-α type I receptor in ischemic mouse brain, localized to neurones and astrocytes within the infarct at 12–24 hours and peri-focal astrocytes at 4 days postinsult (Yin et al., 2004). 2. Anti-inflammatory Cytokines: IL-10 and TGF-b IL-10 mRNA production is upregulated rapidly after ischemia in the rat brain, with peak expression reported at either 6 or 24 hours according to two separate studies (Li et al., 2001; Zhai et al., 1997), while clinical data from the CSF of TBI patients support a putative acute-phase expression profile, with peak protein detection at 24 hours postadmission (Hayakata et al., 2004). Similar data are provided by observations from clinical stroke, which show increases in IL-10 levels up to 3 days (Tarkowski et al., 1997). Although TGF-β mRNA and immunoreactivity are seen 6–24 hours after ischemic (global and focal) and traumatic injury in rats, significant expression is not observed until 2–3 days later (Lehrmann et al., 1995). This expression is localized to microglia and macrophages in ischemic injury and astrocytes in mechanical trauma (Lehrmann et al., 1998; Logan et al., 1994). While TGF-β expression has dissipated by 7 days in global ischemic and traumatic insults, this represents the temporal peak after focal ischemia and TGF-β immunoreactivity persists in the infarct border of such injury for up to 3 months (Lehrmann et al., 1998). Interestingly, Vivien et al. (1998) demonstrate that while TGF-β mRNA production is elevated in the ischemic mouse cortex 1–3 days after the insult, expression of the TGF-β type II receptor (TβII) is significantly downregulated over the same time period, suggesting a negation of any increase in TGF-β levels. However, although Fee et al. (2004) report unchanged TβII ir 1 day after cryo-traumatic cortical injury in support of these data, this expression is significantly increased at 4–7 days, primarily in endothelial cells. Elevated intrathecal TGF-β levels are also a feature of clinical TBI (Morganti-Kossmann et al., 1999).

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B. Experimental Evidence of a Role for Cytokines in CNS Injury Alterations in cytokine expression as described above may simply reflect a response to the emerging tissue damage rather than a direct causative role. Accordingly, several approaches have been used to further elucidate a pathophysiological role in the brain for a number of cytokines. The majority of studies have employed animal models of acute brain injury, most notably focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, and as such, this section will focus on the findings which have emerged from these studies. 1. Pro-inflammatory Cytokines Administration of recombinant IL-1β by intracerebroventricular (i.c.v.) or intrastriatal infusion exacerbates ischemic brain damage induced by transient and permanent MCA occlusion in rodents (Loddick and Rothwell, 1996; Stroemer and Rothwell, 1998; Touzani et al., 2002; Yamasaki et al., 1995). Recently, we have further observed that peripheral (intraperitoneal) administration of IL-1β similarly exacerbates ischemic damage after transient MCA occlusion (our unpublished data), supporting an important role for periph-

eral inflammation in modulating outcome to ischemic brain injury (Figure 2). In contrast, administration of IL-1RA causes a marked reduction in ischemic brain damage when administered i.c.v. (Mulcahy et al., 2003; Loddick and Rothwell, 1996; Relton and Rothwell, 1992; Touzani et al., 2002), intrastriatally (Stroemer and Rothwell, 1997), or systemically (Garcia et al., 1995; Relton et al., 1996), suggesting a role for endogenous IL-1 in the development of ischemic brain damage. Furthermore, adenoviral-mediated overexpression of IL1RA also attenuates focal ischemic brain damage (Betz et al., 1995; Yang et al., 1998a). Interestingly, inconsistent effects of chronic (transgenic) overexpression of human IL-1RA in mice have been demonstrated. Tehranian et al. (2002) reported an improved neurological outcome after closed head injury, whereas Oprica et al. (2004) did not observe a neuroprotective effect after permanent MCA occlusion. This may reflect experimental differences or compensatory changes in IL-1 expression due to chronic blockade of IL-1 receptors. Simultaneous deletion of both the IL-1α and IL-1β genes results in a marked reduction in damage caused by transient MCA occlusion; however, deletion of IL1α or IL-1β alone does not have any impact, implying an important role for both IL-1 ligands and again suggesting that compensatory changes in the IL-1 system

FIGURE 2 Exacerbation of focal ischemic brain damage by peripheral IL-1β. Coronal brain sections stained with cresyl violet demonstrate areas of viable tissue (deep blue/purple) and infarcted tissue (pale blue/white). Images on the top row show typical damage 24 hours after transient MCA occlusion (30 minutes) from a mouse treated with vehicle (ip) at the onset of occlusion. Administration of IL-1β (4 μg/kg) markedly increases the extent of the infarct (bottom row), incorporating cortical tissue that is undamaged after vehicle treatment.


may accompany its manipulation (Boutin et al., 2001). Indirect evidence of a role for endogenous IL-1 in acute brain injury has also emerged from studies in which the actions of IL-1β converting enzyme (ICE) have been inhibited. Administration of the ICE inhibitors z-VAD. FMK, YVAD.CMK, or z-DEVD.FMK reduces infarct volume after transient or permanent MCA occlusion (Hara et al., 1997b; Loddick et al., 1996). Similarly, mice expressing mutant, inactivating forms of the ICE gene demonstrate reduced infarct volumes (Hara et al., 1997a; Friedlander et al., 1997). It is tempting to speculate that the neuroprotective effects of ICE inhibition are due to a reduction in the production and release of mature IL-1; however, it cannot be excluded that attenuation of apoptotic cell death is the primary neuroprotective mechanism. Further indirect evidence for a pathogenic role for IL-1 in the brain was recently provided by Basu et al. (2005), who demonstrated that mice deficient in the IL-1 type I receptor (IL-1R1) showed an improved outcome after a hypoxic-ischemic insult. IL-18 shares several structural and functional similarities with IL-1 and is also cleaved by ICE to generate the mature, bioactive ligand (Ghayur et al., 1997; Gu et al., 1997). IL-18 deficient mice display reduced infarct volume and reduced white matter injury in response to neonatal hypoxic-ischemic injury (Hedtjarn et al., 2002, 2005). Furthermore, inhibition of IL-18 activity by a neutralizing binding protein improved neurological outcome after closed head injury (Yatsiv et al., 2002). However, deletion of the IL-18 gene was not associated with any difference in the extent of ischemic damage after transient MCA occlusion (Wheeler et al., 2003). Since brain damage was assessed later after neonatal injury (up to 7 days) compared to MCA occlusion (24 hours), the above discrepancies may reflect the delayed expression profile of IL-18, which was shown to begin at 48 hours and peak at 7–14 days after injury (Jander et al., 2002) and may therefore contribute to damage in the post-acute phase. Several strategies have been employed to investigate the role of TNF-α in acute brain injury. Administration (i.c.v.) of TNF-α produces a dose-dependent exacerbation of ischemic damage induced by permanent MCA occlusion in spontaneously hypertensive rats (Barone et al., 1997). In contrast, administration of compounds that suppress TNF-α synthesis have shown protective effects in models of focal ischemia and closed head injury (Leker et al., 1999; Meistrell et al., 1997; Shohami et al., 1996), although their actions may, in part, be mediated by their NMDA antagonist and antioxidant properties. Recently, Wang et al. (2004) achieved more selective suppression of TNF-α production by inhibiting TNF-α converting enzyme and dem-

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onstrated a marked reduction in focal ischemic damage. Numerous studies have inhibited endogenous TNF-α bioactivity by administration of TNF-α neutralizing antibodies (Barone et al., 1997; Hosomi et al., 2005; Lavine et al., 1998; Yang et al., 1998b) or TNF-α binding protein (TNFbp), a soluble fragment of the TNF-α cellsurface receptor (Dawson et al., 1996; Nawashiro et al., 1997; Shohami et al., 1996), which have shown consistent neuroprotection after focal ischemia and brain trauma. In contrast to the above studies showing deleterious effects of TNF-α in acute brain injury, studies in mice deficient in TNF-α receptors have produced conflicting findings. Bruce et al. (1996) reported that ischemic damage and oxidative stress were significantly greater in mice lacking the p55 and p75 receptors, suggesting acute protective effects of TNF-α. Deletion of the p55 receptor alone, but not the p75 receptor, also resulted in reduced ischemic brain damage (Gary et al., 1998), indicating that neuroprotective actions of TNF-α may be primarily p55 receptor-mediated. Scherbel et al. (1999) found that TNF-α knockout mice had a poorer chronic outcome after cortical contusion injury although acute recovery 24– 48 hours after injury was improved in the knockout mice, suggesting time-dependent biphasic effects of TNF-α. Thus, it appears that acute and chronic suppression of TNF-α activity results in divergent effects on the response to acute brain injury. Conflicting results have also been obtained regarding the role of IL-6 in acute brain injury. A protective role for IL-6 was suggested by Loddick et al. (1998), who demonstrated that administration of recombinant IL-6 (i.c.v.) significantly reduces infarct volume after permanent MCA occlusion. Astrocyte-targeted expression of IL-6 is also protective after a focal acute brain injury (Penkowa et al., 2003a, 2003b). Increased oxidative stress and neurodegeneration after excitotoxic lesion in IL-6 deficient mice further suggested a neuroprotective role for endogenous IL-6 (Penkowa et al., 2000, 2001). In contrast, infarct volume and neurological deficit induced by transient MCA occlusion were similar in IL-6 deficient and wild-type mice, suggesting that endogenous IL-6 is not directly involved in the development of focal ischemic brain damage (Clark et al., 2000). Similarly, neurological recovery after experimental traumatic brain injury was not affected in IL-6 deficient mice (Stahel et al., 2000) or by administration of a monoclonal neutralizing antibody to IL-6 (Marklund et al., 2005). 2. Anti-inflammatory Cytokines Consistent findings of a neuroprotective and antiinflammatory role for IL-10 have emerged. Adminis-

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tration and adenoviral-mediated overexpression of IL-10 ameliorated ischemic damage after both focal and global cerebral ischemia (Ooboshi et al., 2005; Spera et al., 1998), suggesting neuroprotective effects for this cytokine. Subcutaneous but not i.c.v. injection of IL-10 also improves neurological outcome after experimental TBI (Knoblach and Faden, 1998). Furthermore, infarct volume is smaller in IL-10 deficient mice compared to wild-type mice, indicating a neuroprotective function for endogenous IL-10 after ischemic brain injury (Grilli et al., 2000). Induction of IL-10 expression by CD4+ T-cells was also shown to be responsible for the anti-ischemic effects of myelin oligodendrocyte glycoprotein vaccination (Frenkel et al., 2003, 2005). Evidence has also emerged supporting TGF as an endogenous neuroprotectant. TGF-β1 deficient mice are more susceptible to excitotoxic injury in vivo and in vitro, and astrocytic overexpression of TGF-β1 protected against excitotoxic damage (Brionne et al., 2003). Administration of a TGF-β antagonist exacerbated excitotoxic injury and ischemic damage after transient MCA occlusion, further suggesting that TGF-β may limit the extent of acute brain damage (Ruocco et al., 1999). In addition, infarct volume is increased by 300% in mice deficient in persephin, a member of the TGF-β superfamily, and application of recombinant persephin markedly reduces ischemic damage (Tomac et al., 2002). Several studies have also shown that administration of exogenous TGF-β attenuates ischemic damage induced by global or focal cerebral ischemia, or excitotoxic damage (Boche et al., 2003; Henrich-Noack et al., 1996; Prehn et al., 1993). Similarly, exogenous TGF-α also reduces infarct volume after focal ischemia (Justicia et al., 2001). Thus, there is ample evidence to suggest that several cytokines are involved directly in the pathogenesis of brain damage after acute injury. Evidence indicates that IL-1 is detrimental to outcome and that IL-10 and TGF-β are neuroprotective. Inconsistent findings for other cytokines, notably IL-6 and TNF-α, allude to potential divergent effects of these mediators. Such discrepancies may reflect model-specific effects and associated differences in the spatial and temporal expression and sites of action of cytokines after brain injury. An inflammatory response is an integral component of regenerative processes in the periphery and, therefore, it will be important to further establish if some cytokines could have opposing acute and chronic actions in the aftermath of CNS injury as demonstrated by Scherbel et al. (1999). Such studies may also reconcile the neuroprotective effects of some cytokines (e.g., IL-1, TNF-α) observed in vitro with observations from

in vivo studies that generally indicate neurotoxic actions.

C. Clinical Evidence of a Role for Cytokines in CNS Injury and Disease Many experimental strategies to investigate the role of cytokines in brain injury and disease are evidently not feasible or ethical in humans. Therefore, clinical evidence for the involvement of cytokines in brain pathologies is more scarce and derived mostly from studies of expression (see “Evidence of Cytokine Overexpression after CNS Injury”). However, in a subset of these studies, associations between the level of expression and functional outcome have been investigated. In addition, there is emerging evidence that polymorphisms in some cytokine genes may be associated with increased susceptibility to neurodegenerative disease. 1. Stroke and Traumatic Brain Injury Numerous studies have consistently observed elevated plasma and CSF IL-6 levels after stroke and TBI in humans (see “Evidence of Cytokine Overexpression after CNS Injury”). Striking associations between IL-6 levels early after stroke and both infarct volume and long-term neurological outcome have been consistently shown (Fassbender et al., 1994; Mazzota et al., 2004; Tarkowski et al., 1995; Waje-Andreassen et al., 2005), suggesting that IL-6 may be an important factor in determining the pathological and neurological outcome after stroke. Conversely, such correlations may simply reflect that IL-6 is a marker of the severity of initial brain damage. A similar IL-6 response has also been observed consistently after traumatic brain injury. However, in contrast to the data from stroke patients, increased IL-6 levels appear to correlate with improved neurological outcome in traumatic brain injury, suggesting a possible neuroprotective effect for this cytokine (Singhal et al., 2002). There is little evidence of correlations between the levels of other cytokines after acute brain injury and acute damage or long-term outcome. There is little evidence showing that IL-1 levels are altered after acute brain injury, although it has been reported that plasma IL-1 levels following thrombolytic treatment of stroke patients correlate with clinical outcome at 3 months (Mazzota et al., 2004). The failure to correlate IL-1 with injury severity or outcome may reflect the fact that IL-1 expression is confined to local tissue injury but is rarely detected in extracellular fluids. Conflicting data also exist regarding the association between serum TNF-α levels after stroke and infarct size or neurological outcome (Intiso et al., 2004; Zaremba et al., 2001;


Zaremba and Losy, 2001). Interestingly, reduced levels of the anti-inflammatory cytokine IL-10 after stroke correlates with early decline in neurological function (Vila et al., 2003), which is consistent with the neuroprotective effects of IL-10 observed in animal models (see “Anti-inflammatory Cytokines”). 2. Alzheimer’s Disease Genetic polymorphisms are now recognized as important elements in modulating susceptibility to and outcome from CNS injury and disease. Recently, polymorphisms in cytokine genes have been identified which may modulate the response to acute brain injury and also the risk of developing Alzheimer’s disease (AD), further implicating a pathogenic role for cytokines in neurological dysfunction (McGeer and McGeer, 2001; Waters and Nicoll, 2005). In a study assessing disability 3 months after ischemic stroke, patients with the -174G/G genotype in the IL-6 promoter region were more likely to be severely disabled (Greisenegger et al., 2003). IL-6 gene polymorphisms are also associated with risk of cerebrovascular disease (Chamorro et al., 2005; Flex et al., 2004; Pola et al., 2003; Revilla et al., 2002). Furthermore, there is growing evidence that polymorphisms in several cytokine genes, including IL-1 (Grimaldi et al., 2000; Kolsch et al., 2001; Nicoll et al., 2000; Rebeck et al., 2000), IL-1RA (Seripa et al., 2005), IL-6 (Arosio et al., 2004; Licastro et al., 2003; Papassotiropoulos et al., 1999; Pola et al., 2002), TNF-α (Alvarez et al., 2002; Culpan et al., 2003; Laws et al., 2005; Ma et al., 2004), and IL-10 (Arosio et al., 2004; Infante et al., 2004; Lio et al., 2003; Ma et al., 2005), modulate the risk of developing AD. Most of these polymorphisms reside within the promoter region of the gene, suggesting that cytokine expression levels may be a key factor in modulating risk of AD, and further supporting a role for cytokines in the pathophysiology of acute brain damage and chronic neurodegeneration.

III. CYTOKINES IN REPAIR AND RECOVERY AFTER CNS INJURY The repeated failure of putative neuroprotective strategies in recent years has led to growing attention to, and endorsement of, post-acute physical remodeling and neuromodulation as a route to improved clinical outcome. This shift in utilitarian goals has led to greater interest in the possibility that cytokines may exert significant influence upon such processes, a contention for which circumstantial evidence has existed for some time. Foremost among such evidence are the

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observations of chronic cytokine upregulation within peri-infarct regions of extensive physical reorganization, and their well-established involvement in psychopathological phenomena. Unfortunately, while several interventional studies have added weight to these arguments, the promiscuity of cytokines and the plurality of their observed effects limit the significance of such findings. Overall much of the data, reviewed here, indicate that cytokine coordination of physical remodeling would arise primarily indirectly via glial activation, while putative psychomodulatory effects would result from direct modulation of neural activity and synaptic plasticity.

D. Cytokines in Physical Reorganization and Reparative Processes Glial activation is a well-established response to pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α, which stimulates cytotaxis towards injured tissue and the release of associated molecules such as matrix metalloproteinases, growth factors, and of course, cytokines themselves. The general view is that glial activation is a detrimental facet of CNS injury, based mainly on evidence that microglia may contribute to acute neurodegeneration and the long-held convention that astrocytic glial scarring, coordinated by TGF-β, impedes physical reorganization of neural connections (Fawcett and Asher, 1999). Yet such processes mimic the mobilization of macrophages and fibroblasts in peripheral tissues, mechanisms which, while imperfect, are a vital aspect of clearing and containing cytotoxic necrosing tissue, which may be of specific importance within the brain. Whatever the net contribution of these gross aspects of glial activation, IL-1, TNF-α, and IL-6 activated glia have been demonstrated to release multiple growth factors including vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and nerve growth factor (NGF) (Carman-Krzan et al., 1991; Fee et al., 2000; Juric and Carman-Krzan, 2001; Mason et al., 2001). VEGF is critical to the initiation and coordination of angio- and ateriogenesis in recovering tissue, while NGF and IGF1 promote neurite outgrowth, dendritic spiking, and mobilization of neural stem cells. Also, IL-1 and TNF-α induce activation and migration of oligodendrocyte precursor cells, which may reflect the need for concomitant neural regeneration and remyelination (Arnett et al., 2001; Mason et al., 2001). This last observation may also go some way to explain the paradoxical response of TNF-α KO mice to cerebral ischemia, characterized by reduced acute injury but improved long-term functional outcome.

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E. Psychopathological and Functional Influences of Cytokines The psychomodulatory effects of cytokines, especially IL-1, are now well established, with significant influences upon mood, learning, memory function, and sleep patterns in rodents reported, while clinically depressed patients exhibit gross perturbations of circulating cytokine levels (Capuron and Dantzer, 2003; Deboer et al., 2002; Hogan et al., 2003; Kenis and Maes, 2002; Manfridi et al., 2003). The contention that such mechanisms may have a significant role in functional recovery after CNS injury is supported by the conjunction of chronic disturbances in cytokine expression with clinical reports of psychological phenomena, consistent with neurochemical imbalance and the experimentally observed psychological effects of cytokines. In particular, clinical depression and impaired motivation, associated with worsened functional outcome, are common in stroke patients and mirror IL-1, IL-6, and TNF-α induced sickness behavior (adaptive behavioral quiescence), sleep disruption, and anhedonia seen in the rat (Kelley et al., 2003; Konsman et al., 2002; Kronfol and Remick, 2000; Parnet et al., 2002). Similarly, IL-1 dependent impairment of task acquisition and memory function, as observed in rodents, may have a significant influence upon the critical process of compensatory “re-wiring” of neural networks (Aubert et al., 1995). While there is much interest in these observations, the subject awaits a thorough analysis and direct evidence of a causative link between cytokine overexpression and functional outcome before any conclusions can be made.

IV. MECHANISMS OF CYTOKINE ACTIONS A full discussion of the known or potential mechanisms of action of each cytokine in neuronal injury and repair is outside the scope of this review. For some, the mechanisms of their contribution to acute injury are beginning to be unraveled (Allan and Rothwell, 2001), but appear complex. Cytokines can act on all neurons and brain cells and contribute to recruitment of peripheral immune cells, but their primary target appears to be glia. There are numerous examples by which glia can influence neuronal survival or death by modification of release of neurotoxins, such as glutamate; release of neuroprotective factors (e.g., growth factors or neurotrophins); and modification of cell matrix (Nedergaard et al., 2003). Cytokines can also exert potent influences on vascular endothelial cells to induce release of potential

neurotoxins (Grammas et al., 2004; Skopal et al., 1998), to compromise or damage the blood-brain barrier and to modify adhesion and invasion of immune cells (Blamire et al., 2000; Ferrari et al., 2004). As a further contribution, many cytokines influence physiological systems or systemic functions which are likely to influence neuronal injury and repair. Such effects include induction of fever, which exacerbates neuronal injury (IL-1), and modifications of neuroendocrine, cardiovascular, and immune function and behavioral changes (Rothwell, 2003). Another area of engaging interest is the relationship between CNS and peripheral inflammation and immune activation. Systemic infection is a key risk factor for poor outcome after stroke or brain injury (Grau et al., 1998; Syrjänen et al., 1988). The mechanism of this effect is unknown, but such infection induces expression of brain cytokines (Emsley and Tyrrell, 2002). Similarly, infectious events within the brain can have a profound effect on systemic inflammation.

V. CONCLUSIONS AND THERAPEUTIC POTENTIAL There is now extensive evidence to implicate cytokines in acute neuronal injury and chronic neurodegenerative disease and further indications of their contributions to repair, recovery, and plasticity. However, the precise role of many cytokines, their mechanisms of action, and indeed whether their effects are wholly beneficial or detrimental remain to be fully elucidated. This is not unexpected given the diversity of action of these proteins and the fact that their key actions as modulators of inflammation and immune activation have both good and bad impacts—these processes are critical in host defense responses but major contributors to disease and tissue injury when activated inappropriately. For several cytokines their impact on functional outcome may depend on dose or timing—with dual roles in early injury and later repair processes. The most extensive evidence for a direct contribution to CNS injury exists for IL-1, where numerous approaches by several independent groups indicate that IL-1 is a key mediator of acute experimental brain injury. At present the most attractive intervention to limit IL-1 actions is its endogenous antagonist, IL-1RA. This is quite a large protein (17 kDa), but it is effective in protecting against neuronal injury in rodents when administered peripherally and penetrates CSF in subarachnoid hemorrhage patients (our unpublished data). We have conducted a small Phase II random-


ized, placebo controlled trial of IL-1RA in acute stroke patients (Emsley et al., 2005). The data indicate that IL-1RA is safe and inhibits the peripheral inflammatory response, and clinical outcomes in this small group were promising. It is likely that in the future IL-1RA and other cytokine-modulating strategies may prove beneficial in acute and chronic CNS injury and repair.

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C H A P T E R

20 The Interaction between Brain Inflammation and Systemic Infection LEIGH M. FELTON AND V. HUGH PERRY

(prion diseases, Alzheimer’s disease). We also describe the impact of repeated systemic infection on the immunological status of the mammalian CNS, and summarize evidence demonstrating that priming of microglia, and other glia, by prolonged or repeated exposure to pro-inflammatory mediators can impact on the progression, severity, and clinical expression of subsequent CNS disease.

I. INTRODUCTION 429 II. NEUROENDOCRINE-IMMUNE INTERACTIONS IN HEALTH AND DISEASE: CYTOKINE EXPRESSION IN THE CNS 431 III. ACUTE INFLAMMATION OF THE CENTRAL NERVOUS SYSTEM 433 IV. IMMUNE-MEDIATED DISEASE OF THE CENTRAL NERVOUS SYSTEM: MULTIPLE SCLEROSIS 435 V. CHRONIC NEURODEGENERATION AND MICROGLIAL PRIMING 439 VI. SUMMARY 444

I. INTRODUCTION The immunological status of the central nervous system (CNS) is highly regulated in order to maintain the precise homeostasis that is required for its normal day-to-day functioning. Until recently, the parenchyma of the CNS, comprising in mammals the brain and the spinal cord, was considered to be an “immunologically privileged” site, being isolated from circulating leucocytes and potentially damaging soluble mediators by a highly impermeable bloodbrain barrier (BBB), and being devoid of cells capable of antigen presentation, such as macrophages and dendritic cells. However, we now know that the CNS is capable of mounting both acute and chronic inflammatory responses, albeit atypical, in response to tissue injury, infection, and disease (Perry et al., 1997). A key feature of inflammation is the expression and release of pro-inflammatory and anti-inflammatory mediators; these include cytokines, chemokines, eicosanoids, and acute phase proteins. The contribution of these mediators to inflammation and tissue repair

ABSTRACT In this chapter we consider the biological mechanisms underlying central cytokine expression, discuss their physiological relevance in the context of systemic inflammation, and contemplate the recent evidence demonstrating that pro-inflammatory cytokines can exacerbate neuronal death and inflammation in rodent models of CNS disease. We also describe the bi-directional nature of communication between the CNS and peripheral immune system, placing particular emphasis on the acute phase response to brain injury, and the complex role of the liver. In order to provide a comprehensive review, we have focused on a number of pro- and anti-inflammatory cytokines, chemokines, and lipophilic mediators of inflammation; and describe how their expression may exacerbate tissue injury in acute brain injury, multiple sclerosis (MS), and chronic neurodegenerative disease PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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is complex and has been best characterized in models of peripheral infection. However, many of the inflammatory mediators that are synthesized and released by leucocytes in response to inflammatory stimuli in the periphery have also been detected in the CNS (Allan and Rothwell, 2003; Bajetto et al., 2001; Dantzer et al., 1998; Felton et al., 2005; Perry et al., 2002; Perry et al., 2003). The source of these mediators has yet to be fully determined, but the use of in vitro and in vivo models of CNS disease has confirmed that many cell types specific to the CNS are capable of their synthesis; these cell types include glia (such as microglia and astrocytes), perivascular macrophages, endothelial cells of the cerebral vasculature, and specific subpopulations of neurones. A number of these cell types, specifically cells of monocytic lineage, have been shown to upregulate their expression of MHC class antigens in response to inflammatory stimuli, and to actively participate in antigen presentation, thereby initiating both acute and chronic inflammatory responses within the brain and spinal cord, and contributing directly to the expression of inflammatory mediators (Aloisi, 2001). In a number of models of CNS disease, the expression of chemokines within the extracellular milieu of the CNS results in leucocyte recruitment to the parenchyma, which may further exacerbate the ongoing inflammation, and contribute to inflammatory mediator expression (Bajetto et al., 2001). The endogenous expression of pro-inflammatory cytokines within the CNS occurs in humans as a consequence of a wide range of disease states. Despite this, the precise roles and contributions of individual inflammatory species has remained elusive, possibly as a consequence of the extensive overlap and redundancy in their biological functions. Individual proinflammatory mediators have often been described as having deleterious effects in the acute stages of injury/disease, thereby exacerbating the ongoing pathology (Allan and Rothwell, 2003), but beneficial effects in the later stages of disease through promoting mechanisms of tissue repair (Kalehua et al., 2004). Furthermore, although many of the archetypal inflammatory diseases of the CNS, such as multiple sclerosis (MS), cerebral ischemia, and acute trauma, result in pro-inflammatory cytokine expression, there are a number of examples of acute injury and chronic neurodegenerative disease that are associated with a very low expression of proinflammatory mediators, and which demonstrate an almost exclusively anti-inflammatory profile (Perry et al., 2002). With the exception of a small number of cytokines and eicosanoids, constitutive expression of inflamma-

tory mediators within the healthy CNS is very low. However, it has been shown that the expression of some cytokines, and other downstream mediators of inflammation, can be elevated within the CNS in the absence of concurrent CNS pathology. For example, elevated expression of cytokine mRNA and protein has been demonstrated in well-defined regions of the brain and spinal cord as a result of systemic inflammation (for review, see Dantzer et al., 1998), and as a consequence of the natural course of aging (immunosenescence) (Felzien et al., 2001; Hacham et al., 2002). There is now a substantial body of evidence confirming that elevated levels of pro-inflammatory cytokines in peripheral tissues and the circulation can give rise to de novo synthesis of the same mediators within the CNS (Dantzer et al., 1998; Laye et al., 1994). This interaction between the peripheral immune system and CNS has been best characterized in rodent models of systemic infection, and it is now clear that this highly stereotyped physiological interaction forms an important part of the acute phase response to tissue injury and infection. Although such brief, intermittent, and tightly regulated increases in cytokine expression are unlikely to give rise to pathology in the healthy CNS, it is important for us to consider the consequences of such a response if brain tissue is already compromised by ongoing inflammatory disease or acute injury. In this chapter we will consider the biological mechanisms that underlie the expression of brain cytokines, discuss their physiological relevance in the context of systemic inflammation, and contemplate the recent evidence demonstrating that proinflammatory cytokines can exacerbate neuronal death and inflammation in rodent models of CNS disease. We will also describe the bi-directional nature of communication between the CNS and the peripheral immune system, placing particular emphasis on the acute phase response to brain injury and the complex role of the liver. In order to provide a comprehensive review, we will focus on a number of pro- and anti-inflammatory cytokines, chemokines, and lipophilic mediators of inflammation; and describe how their expression may contribute to tissue injury in acute brain injury, multiple sclerosis (MS) and chronic neurodegenerative diseases (Alzheimer’s disease). We will also describe the impact of repeated systemic infection on the immunological status of the mammalian CNS, and summarize evidence demonstrating that the “priming” of microglia, and other glia, caused by prolonged or repeated exposure to pro-inflammatory mediators, can impact on the progression, severity, and clinical expression of subsequent CNS disease.

20. The Interaction between Brain Inflammation and Systemic Infection

II. NEUROENDOCRINE-IMMUNE INTERACTIONS IN HEALTH AND DISEASE: CYTOKINE EXPRESSION IN THE CNS A. Infection and Inflammation When living tissue is attacked by physical, chemical, microbial, or viral agents, the body responds by mounting an inflammatory response. Inflammation is an all-encompassing term describing the complex responses mounted by a host organism that aim to protect tissue and promote healing and repair. In response to systemic infection, by either viral or bacterial pathogens, two arms of the host immune system are activated; these comprise a non-specific arm, driven by the innate immune system, and an adaptive arm, known as acquired immunity. Perhaps the simplest and most studied inflammatory response in humans and rodents is that mounted against infection by Gramnegative bacteria, which involves the innate immune system. The cell wall of Gram-negative bacteria is encompassed by an outer membrane, the outer leaflet of which includes a complex family of endotoxic macromolecules, the lipopolysaccharides (LPS). When these bacteria enter the body, the LPS of the outer membrane are recognized as non-self antigens by Toll-like receptors expressed by specialized phagocytic cells of the host (Beutler, 2002; Hajjar et al., 2002; Konsman et al., 2002). These cells, the tissue macrophages, are the first line of defense against infection by pathogenic organisms, and their activation results in the expression and local release of inflammatory mediators such as cytokines, chemokines, and prostaglandins. The main pro-inflammatory mediators that drive the initial host response to infection are the cytokines IL-1β, IL-6, and TNF-α (Dantzer et al., 1999). These small peptides are considered to be pro-inflammatory because they orchestrate the early immune response to infection and injury by communicating with cells of the host immune system, attracting them to the site of infection/injury and causing them to become activated and responsive. Pro-inflammatory cytokines also drive the expression of other inflammatory mediators, such as chemokines, eicosanoids, and acute phase proteins, which mediate other aspects of the inflammatory response, and which contribute to tissue repair and a return to homeostasis. An in-depth discussion of the biological actions of all of these cytokines is beyond the scope of this review, so we refer the reader to the following websites; http://www .copewithcytokines.de/cope.cgi and http://www .weizmann.ac.il/cytokine/.

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B. Neuroendocrine-immune Interactions and “Sickness Behavior” Much of the early research that was aimed at characterizing the biological mechanisms that underpin the host response to tissue injury and infection focused on identifying the contribution of individual inflammatory mediators to the cellular response mounted at the site of infection. However, in 1988, in his paper titled “The biological basis of sick animals,” the veterinary surgeon Benjamin Hart proposed that when mammals become infected by a pathogenic organism, in addition to mounting a cellular inflammatory response, they demonstrate a highly coordinated set of behavioral and metabolic changes, which he collectively termed “Sickness Behavior” (Hart, 1988). In his original hypothesis Hart stated that these changes in behavior and metabolism were not simply a detrimental consequence of the infection and associated inflammation per se, but that they represented a strategic reorganization of the host’s motivational priorities to aid the body’s immune system in fighting off infection, and in promoting tissue repair and recuperation. The behavioral and metabolic changes described in the original hypothesis of sickness behavior included the development of a febrile response, locomotor hypoactivity, and anorexia. Throughout the 1990s much research was subsequently undertaken to identify the spectrum of behavioral and metabolic changes that occur in humans and other mammals as a result of infection, and the list of nonspecific symptoms of ill health that fall under the umbrella of “sickness behavior” has grown significantly (Figure 1). Much of this research was also targeted at characterizing the mechanisms and routes of communication by which the peripheral immune system is able to communicate with the CNS to give rise to these complex changes in behavior, metabolism, mood, and motivation (neuroendocrine-immune interactions). A number of routes of communication have now been proposed and are described in detail in other chapters of this book. These include (1) passive diffusion of cytokines into the brain parenchyma at sites lacking a functional blood-brain barrier and/or active transport of cytokines across the cerebral vasculature and into the brain; (2) activation of neural afferents by cytokines; and (3) self-induction of cytokine synthesis within the brain, mainly by the resident macrophages of the brain and cerebral vasculature.

C. Central Cytokine Expression and Neuroendocrine-immune Interactions The contribution of neuroendocrine-immune interactions to the development of sickness behaviors in

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FIGURE 1 Summary of the metabolic, behavioral, and cognitive changes associated with sickness behavior.

mammals has received much interest over recent years, but a single unifying pathway has yet to be described; it appears that all of the proposed routes of communication described to date (see above) act in synergy to coordinate the complex neurophysiological changes that underpin the expression of sickness behavior. However, despite marked differences in their underlying biology, the actuation of each of these pathways is similar, being dependent on the de novo expression of pro-inflammatory cytokines and related inflammatory mediators in both the periphery and CNS. Peripheral challenges with LPS can induce the expression of pro-inflammatory cytokines within specific compartments of the CNS (Dantzer et al., 2000; Dantzer and Wollman, 1998; Laye et al., 1994). For example, intravenous (i.v.) and intraperitoneal (i.p.) challenges with LPS in rats have been reported to induce the expression of cytokines and their cognate receptors by many cell types throughout the brain (Breder et al., 1994; Gatti and Bartfai, 1993; Muramami et al., 1993) (for review, see Rothwell and Luheshi, 2000). In addition to cytokines, expression of other proinflammatory molecules, such as prostaglandins (PGs) and chemokines, has also been demonstrated in the CNS in response to models of systemic/peripheral infection. For example, upregulated expression of cyclooxygenase-2, the rate-limiting enzyme that metabolizes arachidonic acid to PGs and other eicosanoids, is reported in cells associated with the cerebral vasculature and glia (Lacroix and Rivest, 1998). Constitutive expression of pro-inflammatory cytokines, and other mediators of inflammation, is typically very low in healthy tissues. As discussed in

“Infection and Inflammation,” the acute expression of these mediators in peripheral tissues following infection or tissue injury gives rise to a robust cellular inflammatory response. Although the principal function of such a response is to remove pathogenic organisms, thereby protecting surrounding tissues from further infection, bystander damage to surrounding cells and tissues may occur as a consequence of cellular infiltration and the production of highly damaging superoxides (La et al., 2001; Matsuo et al., 1994; Matsuo et al., 1995). However, in contrast to the situation in peripheral tissues, there is no evidence to suggest that the expression of pro-inflammatory mediators within the healthy CNS parenchyma is damaging in the context of a physiological response to systemic infection/inflammation. This raises two important related questions: (1) What regulatory mechanisms exist within tissues of the CNS to limit the cellular response to the acute expression of pro-inflammatory mediators? (2) Are these mechanisms adequate to protect neurones when the CNS parenchyma is already compromised by ongoing inflammation, trauma, or other disease? In the rest of this chapter we will present and discuss evidence demonstrating that expression of proinflammatory cytokines within the CNS parenchyma can exacerbate tissue injury during acute brain trauma, immune-mediated disease, and chronic neurodegeneration. We will consider mechanisms by which peripheral inflammation and immunosenescence drive glial activation and de novo synthesis of cytokines within the CNS, and review evidence that systemic infection is a risk factor for relapse in immune-


mediated disease, such as multiple sclerosis, and a stimulus for accelerated disease progression in rodent models of chronic neurodegeneration. In order to present a focused discussion, we will concentrate on in vivo models of CNS disease and bacterial infection, with brief reference to in vitro studies where necessary.

III. ACUTE INFLAMMATION OF THE CENTRAL NERVOUS SYSTEM A. Cytokines and Neuronal Death Physical trauma, ischemia, acute neurodegeneration, and infection of neuronal tissues all give rise to inflammation. Within the CNS, this response is mediated almost entirely by the innate immune system and is characterized by vascular and cellular changes including edema, vasodilation, glial activation, and recruitment of neutrophils and monocytes to the parenchymal tissue. In peripheral tissues, the first cells to respond to infection, tissue injury, and disease are the tissue macrophages. The macrophages of the CNS, the microglia, differ dramatically from those found in peripheral tissues, and in a healthy individual they adopt a quiescent phenotype with a highly ramified morphology. However, along with other glia, such as astrocytes, microglia respond rapidly to any changes in their microenvironment, becoming immunophenotypically and morphologically activated, upregulating their expression of cell surface antigens (Kreutzberg, 1996) and synthesizing and releasing a wide range of inflammatory mediators, including cytokines, chemokines, and prostanoids (Aloisi, 2001). Once released, these mediators act to further activate adjacent microglia, astrocytes, and neurones, amplifying the local glial response and driving leucocyte recruitment across the cerebral vasculature by inducing the expression of cell adhesion molecules (CAMs) on adjacent endothelia and by setting up chemotactic gradients. Interestingly, many of the pro-inflammatory cytokines expressed within the CNS in response to acute injury and infection are the same as those that dominate the inflammatory profile of acute injury and infection of peripheral tissues. However, in contrast to the periphery, delivery of the same inflammatory challenge to the brain parenchyma gives rise to only minimal leucocyte recruitment and inflammation (Andersson et al., 1992; Anthony et al., 1997). This relative insensitivity of the brain to acute inflammatory stimuli is important because tissues of the CNS have only a limited capacity to repair and regenerate. Despite this relative resistance of CNS tissues to acute inflammatory stimuli, there is now convincing

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evidence that pro-inflammatory cytokines, such as IL1β, IL-6, and TNF-α, contribute directly to tissue injury and neuronal death during acute trauma (for review, see Allan and Rothwell, 2003). Much of this work has come from studies using rodent models of focal cerebral ischemia and excitotoxic injury. Ischemic injury to the CNS occurs when tissue is deprived of oxygen and/or glucose for a protracted period of time, such as during a stroke. Under these circumstances, damage to tissue and neuronal death occurs in two distinct stages: (1) rapid loss of neuronal tissue in the ischemic core, where blood flow is reduced sufficiently to compromise energy metabolism; and (2) delayed loss of neuronal tissue in the area directly adjacent to the ischemic core (penumra), where blood flow is reduced, but not sufficiently to directly compromise neuronal survival (Dirnagl et al., 1999). Although the precise mechanisms of neuronal death in the penumra region have yet to be fully characterized, excessive glutamate release (excitotoxicity) from dying neurones and oxidative stress have been strongly implicated (Szatkowski and Attwell, 1994). Evidence for a role for inflammation is also convincing; marked leucocyte recruitment, pro-inflammatory cytokine expression, and glial activation occurs throughout the ischemic core and penumra region (Danton and Dietrich, 2003; Dirnagl et al., 1999). Direct evidence for a neurotoxic role of cytokines, such as IL-1β, has been provided by studies using rodent models of cerebral ischemia and experimentally induced excitotoxicity (Allan et al., 2000; Allan and Rothwell, 2003; Relton and Rothwell, 1992). For example, mice in which the genes encoding these cytokines, or their cognate receptors, have been disrupted (knockout mice) are significantly protected from the neuronal death in the striatum that results from occlusion of the middle cerebral artery (MCAo) (Boutin et al., 2001). Furthermore, administration of recombinant IL-1β to the CNS markedly exacerbates neuronal death resulting from MCAo and experimentally induced excitotoxicity. In support of this, abrogation of the activity of endogenous IL-1β by the administration of its endogenous antagonist, IL-1 receptor antagonist (IL-1RA), is protective in these models. Despite this convincing evidence for a neurotoxic action of pro-inflammatory cytokines in the acutely inflamed CNS, we still have only a limited understanding of the molecular mechanisms involved (Allan et al., 2005). To further complicate matters, deletion of certain pro-inflammatory cytokines from mice is detrimental to neuronal survival; for example, deletion from mice of the gene encoding IL-6 results in significantly larger regions of neuronal death in the CNS following MCAo (Loddick et al., 1998). Protection of CNS tissues following MCAo also occurs in mice in

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which expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1), has been deleted (Hughes et al., 2002). In these mice, expression of pro-inflammatory cytokines during MCAo was significantly reduced prior to leucocyte recruitment to the CNS, suggesting that these inflammatory mediators may contribute to neuronal death by pleiotropic mechanisms that could be considered as distinct from their role in driving inflammation. Indeed, certain populations of neurones express receptors for cytokines and chemokines, although the physiological relevance of this has yet to be fully characterized (Cho and Miller, 2002). There are circumstances in which acute trauma and injury to the CNS results in neuronal death in the absence of pro-inflammatory cytokine expression. In our own laboratory, focal cerebral ischemia induced by the microinjection of endothelin-1 into the CNS of rats gave rise to focal neuronal death but in the absence of an acute inflammatory response (Hughes et al., 2003). Neuronal death was unaffected in this model by the administration of recombinant IL-1β and the induction of neutrophil recruitment to the CNS (Felton et al., manuscript in preparation). Thus, it seems that although pro-inflammatory cytokines do affect neuronal survival during acute trauma, this effect is highly dependent on the nature of the insult; the profile of inflammatory mediator expression; and complex interactions between cytokines, neurones, and recruited leucocytes. Of further interest is that acute delivery of cytokines to the healthy CNS, in the absence of concurrent pathology, does not lead to tissue damage despite inducing leucocyte recruitment to the parenchyma and the debilitating expression of sickness behaviors. For example, bolus injection of recombinant IL-1β and TNF-α into the brains of rats induces local glial activation and delayed leucocyte recruitment, but without injury to adjacent neurones or disruption of the BBB (Andersson et al., 1992; Anthony et al., 1997). Similar effects are observed during chronic cytokine expression; for example, adenoviral-mediated transfer of genes encoding human IL-1β into the rodent CNS results in chronic inflammation, lasting up to 2 weeks, but only mild reversible damage to myelin and no loss of neuronal cell bodies (Ferrari et al., 2004); only very high supraphysiological levels of transgene expression give rise to detectable tissue damage and neuronal loss (our unpublished observations). Thus, there is significant evidence that proinflammatory cytokine expression in the CNS compromises neuronal survival following acute trauma. Whether this is a direct consequence of the ensuing inflammatory response and associated bystander damage or a more direct interaction between cytokines,

neurones, and other components of the CNS is still unresolved. Furthermore, the demonstration that cytokine expression in the healthy CNS gives rise to inflammation in the absence of tissue injury and neuronal death suggests that only neurones compromised by concurrent or ongoing pathology are likely to be susceptible.

B. Bi-directional Communication between the CNS and the Peripheral Immune System Despite efforts to identify mechanisms by which peripheral inflammation can modify brain physiology, and vice versa, little consideration has been given to the contribution that the peripheral acute phase response makes to inflammation of CNS tissues following acute trauma or infection. Most studies of neuroendocrine-immune interactions have focused on “inflammatory reflexes” which function to downregulate and control inflammation following damage to or infection of peripheral tissues (for excellent reviews, see Pavlov and Tracey, 2005, and Tracey, 2002). The CNS has been considered as a privileged tissue in terms of its immune status, being relatively protected from the consequences of inflammatory mediator expression, and isolated from the general circulation by a highly impermeable BBB. This may explain to some extent why we are only now beginning to consider the impact on CNS pathologies of bi-directional communication between the peripheral immune system and brain. It is now recognized that inflammation and injury to CNS tissues gives rise to a peripheral inflammatory response, suggesting that descending pro-inflammatory pathways of brain-to-immune communication are activated in response to these pathologies. For example, elevated levels of acute phase proteins (APPs), cytokines, and chemokines have been reported in the circulation and liver after bolus injection of recombinant pro-inflammatory cytokines, such as IL-1β and TNF-α, into the brain parenchyma or cerebral ventricles (Campbell et al., 2003; Campbell et al., 2005; De Simoni et al., 1990; De Simoni et al., 1995; Di Santo et al., 1999; Wilcockson et al., 2002), and after spinal cord compression injury in rats (Campbell et al., 2005). They have also been reported in human patients following ischemic stroke; in these studies the extent of the peripheral inflammatory response was positively correlated with disease outcome in terms of infarct area and neurological deficit (Emsley et al., 2003; Smith et al., 2004). However, this observation should be considered with caution, as many of the patients investigated had concurrent peripheral infections which may have elevated circulating cytokines independently of the CNS pathology.


We have investigated the contribution of the peripheral inflammatory response to leucocyte recruitment to the brain and spinal cord parenchyma following acute injury. We have shown that inflammation of these tissues induces rapid increases in hepatic and circulating concentrations of acute phase proteins (APPs), such as serum amyloid A protein (SAA) and serum amyloid P component, and chemokines, in addition to inducing local synthesis at the site of injury (Campbell et al., 2003; Campbell et al., 2005; Wilcockson et al., 2002). APPs have pleiotropic inflammatory actions, contributing to local inflammatory responses by driving processes such as pathogen opsinization, neutrophil activation, clotting, and tissue repair. Expression of these proteins, both at the site of injury and within the liver, occurs rapidly following acute injury or infection, and results in marked increases in their circulating concentrations within minutes to hours (Streetz et al., 2001). Chemokines also play a pivotal role during inflammation, driving leucocyte recruitment to injured tissues (chemotaxis), and activating resident cells of the immune system (Bajetto et al., 2001; Moser and Willimann, 2004). The traditional model of chemokine-driven leucocyte recruitment to injured/infected tissues is based on the generation of local chemotactic gradients which can be detected by circulating leucocytes through specific cell surface receptors. Chemokines are expressed by many cell types in CNS and peripheral tissues in response to inflammatory stimuli. On exposure to chemokines, circulating leucocytes take on an activated phenotype, allowing them to interact with endothelial CAMs, undergo diapedesis, and migrate into adjacent tissues along concentration gradients generated by passive diffusion of chemokine species from their site of synthesis (Rot and von Andrian, 2004). In general terms the CC chemokines are responsible for the selective recruitment of mononuclear cells to sites of inflammation and injury; examples of CC chemokines include CCL2 (Monocyte chemoattractant protein-1 [MCP-1]) and RANTES. The CXC chemokines, such as CXCL-1 (CINC-1 in the rat), drive the selective recruitment of neutrophils to inflamed tissues (Rot and von Andrian, 2004). We have previously reported that transient increases in levels of hepatic and circulating CC and CXC chemokines occur rapidly following acute injury to spinal cord tissue, or after the induction of inflammation in the brain parenchyma of rats. In the case of CINC-1, a CXC chemokine, this increase in expression peaks as early as 2 hours after an intracerebral (i.c.) injection of recombinant IL-1β, occurring earlier than the peak expression of a number of recognized APPs (Campbell et al., 2003; Campbell et al., 2005). The acute peripheral

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expression of these chemokines is also exquisitely sensitive to any injury of spinal cord tissue, and drives dramatic leucocyte mobilization from tissues such as the bone marrow. Moreover, we have recently demonstrated that this mobilization is essential for local leucocyte recruitment to the brain parenchyma induced by bolus injections of recombinant pro-inflammatory cytokines into the striatum of rats: delayed neutrophil and monocyte recruitment to the brain parenchyma that occurred after i.c. injections of recombinant IL-1β and TNF-α, respectively, was abolished by pharmacological neutralization of hepatic and circulating chemokines. Furthermore, artificial elevation of circulating CCL2 significantly increased local monocyte recruitment to the brain parenchyma. As a result of these investigations, we have proposed a novel mechanism by which hepatic chemokine synthesis contributes to local leucocyte recruitment and inflammation of CNS tissues. A summary of this mechanism is illustrated in Figure 2. The experiments described above provide convincing evidence that acute injury and/or infection of CNS tissues induces a peripheral inflammatory response, and that this is essential for leucocyte mobilization into the circulation, and local recruitment of neutrophils and monocytes during experimentally induced inflammation of the CNS parenchyma. The results of these studies point to the presence of rapid mechanisms of crosstalk between the brain and peripheral immune system that promote and amplify local inflammatory responses in tissues of the brain and spinal cord. Intriguingly, none of the inflammatory challenges administered to the CNS parenchyma in these studies led to detectable elevated levels of circulating cytokines (Campbell et al., 2003; Campbell et al., 2005), suggesting that the hepatic response to acute CNS injury may be mediated by an as yet unidentified pathway. Whether these routes of communication are essential for leucocyte recruitment to CNS tissues in response to all forms of acute injury, trauma, and infection warrants further investigation.

IV. IMMUNE-MEDIATED DISEASE OF THE CENTRAL NERVOUS SYSTEM: MULTIPLE SCLEROSIS A. Multiple Sclerosis as an Inflammatory Disease Multiple sclerosis (MS) is the archetypal immunemediated inflammatory disease of the human CNS, and although its etiology is currently unknown, it is widely believed to result from a complex interaction

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Rodent CNS 1

Inflammatory Stimulus Cytokines, chemokines

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Descending signal 6 Liver Circulation 3

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4 Acute Phase Proteins •Opsinization •Protease inhibition •Neutrophil activation •Blood clotting •Tissue repair

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FIGURE 2 (A) Diagrammatic representation of bi-directional communication between the brain and periphery in response to acute CNS injury. (B) Changes in the levels of CC and CXC chemokines in the liver, circulation, and brain following acute CNS injury. Inflammatory stimuli such as acute trauma rapidly result in glial activation and inflammatory mediator expression within the CNS (1). As a result, descending routes of communication between the brain and peripheral immune system are activated (2, 3), giving rise to the rapid synthesis and release of liver acute phase proteins (APPs) (4) and chemokines (5) into the circulation. APPs exert pleiotropic inflammatory effects, driving processes such as opsinization, blood clotting, neutrophil activation, and tissue repair. The rapid but short-lived increases in CC and CXC chemokine levels within the circulation (B) drives leucocyte (neutrophil and monocyte) mobilization from bone marrow and other tissues (6), resulting in acute leucocytosis. Over the following hours, circulating levels of chemokines begin to fall, but expression within the brain parenchyma continues to increase (B). The resulting chemokine concentration gradient drives leucocyte recruitment across the blood-brain barrier and into the injured brain parenchyma (7), generating a focal inflammatory response.

of an individual’s genotype and environmental factors (Compston and Cole, 2002). MS affects both the brain and spinal cord and is characterized by the presence of focal lesions (or plaques) where macrophages and T-cells cross the BBB and invade the adjacent CNS parenchyma (Prat et al., 2002). In some of these lesions the BBB is damaged to the extent that its integrity is compromised; in the clinic these regions of pathology can be visualized by magnetic resonance imaging (MRI) as gadolinium-enhancing lesions. The classical key feature of MS lesions (or plaques) is an extensive loss of myelin, but recent evidence has highlighted that injury to axons occurs in the early stages of disease (Ferguson et al., 1997; Trapp et al., 1998). Although the molecular events underlying this demyelination and axon injury are unclear, the intensity of the inflammatory response in MS lesions correlates well with the extent of axon injury (Ferguson et al., 1997). Animal models that mimic aspects of MS pathology, such as experimental allergic encephalomyelitis (EAE), have proven extremely useful in developing hypotheses on the inflammatory mechanisms and pathways that underpin the development of an MS lesion (Kivisakk et al., 2001). These models have confirmed the involvement of T-cells and macrophages, and the expression of a wide range of inflammatory mediators, including cytokines and chemokines. Although a progressive loss of axonal integrity is a key feature of MS, the expression of neurological impairment often follows a relapsing-remitting course, particularly in the early stages of the disease (Compston and Coles, 2002). The expression of disease exacerbation (relapse) followed by near-complete recovery from neurological impairment (remission) is also reflected in the underlying pathology and inflammation (Figure 3). This relapsing-remitting feature of MS pathology also characterizes a number of inflammatory diseases of peripheral tissues, which include Crohn’s disease (Yang and Lichtenstein, 2002) and rheumatoid arthritis (Elkayam et al., 2002), affecting the bowel and synovial joints, respectively.

B. Systemic Infection as a Risk Factor for Relapse in Multiple Sclerosis The significance of systemic infection as a risk factor for disease relapse in MS was first described by Sibley et al. (1985). In this detailed study, the rate of symptomatic disease relapse in a 7-week “at risk” period was found to be about three times higher in MS patients that had suffered from a systemic infection. The majority of infections in the experimental cohort were upper respiratory tract infections, but it is now accepted that other infections also increase the risk of relapse.


FIGURE 3 Disease progression in multiple sclerosis (MS). A graph showing the relationship between neurological impairment and axonal integrity. Neurological impairment in MS typically follows a relapsing-remitting course in the early stages of disease, despite a progressive loss of axonal integrity. In the later stages, when axonal integrity is markedly reduced, neurological function becomes progressively impaired (secondary progressive disease).

Further evidence that systemic infection is a risk factor for relapse in MS has been provided by studies of disease progression using MRI and positronemission tomography (PET) to visually assess the extent of CNS pathology. In MS, disruption of the BBB occurs in regions of inflammation and demyelination (plaques), and these pathologies can be visualized non-invasively in conscious patients using MRI to track the movement of normally impermeant molecules (such as gadolinium) from the general circulation and into the CNS parenchyma (Jeffery, 2000). In one such study, where relapse was defined by the onset of neurological impairment in combination with changes in the number and extent of gadolinium-enhancing lesions, the incidence of relapse in MS patients with systemic infections was up to 3.8-fold higher in a 7week “at risk” period than in control periods (Buljevac et al., 2002). Interestingly, when disease relapse was assessed in the same study using serial MRI scans around the time of infection, rather than by considering a protracted “at risk” period, there was no evidence of an increase in the number or extent of gadolinium-enhancing lesions. However, the results of such studies should be considered with caution, as they are likely to underestimate the real contribution of systemic infection to disease relapse. MS plaques visualized by MRI represent only a small proportion of the total inflammatory activity in the brains of MS

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patients. For example, PET scanning has revealed microglia activation throughout the brains of MS patients, including regions of the parenchyma in which the BBB is intact (Banati et al., 2000). Furthermore, animal models of immune-mediated disease have demonstrated that inflammation, BBB disruption, and tissue damage can occur independently in the CNS. It has been demonstrated by Newman et al. (2001) that focal delayed-type hypersensitivity (DTH) reactions in the rodent CNS, which are dominated by T-cell and macrophage infiltrates, have an extended phase in which inflammation, axonal injury, and other ongoing tissue damage can occur independently of BBB disruption, as assessed by MRI. To summarize, there is now substantial evidence that systemic inflammation following infection by bacterial or viral pathogens is a risk factor for relapse in MS. Although the majority of this work has focused on identifying risk factors for acute relapse in MS, it is also likely that systemic infection contributes to disease progression in the context of ongoing axonal injury. The nature of the transition from relapsing-remitting disease to secondary progressive disease in MS is currently unknown, but investigations of the impact of risk factors such as systemic infection would clearly be of great interest.

C. Mechanisms of Disease Exacerbation by Systemic Infection An understanding of the cellular and molecular mechanisms by which systemic infection, and the associated peripheral inflammatory response, may contribute to relapse in MS requires us to consider the role of pro-inflammatory mediators, activated glia, and infiltrating leucocytes in lesion pathogenesis. As discussed in “Multiple Sclerosis as an Inflammatory Disease,” recruitment of T-cells and macrophages across the BBB and into the CNS parenchyma is a key feature of the complex pathology associated with MS. Although the etiology of MS is largely unknown, a process of molecular mimicry of self-proteins by viral peptides is considered by some to be key to the resulting neuropathology (Edwards et al., 1998). Presentation of these peptides by the host’s immune system results in an inappropriate inflammatory response in which activated T-cells cross the BBB, target myelin peptides as non-self antigens, and generate a targeted chronic inflammatory response in the CNS. The contribution of systemic infection to disease progression and relapse in MS has therefore been largely interpreted in the context of further T-cell activation and recruitment that would result from elevated expression of pro-inflammatory cytokines in the periphery and CNS (Panitch, 1994)

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and activation of the T-cells by superantigens. For example, exposure of rodents to bacterial products such as LPS and Staphylococcal Enterotoxin A (SEA) and B (SEB), to mimic aspects of peripheral infection, has been shown to exacerbate clinical signs and induce relapse in models of immune-mediated disease such as EAE (Brocke et al., 1993; Schiffenbauer et al., 1993). However, similar responses to those seen with SEB challenges have also been reported after a single injection of TNF-α (Crisi et al., 1995), suggesting that peripheral innate inflammation, rather than antigendriven T-cell activation, is the stimulus for disease exacerbation. Although the mechanisms that result in clinical exacerbation have not been well studied, we do know that brain cytokine expression occurs as a result of systemic infection or inflammation (see “Neuroendocrine-Immune Interactions in Health and Disease: Cytokine Expression in the CNS”). We have studied how systemic inflammation impacts on an immune-mediated local DTH lesion in the brain parenchyma (Broom et al., 2005). A DTH lesion in the brain was evoked by depositing heatinactivated Bacillus Calmette-Guérin (BCG) into the brain parenchyma followed by systemic activation of the immune system with BCG (Matyszak and Perry, 1995). This results in a focal lesion in the brain parenchyma that is dominated by T-cells and macrophages with damage to the BBB, myelin, and axons. Over time the BBB damage resolves, but the inflammatory focus persists for many weeks. Using MRI and systemic administration of gadolinium, we have shown that peripheral challenge with bacterial endotoxin can give rise to a local transient breakdown of the BBB at the focus of the DTH lesion (Broom et al., 2005). Although the biological mechanisms underlying the reactivation of DTH lesions during systemic inflammation have yet to be characterized, the re-opening of the BBB observed in these rats clearly illustrates that systemic inflammation may impact on an immune lesion that is present behind an intact BBB. There is an extensive body of evidence to show that systemic inflammation can also give rise to the expression of pro-inflammatory mediators in regions of the CNS that have a fully functional BBB. When healthy rodents are exposed to very aggressive high-dose peripheral challenges with LPS, akin to levels associated with septic shock in man, expression of inflammatory cytokines has been observed throughout the CNS; studies using immunocytochemistry and in situ hybridization have demonstrated expression of cytokines by cells of the cerebral vasculature (endothelial cells and perivascular macrophages), meningeal macrophages, and parenchymal microglia. In related studies, the temporal and spatial expression pattern of

these cytokines within tissues of the CNS was also observed for other markers of inflammation, such as I-κβ, COX-2, CD-14, and cytokine receptors (for review, see Rivest, 2003, and Rivest et al., 2000). Although very aggressive challenges with LPS are required to induce detectable cytokine expression in the brains of healthy rats and mice, this may not be the case if tissue has already been compromised by a previous inflammatory reaction. Although some progress had been made by studies using immunocytochemistry and in situ hybridization, identification of the precise cellular sources of brain pro-inflammatory cytokines following systemic infection has proven difficult due to the limited spatial resolution of these approaches. In our laboratory, we have been particularly interested in the contribution that CD-163–positive macrophages make to brain cytokine synthesis during systemic infection. Although these are a minority population of brain macrophages, which comprises perivascular macrophages (PVMs) and meningeal macrophages (MMs), their strategic location might reflect a potentially important role in immune-to-brain signaling. PVMs are located throughout the cerebral vasculature and occupy a unique position at the BBB, lying between the glia limitans (formed by astrocytic end feet) and the basal lamina of cerebral endothelial cells (for review, see Schiltz and Sawchenko, 2003), whereas MMs are situated at the brain-cerebrospinal fluid interface (Figure 4). We now know that both PVMs (Elmquist et al., 1997; Schiltz and Sawchenko, 2002) and MMs (Garabedian et al., 2000) respond rapidly to elevated levels of circulating inflammatory mediators. Given the ideal locale of these cells to act as a relay mechanism between the circulation and brain parenchyma, we have been investigating whether they contribute to brain cytokine expression in a well-characterized model of peripheral infection. To do this, we have taken an in vivo approach in which CD-163–positive macrophages are depleted from the brains of rats using a selective toxin, sodium clodronate. Although not toxic at low concentrations, sodium clodronate induces apoptosis when actively concentrated in the cytoplasm of living cells (van Rooijen et al., 1996). In order to deplete CD-163–positive macrophages, sodium clodronate–filled liposomes were delivered into the cerebral ventricular system of anesthetized rats. These macrophages are the only cell type in the healthy CNS that is constitutively phagocytic, and as a result these are the only cells to take up the toxin and be depleted (van Rooijen et al., 1996). This approach has previously been utilized to demonstrate an important role for PVMs and MMs in driving leucocyte recruitment to the CNS during pneumococcal meningitis (Polfliet et al., 2001). However, our


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FIGURE 4 CD-163–positive macrophages in the rat brain. Photomicrographs of rat brain sections stained with a primary antibody (ED2) raised against the CD-163 antigen. (A) CD-163–positive perivascular macrophages (dark cells; arrows) lying between the glia limitans and the basal lamina of endothelial cells in the striatum of a healthy rat. (B) CD-163–positive meningeal macrophages (dark cells; arrows) lying at the braincerebrospinal fluid interface of a healthy rat. Scale bars = 50 μm.

investigations have failed to demonstrate a reduction in brain cytokine mRNA expression after an i.v. challenge with LPS in rats in which CD-163–positive macrophages had been depleted (Galea et al., manuscript in preparation). There are a number of possible explanations for this, but the most likely is that cerebral endothelial cells are capable of de novo cytokine synthesis in the absence of PVMs and MMs; endothelial cells outnumber PVMs throughout the CNS (25 : 1 in the striatum and 4 : 1 in the cortex), and have long been recognized as “brain sensors” of peripheral inflammation (Schiltz and Sawchenko, 2003). Thus, although there is a wealth of evidence showing that CD-163–positive PVMs and MMs do express cytokines (Garabedian et al., 2000; van Dam et al., 1992), their contribution to overall de novo brain cytokine synthesis is overshadowed by endothelial cells. However, whether endothelial-derived cytokines can cross the cerebral vasculature and penetrate the parenchyma remains to be demonstrated. Furthermore, our investigations to date have been limited to studies of brain cytokine expression in response to systemic infection in rats that are otherwise healthy. As observed for microglia, PVMs are reported to be upregulated in number and activation state in the CNS during and following immune-mediated disease (Fabriek et al., 2005). Thus, the contribution of these cells to brain cytokine synthesis may become significant if brain tissue is already compromised by ongoing pathology.

V. CHRONIC NEURODEGENERATION AND MICROGLIAL PRIMING Despite convincing evidence that inflammatory processes contribute to tissue pathology in acute CNS injury and disease, there are circumstances in which overt pathology of brain tissues can occur in the absence of a “typical” innate or acquired immune response. “Atypical” inflammatory responses are a characteristic feature of pathologies resulting from chronic neurodegenerative diseases, the archetypal example of which is Alzheimer’s disease (AD). AD is highly prevalent and the most common cause of cognitive decline in the elderly human population. The key features of AD pathology are non-specific deposition of Aβ-amyloid protein and the formation of neurofibrillary tangles within degenerating neurones. Although inflammatory mediator expression has been reported in postmortem brains of human AD patients and in some rodent models of chronic neurodegeneration, its contribution to disease progression has yet to be established (Akiyama et al., 2000). Much of the early research into the “inflammatory component” of AD was fueled by epidemiological studies demonstrating a significant protective effect of non-steroidal antiinflammatory drugs (NSAIDs) on disease onset and/ or progression (in t’ Veld et al., 2001; McGeer and Rogers, 1992; McGeer et al., 1996; Perry et al., 2003). It has been argued, however, that inflammation per se

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does not contribute to disease progression in AD. For example, AD pathology does not give rise to leucocyte recruitment to the brain parenchyma, as evidenced by the absence of immune cells in the perivascular space of blood vessels supplying CNS tissues; the cellular profile of AD is dominated instead by a process of microglial activation (Akiyama et al., 2000). Furthermore, clinical trials attempting to repeat the results of earlier NSAID studies using new and highly efficacious inhibitors of cyclooxygenase-2, a key enzyme in prostaglandin biosynthesis, or other antiinflammatory agents have proven unsuccessful (Aisen, 2002a; Aisen, 2002b; Aisen et al., 2002). Moreover, many patients with AD have systemic infections at the time of death (Beard et al., 1996), and we have to consider that this could significantly contribute to much of the pro-inflammatory mediator expression reported in postmortem brains. Interestingly, in our own studies, we have demonstrated that AD patients who have had a systemic infection, and/or elevated levels of circulating inflammatory cytokines, show a more rapid rate of cognitive decline over the following 2 months than patients that remain otherwise healthy (Holmes et al., 2003); these results were interpreted as evidence that peripheral inflammatory responses mounted against systemic infections have the potential to exacerbate or accelerate disease progression in AD. In related epidemiological studies, systemic infection has also been identified as a significant risk factor for the onset of dementia in the elderly, an important clinical symptom of AD (Dunn et al., 2005). In order to further investigate the contribution of inflammation to disease progression in AD, many research groups have turned to rodent models. However, although it is possible to induce aspects of AD-associated pathology in rodent brains, a reliable in vivo model has yet to be developed. As a result, we have turned our attentions to other models of chronic neurodegenerative disease. The transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of chronic fatal neurodegenerative diseases that share many pathological features with AD, and can be efficiently modeled in experimental rodents. Examples of prion disease include Creutzfeldt-Jakob disease (CJD) in humans, and bovine spongiform encephalopathy (BSE) in cattle. These diseases are either genetic, sporadic, or infectious in origin, and the causative agent is a protein (PrPSc) that is capable of inducing a conformational change in a normal host protein (PrPc), converting it into a protease-resistant likeness of itself (Prusiner, 1982). Despite the widespread expression of PrPc throughout the body (Aguzzi and Weissmann, 1997), and its conversion to PrPSc during disease, pathology

appears to be strictly isolated to the CNS. Much of our own research of the mechanisms underlying the pathology of prion disease has come from murine models in which mice have been intracranially inoculated into the hippocampus with brain homogenate of an infected animal. One example of this is the ME7 model (Betmouni et al., 1996; Williams et al., 1994), which has an incubation period of approximately 20 weeks (onset of clinical disease) and invariably results in death 2 to 3 weeks later. CNS pathology in this model reflects nearly all aspects of human prion disease. The first signs of disease appear within the hippocampus, well before the onset of clinical disease, and are characterized by synaptic loss, microglial activation, and deposition of PrPSc protein (Figure 5). Neuronal loss in this model typically occurs in the CA1 layer of the hippocampus and dorsal thalamic nuclei, but not until much later in the course of the disease (from week 18), when aspects of pathology have spread throughout the limbic system (Cunningham et al., 2003; Jeffrey et al., 2000). Interestingly, the inflammatory response to prion disease is highly atypical, being dominated by an immunophenotypical activation of microglia, but in the absence of marked pro-inflammatory cytokine expression. The pro-inflammatory cytokines that typically dominate acute inflammatory lesions, such as IL-1β, IL-6, and TNF-α, appear to be virtually undetectable in prioninfected brains (Cunningham et al., 2002; Walsh et al., 2001). The only mediators to have been shown to be consistently and significantly upregulated are acute phase proteins, transforming growth factor β1 (TGFβ1) (Cunningham et al., 2002), and the eicosanoid prostaglandin-E2 (PGE2) (Cunningham et al., 2005c; Minghetti et al., 2000; Walsh et al., 2000). TGFβ1 and PGE2 have well-defined anti-inflammatory properties in the CNS (McDonald et al., 1999; Zhang and Rivest, 2001), and are the principal mediators released by macrophages during phagocytosis of apoptotic cells (Fadok et al., 1998) (for review, see Perry et al., 2002). This has led to the suggestion that microglia in prion-infected brains are “primed” as a consequence of phagocytosing degenerating synapses and processes of neurons, which appear to degenerate in a manner akin to apoptosis (Cunningham et al., 2002; Perry et al., 2002). The nature of the triggers for microglial priming and neuronal apoptosis in prion-infected brains is still an area of much debate. Thus, taken together, the evidence suggests not only that the inflammatory response to prion disease is atypical, but that it may be relatively benign; in support of this, many manipulations of the immune system have failed to modify disease progression when prion agents are delivered directly into the CNS (Mabbott et al., 2000).


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FIGURE 5 Pathological changes in the brains of mice with prion disease. Microglia (FA11 staining) in the dorsal hippocampus of control mice (A; arrow), and mice inoculated with ME7 prion agent (B; arrows). Note the increase in the number of microglia in the brains of prion-diseased mice (B), and their activated morphology (B inset); microglia in the healthy CNS have a highly ramified morphology (A inset) characteristic of their quiescent phenotype. Synaptophysin staining in the dorsal hippocampus of control mice (C), and mice inoculated with ME7 scrapie (D); note the reduced synaptophysin staining, indicative of synaptic loss, across the hippocampus of prion-diseased mice (D). All prion-diseased tissue is taken from mice 19 weeks after inoculation with ME7 scrapie. Scale bars = 50 μm (A and B), 20 μm (A and B insets), 200 μm (C and D).

We have become interested in the contribution that “primed microglia” make to disease progression in the ME7 model of prion disease, particularly in the context of disease exacerbation during systemic infection. We have previously proposed that microglia primed by ongoing neuropathology may be more responsive to peripheral inflammatory responses than quiescent microglia, producing more potentially damaging inflammatory mediators in response to models of systemic infection (Figure 6) (Perry et al., 2003): Indeed, expression of the pro-inflammatory

cytokine, IL-1β, is much exaggerated in the brains of mice inoculated with ME7 after peripheral or intracerebral challenge with LPS (Combrinck et al., 2002). Similar elevated cytokine responses have also been reported in the brains of elderly (16 month) TgAPPsw mice, which display aspects of AD-associated pathology (Sly et al., 2001). Further evidence has come from studies of behavior in prion-diseased mice. Although clinical symptoms of disease, such as gross locomotor retardation, piloerection, and urinary incontinence, do not occur until late in the course of ME7 prion

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FIGURE 6 Microglial activation/priming responses. In a healthy CNS, microglia take on a quiescent phenotype with a highly ramified morphology and low expression of cell surface antigens (A). These quiescent cells respond rapidly to acute CNS injury or systemic infection, taking on an activated phenotype (B), upregulating their expression of cell surface antigens and pro-inflammatory mediators, thereby driving a local inflammatory response. In response to chronic neurodegeneration, or as a natural consequence of aging, quiescent microglia (A) do not become activated and are instead “primed” (C). Although primed microglia appear morphologically very similar to their fully activated counterparts, they express mediators that exert anti-inflammatory effects within the CNS; examples include TGF-β and PGE2. When these cells are exposed to a subsequent inflammatory stimulus, such as that which may occur during acute trauma or systemic infection, they switch to an activated phenotype (B), synthesizing and releasing pro-inflammatory mediators, such as IL-1β. In a CNS already compromised by pathology, such as that resulting from a chronic neurodegenerative condition, this expression of pro-inflammatory mediators may directly contribute to tissue injury and give rise to a heightened expression of sickness behaviors.


disease (approximately week 18), formal behavioral testing has revealed a full battery of pre-clinical changes in species-stereotyped (“instinctive”), affective, locomotor, and nesting behaviors, and early changes in cognitive function (Cunningham et al., 2003; Felton et al., 2005). The onset of these subtle behavioral changes coincides with the first detectable loss of synapses from the dorsal hippocampus, and long before overt neuronal loss can be detected (Cunningham et al., 2003). The onset of these behavioral dysfunctions also coincides with the first signs of microglial activation/priming and PrPSc deposition in the dorsal hippocampus and adjacent thalamic and cortical tissue. The pathological mechanisms underlying these complex behavioral changes have been discussed at length elsewhere (Cunningham et al., 2005a). Interestingly, the temporal profile of these pre-clinical behavioral dysfunctions is highly ordered, their onset occurring in the same order even where different strains of prion agent that induce distinct pathologies have been compared (Bruce et al., 1991; Cunningham et al., 2005b). This has led to the suggestion that these distinct rodent models of prion disease share a common early neurodegenerative pattern that has yet to be fully identified. Furthermore, the highly ordered temporal profile of regional neuropathology, glial activation, and pre-clinical behavioral dysfunction in rodent prion disease has allowed us to use formal behavioral testing as a powerful non-invasive tool for following disease progression (Felton et al., 2005). This approach has many benefits over traditional studies that have investigated pathology alone; for example, using long-term assessment of behavioral dysfunction in a single prion-diseased mouse provides us with invaluable information about the temporal evolution of neuronal dysfunction and pathology, whereas immunocytochemical analysis of neuronal death and/or inflammation at a single time-point provides us with only a “snapshot” of the disease process. In addition to mounting exaggerated cytokine responses to systemic infection, prion-diseased mice demonstrate exaggerated sickness behavior in response to i.p. challenges with LPS (Combrinck et al., 2002): Mice challenged with LPS 19 weeks after i.c. inoculation with ME7 showed greater, more prolonged febrile responses and suppression of locomotor activity in a novel open-field, when compared to control mice that had been inoculated with a normal brain homogenate control. Both of these exaggerated sickness responses are consistent with elevated expression of brain pro-inflammatory cytokines, such as IL-1β, which are detrimental to neuronal survival in the many models of CNS pathology.

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In the terminal stages of disease, from weeks 18 onwards, mice infected with ME7 prion disease develop significant and debilitating clinical symptoms, including urinary incontinence and associated inflammation of the anogenital area and of the underbelly. In a recent series of experiments, we investigated differences in prion-induced pathology, inflammation, and behavioral dysfunction in mice in which the gene encoding the chemokine, monocyte chemoattractant protein-1 (MCP-1), had been ablated (Felton et al., 2005); this inflammatory mediator is an important monocyte chemoattractant and activator of microglia, and its expression is markedly upregulated in the brains of mice early in the course of prion disease. Interestingly, mice deficient in MCP-1 (MCP1−/− mice) lived significantly longer (10–15%) than their wild-type controls, and in most cases failed to develop typical clinical symptoms of disease, including anogenital and abdominal inflammation; most MCP-1−/− mice were terminated due to severe weight loss in the absence of any other clinical symptoms. However, despite these differences in the onset of clinical disease and survival, no protection against the usual neuronal death, microglial activation, synaptic loss, or PrPSc deposition was observed in the brains of these mice; neither was there protection against the early behavioral deficits in speciesspecific behaviors. It is possible that that the delay in transition to terminal disease observed in MCP-1−/− mice may be related to the absence of peripheral inflammation that is normally associated with prolonged urinary incontinence in wild-type mice. Although the absence of this chemokine did not protect against early pathological changes in the brains of MCP-1−/− mice, it is possible that it acted to downregulate inflammation at the site of incontinence. Whether the absence of a peripheral inflammatory response in MCP-1−/− mice contributes to the observed increase in survival time is under investigation. Despite the evidence from our behavior studies that peripheral inflammation/systemic infection may contribute to disease progression in rodent models of chronic neurodegeneration, it has proven difficult to demonstrate this at the level of pathology. One possible explanation for this is that inflammation resulting from acute challenges with LPS is very short-lived (12–24 hours); this is also reflected in the associated sickness behavior, which typically resolves within 24 hours (Combrinck et al., 2002). However, we now have evidence from our own laboratory that a secondary peripheral challenge with LPS can significantly increase the rate of neuronal death in the CA1 dorsal hippocampus in mice with late-stage prion

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disease (Cunningham et al., 2005). Furthermore, a recent study by Nguyen et al. (2004) has described exacerbation of a chronic neurodegenerative disease by repeat peripheral challenge with LPS: In a transgenic mouse model of amyotrophic lateral sclerosis (ALS), it was demonstrated that repeat i.p. injections of LPS in the presymptomatic stages of disease exacerbated CNS inflammation and motor neuron pathology, and reduced survival time. Thus, using approaches in which formal behavioral testing has been combined with studies of CNS pathology and inflammation, we have provided evidence that systemic infection may exacerbate clinical disease and/or pathology in rodent models of chronic neurodegeneration. The results of these investigations are consistent with the results of preliminary clinical studies of human AD patients, which have identified systemic infections and peripheral inflammation as a significant risk factor for accelerated disease progression (Dunn et al., 2005; Holmes et al., 2003). The mechanisms of neuronal death that underlie clinical disease in human neurodegenerative conditions are largely unclear. However, in the context of disease exacerbation during systemic infections, exaggerated cytokine expression by primed microglia has been strongly implicated. As discussed in previous sections, expression of pro-inflammatory cytokines within the brain parenchyma is deleterious to neuronal survival in a wide range of disease processes, although the precise mechanisms involved are unclear. This hypothesis has led to much concern recently with the demonstration that primed microglia, and other inflammatory markers such as cytokines and chemokines, can be found in the brains of healthy aged humans and rodents (Godbout et al., 2005; Perry et al., 1993; Perry et al., 2003; Sheng et al., 1998). Indeed, it is now well established that progressive microglial activation/priming forms a natural part of the aging process (immunosenescence), possibly resulting from repeated lifetime exposure to peripheral infections. In a recent paper by Godbout et al. (2005), it was demonstrated using behavioral analysis and gene expression profiling that aged healthy mice mount significantly greater brain inflammatory responses and sickness behaviors to peripheral challenges with LPS. That these responses would be even more exaggerated, and potentially damaging, if brain tissue was already primed by ongoing pathology seems most likely. Given the steady increases in life expectancy that we are now experiencing, the impact of immunosenescence on the progression of chronic neurodegenerative diseases, and on the clinical outcome of acute

brain injury and other pathologies, deserves further investigation.

VI. SUMMARY In this chapter we have discussed mechanisms by which peripheral inflammation resulting from systemic bacterial or viral infections can impact upon pathology and clinical progression of CNS diseases. An important physiological response to systemic infection is the expression of pro-inflammatory cytokines within the brain and the development of sickness behaviors, which help the body to fight off infection, recover, and recuperate. However, there is now substantial evidence that aging, perhaps as a result of repeated lifetime exposure to systemic infections or as a consequence of early subtle stages of neurodegeneration, can result in a “priming” of microglia within the brain parenchyma. Primed microglia are responsive to subsequent inflammatory challenges, switching their phenotype to a more activated state, and there is evidence that this may by deleterious to neuronal survival in chronic neurodegenerative diseases. There is also strong evidence from clinical and in vivo studies that brain cytokine expression resulting from systemic infection is detrimental to neuronal survival during acute trauma. Furthermore, systemic infections have been identified as an important risk factor for disease relapse and progression in MS patients, and there is circumstantial evidence that this is also associated with brain cytokine expression. Thus, it is clear that systemic infections have the potential to impact deleteriously on tissues of both the periphery and CNS, and that this may be more marked in individuals, such as the aged, where the immune system has been “primed” by previous inflammatory events. In terms of the underlying biological mechanisms, a role for pro-inflammatory cytokines appears promising. Given this, and the strong evidence for bi-directional communication between the peripheral immune system and brain tissues during systemic infections, it would appear that the “immunological privilege” once believed to be afforded to CNS tissues is far from absolute. These observations are clearly of great clinical significance.

Acknowledgments We should like to thank Dr. Ian Galea, Dr. Tracey Newman, and Dr. Colm Cunningham for their help with the preparation of this manuscript and for allowing the inclusion of preliminary results from their ongoing investigations. We should also like to thank Prof. C. Anthony for critical appraisal of this manuscript, and acknowledge the Wellcome Trust, MRC, BBSRC, MS Society, and Nurin Ltd. for funding much of the work described.


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P A R T

III BEHAVIOR AND IMMUNITY MICHAEL R. IRWIN, LOS ANGELES, CA

INTRODUCTION Recent years have seen increasing public health attention to the contribution of psychosocial factors, behaviors, and behavioral disorders to chronic disease and health. In two separate reports from the Institute of Medicine, Health and Behavior (Institute of Medicine, 1982) and Health and Behavior (Institute of Medicine, 2001), a range of research aimed at understanding the interplay among biological, behavioral, and social factors in health and disease was identified and integrated. Indeed, this broad psychosomatic research has shown many reciprocal links among the central nervous system, which recognizes and records experiences; the endocrine system, which produces hormones that govern many bodily functions; and the immune system, which organizes responses to infections and other challenges. Similarly, it is recognized that specific behaviors are associated with increased risk of specific diseases and related conditions, which has led to an increasing acceptance of the fact that depression, for example, is linked to cardiovascular disease and to other health problems (Irwin, 2002). Recent research not only documents the importance of these behavioral factors, but also reveals some of the mechanisms involved. In other words, psychosocial factors influence health directly through biological mechanisms and indirectly through an array of behaviors. This Part addresses the myriad ways that behaviors and health are inter-related with an emphasis on the immunological mechanisms that underlie these interactions. As such, this series of chapters presents current knowledge about the influence of behaviors and behavioral disorders on the immune system, and about the impact of interventions on immune mechanisms with potential attendant improvements on health through modifying behaviors, emotions, or social relationships.

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The immune system is highly integrated with other physiological systems. It is sensitive to virtually every hormone, and sympathetic, parasympathetic, and sensory nerves innervate the organs of the immune system. In turn, the nervous, endocrine, and immune systems communicate bi-directionally through common hormones, neuropeptides, and cytokines. Hence, one conceptual theme for this collection of chapters is that behavioral responses are key in the activation of neuroendocrine and autonomic pathways, which in turn modulate the immune system with implications for increasing susceptibility to a variety of diseases. The importance of individual variability in disease susceptibility and health has been previously recognized (Ader, 2005), and this theme is similarly emphasized throughout this Part. People show large differences in resilience to and recovery from illness. Differences in behavioral responses are thought to mediate resilience, the ability to recover from adversity, by altering cellular processes that protect and build cells and tissues. A critical question raised by this research is whether positive emotions or behavioral interventions that target emotional processes or biological mechanisms including immunity might drive reserve capacity or resistance to the damaging effects of stressors on health. The generation of individual behaviors that influence morbidity and mortality occurs within a frame of development. Cumulative experience, adaptive plasticity, physical and social exchange with surrounding environments, and genetic predisposition interact to influence development and the biological and behavioral processes that preserve health or lead to human disease throughout life. Whereas developmental status is a continuing factor in health outcomes over life, in Chapter 21, Coe and Lubach aptly focus on the challenges faced during early infant development, given the heightened salience of this period for immediate and long-term health responses. These critical times of development from conception to weaning encompass extremely rapid biological and psychosocial changes, which have the potential to determine set points for homeostatic systems and the trajectories for subsequent biobehavioral functioning that can have long-term effects on health. For example, the handling of neonatal rats by experimenters leads to reduced emotionality and stress hormone reactivity throughout life, whereas prenatal stress increases emotionality and stress hormone reactivity throughout the life of the animal. Individuals develop and live in social systems, in which people influence and are influenced by their social networks. A full understanding of the interactions between health and behavior requires consideration of the separate levels of social interactions and the interplay among them. As articulated by Laudenslager and Kennedy in Chapter 22, social organization of many mammalian species creates dominance hierarchies, which alter behavioral responses, neuroendocrine activation, and immunity. Importantly, relational strengths among humans, such as positive ties with parents during childhood or intimate ties with a spouse during adulthood, can mitigate the negative consequences of social and environmental challenges. Most behaviors are not randomly distributed in the population, but are socially patterned and often occur together. Lower mortality, morbidity, and disability among


socioeconomically advantaged people have been observed for hundreds of years, using various indicators of socioeconomic status and multiple disease outcomes. In Chapter 23, Chen and Miller provide a solid body of evidence that social integration, the quality of social ties, and extent of social support are critical in influencing disease processes and mortality, possibly via the cumulative effects of socioeconomic status on immune mechanisms relevant to inflammatory disorders. Behavioral disorders that encompass a constellation of abnormal behavioral processes along with changes in psychosocial factors and biological mechanisms yield some of the most compelling support for the contributions of behavior to health. Major depressive disorder, which exceeds a lifetime incidence of 10%, is a potent risk factor for disease morbidity, with depressed persons showing a mortality rate twice that found in non-depressed persons. As discussed by Capuron, Miller, and Irwin in Chapter 24, altered functioning of the immune system is implicated as a mechanism that might contribute to medical morbidity of major depressive disorder including risk of infectious disease as well as inflammatory disorders. Moreover, much evidence has shown that anxiety and depressive disorders are often a frequent mental health outcome of traumatic stress exposure, and it is also reasonable to ask whether the persistent behavioral sequelae of severe stress, as found with post-traumatic stress disorder (PTSD), contributes toward similar biologic dysregulation. The composite alterations of immune function, as found in major depression, PTSD, and chronic stress (as reviewed in Part II), are striking, which together raise the possibility of a single, common adaptive response among brain, behavior, and immunity. Less clear is whether these immunological effects are of clinical significance. Longitudinal research strategies that target vulnerable “at risk” populations and utilize immune biomarkers that are biologically relevant to specific disease processes need to be exploited in order to investigate the degree to which behavioral disorders influence the expression or progression of disease. Communication between the brain and the immune system does not flow only from the brain to the immune system, but also from the innate immune system to the brain. As discussed elsewhere in this book, immune-to-brain communication pathways induce a cascade of cellular and molecular events in the central nervous system, which have behavioral consequences. Capuron, Miller, and Irwin also discuss brain effects of proinflammatory cytokines due to the activation of the innate immune system that prevails in depressed subjects, and whether these mechanisms lead not only to functional changes in the brain but also to structural alterations. In addition, an exceedingly relevant extension of this hypothesis concerns the involvement of immunological and immunopathological mechanisms in the etiopathogenesis of schizophrenia. As reviewed by Rothermundt and Arolt in Chapter 27, three hypotheses are being critically examined—namely, the infectious hypothesis, autoimmune hypothesis, and the Th1/Th2 imbalance hypothesis. Future research directions are discussed to carry forward evidence that schizophrenia is associated with abnormalities in microglia, astroglia, antibodies against brain structures and neurotropic viruses, blood-brain barrier, humoral immunity, cellular immunity, and cytokines.

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Alterations in other behaviors such as increases in alcohol consumption, poor sleep, and inadequate physical activity frequently co-occur with behavioral disorders including depression and PTSD; and each of these adverse behaviors has been recognized as unhealthful. Indeed, excessive alcohol consumption, other substance abuse, unhealthy dietary habits, and sedentary lifestyles are among the most health-compromising behaviors identified and targeted for modification or prevention with consequent benefit to the public health. Further chapters in this Part focus on the influence of these behaviors on immune system responses. Alcohol abuse is one of the top contributors to death in the United States, after tobacco use and diet and activity patterns. Approximately 100,000 deaths are related to alcohol consumption in the United States each year, which translates into 15% of potential years of life lost before the age of 65. The relationship between mortality, alcohol-induced illnesses, and alcohol consumption is primarily attributable to differences in the amount, duration, and patterns of alcohol consumption, as well as to differences in genetic vulnerability to particular alcohol-related consequences. In Chapter 26, by Schleifer, there is focus on the consequences of alcohol on the immune system at the upper end of a U-shaped relationship, with much evidence indicating that the alcohol alters immunity in part by its effects on neuroendocrine and autonomic pathways and on behaviors including the induction of poor sleep. In 2004, the National Sleep Foundation in the United Stated specifically promoted the importance of sleep for good health, an emphasis that is well justified by the striking prevalence of sleep problems with about one quarter of the population of the United States reporting complaints of insomnia, as well as population-based data showing steady decreases in sleep amounts in the last century. Moreover, insomnia is increasingly implicated as a predictor of cardiovascular and other mortality, particularly in vulnerable older adults. Despite these robust epidemiologic data, little is known about the physiological consequences of poor sleep nor the reasons that sleep is necessary for maintenance of health. In Chapter 28, by Opp, Born, and Irwin, the bi-directional relationships between sleep and the immune system are examined. The action of the proinflammatory cytokine network on sleep and the consequences of sleep in the modulation of the immune system, particularly innate immunity, are discussed. The co-morbidity between insomnia and chronic inflammatory disorders illustrates an area that can be exploited to understand the reciprocal influence among inflammatory cytokines and sleep on disease progression. In Chapter 31, Nieman examines the role of physical activity on immunity. Only 11% of the United States adult population report regular, vigorous physical activity for 20 minutes or longer more than twice each week. The major decreases in physical activity over the last decade are thought to contribute to the explosion of obesity in the United Studies. Studies show that men and women who are physically active have, on the average, lower mortality than people who are inactive; in contrast, a sedentary lifestyle has been linked to 23% of deaths from major chronic diseases. However, physical activity does not need to be vigorous to be beneficial to health, and even small increases have been associated with measurable health benefits for people who are inactive.


Against this backdrop, Nieman summarizes growing evidence that physical activity influences immune function and as a consequence, risk of certain types of infection including the most common of all, upper respiratory tract infections. However, emerging evidence also counters that prolonged and intensive exertion, in contrast to moderate physical activity, causes a downregulation of immunity, possibly by activation of stress response pathways. Variability in individual responses remains one of the common research themes linking behaviors and immunity (Ader, 2005), and the opportunities for research that exploits individual differences is further examined. In Chapter 29, Kemeny elegantly argues for a research strategy that is increasingly focused on psychological states and emotions that are posited to be the final links in brain regulation of physiological pathways and immune responses. Emotions, in contrast to longer-term affective states such as moods, are short, intensely felt, affects that are associated with distinctive facial expressions and behavioral predispositions. In other words, emotions serve to orchestrate the onset and coordination of a behavioral response to specific eliciting conditions (e.g., fear in response to threat of attack). Extensive research also documents that expression of emotions has beneficial effects on both emotional and physical well-being. Successful regulation of emotion appears to involve the controlled and modulated expression and release of feelings in ways that contribute to an increased understanding of those emotions and their meaning, which in turn can have immunologic consequences and even impacts on clinical outcomes of inflammatory disorders such as asthma and rheumatoid arthritis. As discussed by Kemeny, the specific mechanisms through which regulation of emotional expression affects immunity and possibly health are not fully understood and are the subject of continuing research. Nevertheless, the regulated expression of emotion is a potentially important component of interventions to change health. Behavior can be changed, and behavioral interventions can successfully teach new behaviors, alter behavioral response to stressors, and improve affective states (e.g., depressed mood). A critical challenge is to determine whether behavior changes can be maintained over time, and whether behavioral interventions can improve health outcomes and alter disease progression. Moreover, there are limited data on the influence of behavioral interventions on salient physiological pathways including immune responses, which may be relevant for infectious or inflammatory disease processes. Given this growing interest, the timely chapter by Antoni, Schneiderman, and Penedo (Chapter 32) provides insight into the empirical base demonstrating the influence of behavioral interventions on immune system functioning. Whereas relevant mechanisms have not been fully explored, it is thought that these approaches primarily act through proximal behavioral pathways in targeting emotional and affective responses with an emphasis on teaching new cognitive approaches to stress management. As a further extension and explicit test of the interplay between behavioral changes and the immune system, Pacheco-López, Niemi, Engler, and Schedlowski address whether immune response can be augmented by behavioral conditioning in Chapter 30. Building upon the seminal work by Ader and Cohen, this chapter provides a conceptual

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context for classical conditioning and presents empirical findings to demonstrate the adaptive capacity to associate specific stimuli (e.g., environmental cues) with certain immune responses. Given that both innate and adaptive immune responses can be enhanced by behavioral conditioning, potential clinical applications of this approach require investigation, with a focus on long duration efforts.

References Ader, R. (2005). Integrative summary. In K. Vedhara and R. IM (Eds.), Human psychoneuroimmunology (pp. 343–349). Oxford: Oxford University Press. Institute of Medicine. (1982). Health and behavior: frontiers of research in the biobehavioral sciences. Washington, D.C.: National Academy Press. Institute of Medicine. (2001). Health and behavior: the interplay of biological, behavioral, and societal influences. Washington, D.C.: National Academy Press. Irwin, M. (2002). Psychoneuroimmunology of depression: clinical implications (Presidential Address). Brain Behav., and Immun., 16, 1–16.

C H A P T E R

21 Mother-infant Interactions and the Development of Immunity from Conception through Weaning CHRISTOPHER L. COE AND GABRIELE R. LUBACH

I. INTRODUCTION 455 II. FETAL-INDUCED IMMUNE CHANGES IN THE MOTHER 457 III. PLACENTAL TRANSFER OF MATERNAL ANTIBODY TO THE FETUS 459 IV. ONTOGENY OF FETAL IMMUNITY 461 V. SOME IMPORTANT NUANCES OF IMMUNITY IN THE NEONATE 463 VI. SUSTAINED EFFECTS OF PRENATAL CONDITIONS ON LYMPHOCYTE FUNCTIONS 465 VII. INFLUENCES OF THE EARLY REARING ENVIRONMENT ON IMMUNITY 467 VIII. IMMUNE RESPONSES TO SOCIAL STRESSORS AND DISTURBANCE IN OLDER INFANTS 467 IX. CONCLUSIONS 469

processes are critically involved in the initiation and maintenance of pregnancy. Moreover, immune activation and disturbances of immune development are sometimes associated with cascading effects on brain and neuroendocrine physiology. As a consequence, the origins of health and immune-related disease can frequently be traced back to events that occurred in the womb and during early infancy.

I. INTRODUCTION Immune processes play a critical role in ensuring the survival and well-being of the developing baby. From the moment of implantation, the embryo must manipulate the mother’s immune responses to maintain the pregnancy. After birth the baby’s own immune responses then become essential for facilitating a successful adaptation to the rearing environment. Navigating this series of potentially treacherous hurdles requires a mixture of pre-programmed immune events, which appear to anticipate the baby’s needs, as well as some flexibility to accommodate unpredictable demands, including the variable level of pathogens in the postnatal world. It is the latter plasticity that is the subject of this chapter. Many studies have now documented that the developmental course followed by the immune system is not rigidly fixed in the immature host, but rather can be significantly affected by the antigens encountered by the young infant. In addition, the immune competence of the developing baby can

ABSTRACT The maturation of immunity in the developing infant highlights the complex interplay between intrinsic and external processes in shaping and regulating host defense. In addition to the programmed ontogeny of lymphoid tissue and cells, a capacity to adapt and learn from environmental priming is needed to provide optimal flexibility and resiliency. This malleability of immune responses during fetal life and infancy allows for a strong influence of pathogen exposure and early life events, including parental behavior. During development, the functions of leukocytes and cytokines also extend beyond protection against infection. Immune PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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be influenced by many other environmental and contextual factors. Exposure to environmental toxicants, maternal alcohol consumption, stress-related hormones, poor nutrition, and inadequate parental care have all been shown to influence the development of immune responses (Chandra, 1975; Weinberg and Jerrells, 1991; Wright et al., 2005). In a very real sense, the development of immunity in a young infant should be viewed as a malleable “learning” process, almost to the same degree as the developing brain. The basic building blocks of the immune system are set, such as the fact that a thymus will develop during the fetal stage. However, the amount and type of cellular activity are strongly influenced by the environmental priming, including the propensity toward a greater or lesser reliance on innate or adaptive immunity, and the set points at which the regulation of immune responses will become established in the mature host. For example, the resting number of B lymphocytes that will circulate in the healthy adult are strongly determined first by in utero processes and then affected further by the types of bacteria that are established in the gut (Cebra, 1999; Freitas and Rocha, 1993). Similarly, we have come to appreciate that early antigen exposure and infection can tilt the balance of immune processes toward or away from the development of allergies and asthma in the atropy-prone child (Warner et al., 2000). These findings are of importance for the field of psychoneuroimmunology (PNI) because they emphasize the significant influence of developmental and psychological processes on immunity (Ader, 1983). It is also of historical interest that a number of the pioneering findings in PNI were based on the observation that stressful events in infancy and disruptions of maternal care had a deleterious effect on later immune responses and resistance to disease (Ader and Friedman, 1965; Solomon et al., 1968). One challenge for the ongoing research today is to determine the full range of environmental events and stimuli that are salient enough to exert these longlasting effects on immunity. A second question of importance concerns the ways in which such persistent changes are mediated and sustained. In many cases, the immediate immune responses to the initiating event, such as a bacterial or viral infection during infancy, may have long subsided. Nevertheless, some lingering alterations remain because of changes that occurred in the infant’s developmental trajectory. Like a rocket on a new path, the altered course of maturation may extend and perhaps even magnify the consequences far beyond the initial responses. For example, injecting young mice and rats just once with endotoxin (lipopolysaccharide, LPS), or jolting it with a high dose

of a pro-inflammatory cytokine like interleukin-1 (IL-1), has been found to have life-long effects on the animal’s emotional reactivity, behavior, and adrenal responses to stress when tested in adulthood (Bilbo et al., 2005; Granger et al., 2001; Hodgson et al., 2001; Shanks et al., 1995). These concepts will be exemplified in this chapter mostly by reference to experiments conducted in our laboratory on the development of immune responses in young monkeys. However, the broader perspectives emanate from seminal papers on many other species, especially on rodents and domesticated farm animals, as well as from research on human infants (Kelley, 1980). The conclusions bear on our understanding of why some children grow up resilient and healthy, whereas others seem to succumb more readily to illness (Meyer and Haggerty, 1962). We have come to appreciate that the origins of cardiovascular and metabolic diseases can be traced back to the fetal period (Barker, 1994; Sayer et al., 1997). A similar viewpoint may also be appropriate for a number of immunerelated conditions, especially ones that involve inflammation like allergies and asthma (Devereux et al., 2001; Erb, 1999; Ferguson et al., 1997). The chapter begins with a brief review of immune processes required for implantation and the maintenance of pregnancy in the gravid female, which are essential to prevent rejection of the fetus. The next section provides a short summary of several formative immune events that occur during prenatal life, including the development of the thymus and the placental transfer of maternal antibody, critical for protecting the baby immediately at term. The rest of the chapter is devoted to a discussion of how events during the fetal and infant stages can elicit effects that continue to be evinced by the mature host. At least in animal models, including rats and nonhuman primates, many gestational stressors and perturbations of the rearing environment can exert a persistent influence on later immune responses (Klein and Rager, 1995). Investigations into the mediating processes often reveal that there were cascading effects that extend beyond immunity and involve parallel changes in both the brain and endocrine system. In many cases, animals that were disturbed during development also show alterations in hormones secreted by the hypothalamic-pituitaryadrenal (HPA) axis (Kay et al., 1998). Thus, understanding the basis for the immune sequelae of experimental manipulations in an immature animal often requires an analysis of multiple systems. Moreover, to say that one has an interest in learning about developmental PNI is analogous to stating a desire to delve more deeply into the numerous antecedents of health and the etiology of disease.

21. Developmental Antecedents of Immunity

II. FETAL-INDUCED IMMUNE CHANGES IN THE MOTHER Alterations in the immune biology of the gravid female become evident almost immediately after conception and must be sustained throughout pregnancy in order to allow the baby to reach term (Wegman et al., 1993). As illustrated in Figure 1, one of the embryo’s first tasks is to manipulate the intrinsic immunology of the uterine endometrium to promote engulfment of the implanting embryo and to prevent rejection. The invading edge of the trophoblasts that will become the mature placenta must actively inhibit certain types of maternal lymphocytes within the uterine tissue (e.g., the gamma/delta T-cells [γδ] and a CD56+ lymphocyte similar to a natural killer [NK] cell in blood) (Rai et al., 2005). This change is accomplished in part by activating uterine macrophages (Mφ) and CD8+ lymphocytes to constrain the other cells. The Mφ simultaneously stimulate the recruitment of maternal capillaries in order to provide a blood supply to the placenta, which they assist in sculpting (i.e., angiogenesis). Some of the mediating mechanisms have been resolved in exquisite detail, including the role of the non-classical human leukocyte antigen (HLA-G) on the trophoblast cells (Slukvin et al., 1998). Its involve-

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ment is intriguing because it must temper the response to the other paternal major histocompatibility complex (MHC) class 1 molecules on fetal cells, one of the primary factors against which the mother’s immune responses would have been directed. In addition, the maternal leukocytes are modulated further by the placentally induced increase in progesterone that is secreted from the ovary (i.e., via the stimulation of the corpus luteum with chorionic gonadotrophin). The complexity of getting these steps just right probably contributes to the high failure rate of early pregnancy. These are also immune pathways through which maternal stress could cause infertility, or affect fecundity in litter-bearing animals. Any physiological process or infectious pathogen that might stimulate the activity of the uterine NK cells could have an adverse effect on the viability of the conceptus. As shown in Figure 1, at this precarious stage, a promoter of the uterine NK response, such as increased interleukin-2 (IL-2), could become an abortifacient (Raghupathy, 1997). However, there has been relatively little systematic research on whether psychological factors do actually act at this level of reproductive immunology. Many more studies have been focused on how environmental and psychological factors may influence

FIGURE 1 Immune alterations induced in the uterine endometrium by the implanting embryo and trophoblast cells. Stimulation of macrophage and CD8+ cells helps to inhibit the responses γδ T- and NK cells and is required for successful implantation. The direction of facilitation (+) or inhibition (−) is shown.

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maternal and fetal immunity later in pregnancy. The conclusions from this large body of research are often erroneously simplified as just indicating that pregnancy is a time of immune suppression for the mother. Figure 2 illustrates that it is more appropriate to characterize pregnancy as a complex shift in immune balance (Makhsee et al., 2001). While some cellular responses in the mother do decline across pregnancy— such as how readily lymphocytes proliferate in culture or the inflammatory response in a test of cutaneous hypersensitivity—others may be enhanced (Sacks et al., 1999). For example, several components of innate immunity and inflammation may appear activated across pregnancy. Neutrophil and white cell counts are higher, and there is more granulocyte-macrophagecolony stimulating factor (GM-CSF) in the blood stream (Belo et al., 2005). The differential suppression or enhancement reflects the degree to which a particular immune reaction might participate in fetal rejection. It was once thought that fetal rejection was not initiated because the uterus is an “immune-privileged” site. Now it appears that the modulation of maternal immunity is really the primary factor. It is also reasonable to hypothesize that this immune modulation would have to be more pronounced in those species that have a hemachorial as compared to an epitheliochorial placenta. As some mammals, including the higher primates, evolved a more invasive placenta with less of a barrier between the maternal and fetal compartments, it would have increased maternal exposure to fetal cells and proteins. Thus, it became necessary to reduce the immune reaction to these fetal tissues exuding paternal antigens, which some have described as like a “tissue allograft” within the womb. Protection against infection must still be sustained. Cytokine production by maternal lymphocytes shifts increasingly away from a Th1 toward a Th2 profile, which enhances humoral immune responses (Veenstra van Nieuwenhoven et al., 2002) (Figure 2). Many studies of pregnant women indicate that a failure to appropriately make this transition, or a delay in its timing, could be a critical factor that contributes to miscarriage and other complications later in pregnancy, including eclampsia (Bates et al., 2002; Gervasi et al., 2001). The immune shift appears to be driven by a number of the key maternal and placental hormones that rise during pregnancy. It was once thought that the dramatic increase in progesterone was the major factor (Siiteri and Stites, 1982). Progesterone is secreted in large quantities from the placenta as pregnancy progresses, and does have immunomodulatory effects. However, other substances, such as alpha-fetoprotein (AFP), may be even more important. AFP is secreted

FIGURE 2 Changes in maternal immunity away from cellular and toward humoral responses, which is required for the maintenance of pregnancy and to prevent fetal rejection. Alpha fetoprotein and a number of hormones, including placental progesterone and CRH, may drive this shift in cytokine responses as pregnancy progresses. Used with permission of Blackwell Publishing.

in abundance from the fetal liver and crosses into maternal circulation, where it has many actions (Gabrant et al., 2002; Tomasi, 1977). The important take-home message for our review is that the immune changes in the mother (1) are substantial, (2) are essential for the viability of the pregnancy, and importantly (3) are driven by the fetus and placenta. Even though the immune changes are occurring within the mother and can be assessed by how her lymphocytes respond in culture—producing less Th1 cytokine (IFN-γ or IL-2) and more Th2 cytokine (IL-4 and IL-10)—the alterations were effected by the fetus for its own well-being. One other ramification of these immune changes for those interested in pregnancy outcomes and infant health is that they may be the vector explaining how the environment can influence the fetus. For example, bacterial infections during pregnancy, and the immune response to them, can be a cause of premature delivery. It doesn’t necessarily require a systemic infection. Women who have high levels of bacteria in their reproductive tract are at greater risk for delivering early (i.e., the gynecological condition of bacterial vaginosis, BV) (Hillier, 2005). High levels of maternal stress have been found to increase the prevalence of BV, perhaps by altering the protective levels of natural bacteria (Lactobacilli), and by inhibiting the ability of mucosal immune responses to contain the resulting bacteremia (Culhane et al., 2002). Increases in pro-inflammatory cytokines could then pose a problem for the maintenance of pregnancy. Research in animals has demonstrated that high levels of inflammatory cytokines, including interleu-

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kin-1 (IL-1) and tumor necrosis factor (TNF-α), can be a cause of pregnancy failure. Rising TNF-α levels in a woman’s blood are considered to be a valuable diagnostic marker of a pregnancy going awry and headed for an early termination (Bates et al., 2002; Makhsee et al., 2001). The placenta is extremely sensitive to the cytokines in maternal circulation, and in response may release its own cytokines and hormones. In the specific case of the monkey and human placenta, cytokines will also drive an increase in its secretion of corticotrophin-releasing hormone (CRH), another warning sign for a premature delivery (MacLean and Smith, 2001; Wadhwa et al., 2001). Some placental cytokines have been found to cross into the amniotic fluid and the fetal compartment. In fact, administering endotoxin (LPS) to a pregnant rat will result in a series of cytokine changes in the placenta and fetal blood, which in turn elevates cytokine production within the fetal brain (Gilmore et al., 2004; Urakubo et al., 2001). A similar type of cascading effect was seen after endotoxin administration to pregnant mice, because cardiovascular functioning in the fetuses was adversely impacted by impaired blood flow to the placenta (Rounioja et al., 2005). These types of findings have led many researchers to advocate that further study of cytokine biology during pregnancy is critical for our understanding of the relationships between stress, reproductive health, and infant development (Coussons-Read et al., 2003). In addition to the clinical significance of bacterial infections for explaining pregnancy complications, there is also a substantial literature on the risks posed by certain viral infections. Beyond viruses known to be teratogenic pathogens in the first trimester, causing fetal malformations and death (e.g., rubella and mumps), there are a number of others that may have more subtle effects on the fetus (Gonik, 1994). Of particular importance today are suspicions about the more virulent strains of influenza because of the new threat of avian flu. During the infamous 1918–19 flu pandemic, more than 50% of the infected pregnant women and babies died (Harris, 1919). But maternal infections with other strains of flu may also be of some concern, even for viable babies (O’Callaghan et al., 1991). A survey of antibody titers in 1,659 pregnant English women in 1993–94 indicated that approximately 11% had been infected with flu at some point during gestation (Irving et al., 2000). Prenatal flu infections have been implicated as a risk factor for a number of neurodevelopmental disorders as well as for later psychopathology, including schizophrenia (Brown et al., 2000; Dammann and Leviton, 1997). Animal models of flu infection have provided some support for this conjecture, even though the flu virus typically does not cross the placenta (Shi et al., 2003). In our research with

nonhuman primates, we are currently studying the consequences of maternal flu infections, with a particular focus on gestational timing because mid-trimester infections have been hypothesized to be of greatest concern. A greater fetal vulnerability at mid-pregnancy is thought to be due to the fact that a baby’s brain regions are still being organized and neurons are in the process of migrating, whereas later they proliferate and make connections in situ.

III. PLACENTAL TRANSFER OF MATERNAL ANTIBODY TO THE FETUS As pregnancy progresses, the maternal immune shift away from Th1- and toward Th2-driven humoral responses has a unique benefit for the developing fetuses of some species. For those mammals that transfer maternal antibody to the baby before term, the humoral bias may help to facilitate the transmission of maternal IgG across the placenta. Table 1 provides examples of species that transfer antibody prenatally as well as ones that do it postnatally in colostrum and breast milk. Higher primates have opted to provide antibody in two installments, transferring the G class primarily across the placenta, while maternal IgA is provided largely after birth in breast milk. About a decade ago, we became interested in how this placental function evolved in humans. Our species has a particularly active transport mechanism, because children are usually born with titers that exceed adult levels, typically between 100–200% of the maternal IgG level (Allansmith et al., 1968). These high antibody titers protect against pathogens previously encountered by the mother and afford an extended period of passive immunity. The availability of this antibody for binding and neutralizing pathogens reduces the need for as

TABLE 1 Representative Species that Provide Maternal Antibody (IgG) Before Birth via the Placenta or Postnatally in Colostrum and Breast Milk.

Kangaroo Pig, cow, horse Rabbit Rat, mouse Monkey, ape Human

Prenatal

Postnatal

yolk sac yolk sac placenta (IgG) placenta (IgG)

via ingestion colostrum, milk nursing nursing milk (sIgA) milk (sIgA)

Note: Humans acquire most IgG across the placenta and then receive sIgA via nursing to protect against enteric pathogens. Premature human babies are thus deficient in IgG; bottle-fed infants receive less sIgA.

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vigorous an immune response by the infant for the first 3–6 months of life. To better understand the transfer process, we conducted a comparative evaluation of representative species across the Primate Order (Coe et al., 1994). Blood samples were obtained from mothers and neonates in five taxonomic groups: prosimian, New World monkey, Old World monkey, ape, and human. Figure 3 shows the results, portraying the neonate’s IgG titer as a percentage of the mother’s level after birth. The galago, a prosimian more like the ancestral primate, is apparently still reliant on a postnatal transfer like that seen in camels, horses, and cows. Infants from the less advanced South American monkey had birth levels averaging between 35 and 40% of the mothers’ IgG. It was not until the rhesus monkey and the chimpanzee that IgG levels at birth began to approach equivalence between mother and baby. Humans go beyond attaining an equilibrium. With our active transport system, maternal IgG is avidly captured by the Fc receptors on the placenta and accumulated on the fetal side (Kohler and Farr, 1966). The importance of the placentally transferred antibody for the human neonate is that relatively little of the additional IgG acquired after birth by ingestion crosses the baby’s gut. Those animal species that provide IgG via colostrum or milk do so while the babies have a more permeable small intestine. Thus, in human and monkey babies, the maternal IgG declines

progressively over the next 3–6 months as the antibody slowly breaks down (Coe et al., 1988a). Infants born with low IgG levels will reach a nadir sooner and will experience a more marked period of hypogammaglobulinemia before they are capable of synthesizing substantial amounts of antibody by themselves (Hosking and Robertson, 1983). Children born premature are especially prone to this transient condition, because they typically have low IgG levels at the time of delivery (Berg, 1968). To track the temporal dynamics of the transfer process back into the fetal period, IgG levels were determined in cord blood samples collected from fetal rhesus monkeys delivered by caesarian-section (Coe et al., 1993). Sampling began at Day 125 postconception and continued through the end of pregnancy, which is typically 169 days. This evaluation revealed that there was a substantial increment in the amount of IgG transferred in the final 2 weeks of pregnancy (Figure 4). The late installment would make functional sense given the goal of extending the benefits as long as possible into the postpartum period. However, the lateness of the placental transfer would mean that any infant born more than a few weeks premature would be deficient. This observation provides one of the explanations for why premature babies are at greater risk for infection. The premature baby would also have to more quickly transition from a reliance on passive immunity to its own immature cellular immune

FIGURE 3 IgG levels at birth in neonates of representative species from five major taxonomic groups across the Order Primates (Coe et al., 1994). Neonate antibody values are portrayed as a percent of adult levels and reveal the progression toward an increased reliance on prenatal transfer. It coincided with the evolution of a more active transport of maternal antibody across the placenta.

FIGURE 4 Progressive increase in maternal antibody in fetal rhesus monkeys (n = 20) from Days 125 to 162 postconception (Coe et al., 1993). The marked increase in the last week demonstrates the lateness of the placental transfer as term approaches (Day 169). Premature infants would thus have low levels of maternal IgG.


defenses. Such a necessity for an accelerated maturation could contribute to an immune predilection for the development of allergies. The immune responses of newborn human babies are usually Th2 dominant. It has been hypothesized that an inappropriate, early initiation of some cellular responses, including genetic biases toward certain cytokine responses (e.g., low IL12 production) might be one of the risk factors for emergence of asthma (Wright et al., 2005). For humans and the other higher primates that actively transfer IgG, this placenta transmission appears to be relatively resistant to gestational stress unless the pregnancy is foreshortened. Maternal stress was also found to not reduce the transfer of specific antibody to herpes simplex (HSV) in mice (Yorty and Bonneau, 2004). Moreover, in this study even low antibody titers were still sufficient to afford protection against HSV in the pups. Experiments on rats and pigs, however, have shown that maternal disturbance can sometimes impact IgG levels in the pups and piglets (Machado-Nedo et al., 1987; Sobrian et al., 1992). In addition, when the effects of gestational stress were investigated in squirrel monkeys—the primate species that transfers only 35% of maternal titers—we also found an effect of recurrent stress across pregnancy (Coe and Crispen, 2000). The finding was quite interesting because of a differential effect seen in the male and female babies. Pregnancy stress was induced by repeated disturbance of the mother’s social relations: moving the gravid monkeys to three different groups during early and mid-gestation. As expected, the male babies from stressed pregnancies had lower-thannormal IgG levels at birth. In contrast, the prenatally stressed female babies actually had higher antibody levels, both above the males as well as even higher than normal female infants from undisturbed pregnancies. It is intriguing to speculate that the placentas of female fetuses had engaged in a compensatory response, enhancing antibody transfer in seeming anticipation of an adverse postnatal world. We have also investigated the effects of antenatal corticosteroid treatments on IgG levels in the rhesus monkey, because it is known that drugs like dexamethasone (Dex) can catabolize antibody (Coe and Lubach, 2005). A 2-day regimen, which is the typical duration used for premature human babies in clinical practice, caused only a modest decline in maternal and fetal IgG levels. However, given that there was any effect of such an acute exposure, it is likely that longer corticosteroid treatments would result in a larger decrease in antibody over time. Repeated courses of Dex or betamethasone are sometimes employed when there is a recurrent threat of early delivery as well as in some premature babies (Cederqvist et al., 1978).

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IV. ONTOGENY OF FETAL IMMUNITY While the placental transfer of antibody is an interesting prenatal process that can affect postnatal immunity, many other aspects of the fetal immune system have already begun to develop in earnest before the third trimester. Moreover, this organogenesis and hematopoiesis within the fetus is likely to be of greater relevance for explaining how the extrinsic world first impinges upon the development of fetal immunity. Considerable information is available on when various components of the immune system become functional. The fetal thymus, for example, develops early in gestation, and in terms of its overall size is actually more prominent by birth than at later points in the life span. For over 100 years, it has been known that human babies are born with a relatively large thymus of about 25 grams, which is comparable to the size of a mature gland in adults, despite the vast differences in body weight (Hammar 1921; Kendall et al., 1980). The developmental course of the thymus postnatally is also predictable (Figure 5). Its size will peak at puberty, which led to some speculation that it was involved in determining age at puberty. In fact, the timing of pubertal maturation can be altered by thymectomy during infancy, and there is still much more to learn about the important life-long relationship between reproductive hormones and thymic function (Grossman, 1989; O’Grady and Hall, 1991). After puberty, the thymus begins a slow but progressive decrease in size, ultimately shrinking sufficiently to contribute to the onset of immune senescence in the aged host (George and Ritter, 1996; Mackall and Gress, 1997; Trainin, 1974). Whether early life events could actually shape the full trajectory of the thymus across the whole life span is

FIGURE 5 Developmental trajectory of the thymus across the human life span. The thymus is extremely large in the neonate, thymic size peaks at puberty, maintains its structure in adulthood and slowly regresses in old age.

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not well understood, but some studies have indicated a link between prenatal events and later thymusdependent functions in adolescence and adulthood (McDade et al., 2001). In addition, it is definitely well established that thymic size is extremely responsive to stress and challenge at all ages (Jeppesen, 2003; DiNaro et al., 2006). Thymo-lymphatic involution was one of the three cardinal signs that Hans Selye identified as indicative of the stressed state in the mature individual (Selye, 1956). Similarly, it has long been known that maternal stress and administration of corticosteroid and estrogenic drugs during pregnancy can cause a marked diminution of thymic size in the fetus (Selye and Marion, 1955). High levels of corticosteroids have a deleterious effect on the structural and lymphoid zones of the thymus, and can directly lyse or stimulate the apoptotic death of immature thymocytes (Godfrey et al., 2000; Hendrickx et al., 1975; Sawyer et al., 1977). Nevertheless, it appears that normal cortisol and corticosterone levels are actually beneficial, and may restrain the premature and inappropriate growth of certain cells. For example, the offspring of adrenalectomized female rats showed an accelerated development of thymic dendritic cells with a mature phenotype (Sacedon et al., 1999). Sex hormones also act on the thymus, and both estrogen and progesterone are very high during pregnancy (Bodey, 2002; Seiki and Sakabe, 1977). The fetus probably plays a major role in stimulating this type of feedback from hormones, because the dehydroepiandosterone (DHEA) from the fetal adrenal serves as the primary precursor for the estrogen made by the placenta. In addition, the androgenic hormones secreted by the male fetus during sexual differentiation may help to explain some of the gender differences in the propensity for autoimmune diseases later in adulthood (Savino and Dardenne, 2000). Androgens tend to have a dampening effect on the thymus (Olsen et al., 2001a, 2001b). Thus, a thymic enlargement is observed following castration of male rats. The development of the thymus during fetal life also coincides with the emergence of T-cells in utero and the initiation of some cellular functions before term. Hematopoietic stem cells migrate from the bone marrow and then grow and differentiate within the optimal microenvironment of the thymus (Bodey, 2002). At the same time, there has to be extensive intrathymic selection of the T-cell repertoires, in particular selecting against thymocytes that would respond inappropriately to self-antigens. This cellular pruning process is critical for preventing immune responses that might result in attacks on healthy tissue or be a cause of later autoimmune disease. In humans, func-

tioning T-cells can be found as early as 10 weeks after conception, based on the capacity to stimulate cord cells in vitro with mitogen (i.e., phytohemagglutinin, PHA) (Warner et al., 2000). Even so, T-cell responses and other aspects of cellular immunity are still not fully mature at birth. By mid-gestation, samples obtained from cord blood indicate that some cells may also have been activated already by allergens. This finding led to the hypothesis that prenatal exposure to certain proteins may be priming those babies who are genetically predisposed to atopic conditions (Devereux et al., 2001). An ex vivo modeling of protein diffusion across whole placentas demonstrated that small molecular weight substances can cross the placenta (especially if less than 500 Da) (Loibichler et al., 2002). Based on these analyses, consumption of eggs or milk by a pregnant woman could allow allergenic proteins like ovalbumin and lactoglobulin to reach the fetus as soon as 2 hours after ingestion. In keeping with this view, surveys of cord blood lymphocytes at term have documented a seasonal variation in the prevalence of babies born with cells activated against pollen (Piccinni et al., 1993; Van Duren Schmidt et al., 1997). These findings of cellular responses to grass and birch trees in the neonate suggested that there had been a prior in utero exposure. Further support for the idea of prenatal priming was garnered by showing that some cat dander can be pulled across the placenta as part of an IgG-bound complex along with the placental transfer of other maternal antibody (Casas et al., 2001). Nevertheless, from an overview perspective, Table 2 shows that it is still most correct to convey that the cellular immune responses of a newborn baby are immature and not yet fully functional. Lymphocytes obtained from cord blood do not proliferate as well as mature adult cells in culture, make less cytokine overall, and show a strong Th2 bias in their pattern of cytokine release (Adkins et al., 1999). Extensive TABLE 2

Distinguishing Features of Immunity in the Neonate

• Innate immunity more established than adaptive immune responses • Low lymphocyte proliferative responses in vitro compared to mature cells • Reduced ability of antigen-presenting cells (APC) • Low cytokine production and sensitivity; Th2 cytokine phenotype • Slower class switching in antibody responses to antigen (IgM to IgG) • Passive immunity from mother (IgG via placenta in monkeys and humans) • Unique CD4+ T-cell suppressor activity

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research has been directed to figure out the reasons for the deficient lymphocyte responses and the cells’ lower production of Th1 cytokines (e.g., IFN-γ). There appear to be many explanations, but an especially important factor is incompetent antigen presentation. When neonatal lymphocytes are incubated in culture along with professional antigen-presenting cells (APC) obtained from adults, a more mature response is elicited. These findings indicate the lymphocyte itself is not dysfunctional in the neonate. But under normal circumstances in vivo, the newborn infant would have to rely more on its innate immune responses, including the complement system, and also the passive immunity obtained via maternal antibody. When induced to produce antibody, the lymphocytes of developing infants are also more likely to make IgM and do not class switch as readily to IgG in the manner of adult cells.

V. SOME IMPORTANT NUANCES OF IMMUNITY IN THE NEONATE While it is essential for the newborn baby to have a sufficient armamentarium to respond to pathogens, an equally pressing need is to limit excessive overreactions. The young host is exposed to a myriad number of antigenic stimuli simultaneously, including a massive invasion of bacteria into the gut. So its immune responses must be tempered. Several decades ago, it was discovered that the young infant has a unique type of CD4+ T suppressor cell that can dampen immune reactions (Papadogianniakis et al., 1990). We took advantage of this observation to investigate whether a history of prenatal disturbance might alter how the newborn monkey responded to the world through a change in the activity of these T suppressor cells (Coe et al., 1996). Rhesus monkeys were generated from undisturbed, control pregnancies as well as from mothers that had been administered daily injections of adrenocorticotrophin hormone (ACTH). ACTH was used because it does not cross the placenta, but would raise the mother’s cortisol to high levels. For 2 weeks from Days 120–133 postconception, ACTH was administered to the gravid female either in the morning or afternoon. At 2 and 6 weeks postpartum, blood samples were then obtained from the babies, and their mononuclear cells (MNC) set up in a suppressor cell assay. Briefly, the infant’s MNC were stimulated with concanavalin A (Con A) for 2 days, after which they were treated with mitomycin C for 45 minutes. The primed cells were then added to heterologous cells from an adult male responding to Con A. Reduction in the proliferation of the responder cells was used as an index of suppressor activity. As can be

seen in Figure 6, monkeys generated from the ACTHtreated pregnancies showed significantly less cellular inhibition. It is also noteworthy that these babies as well as the control monkeys did show the typical agerelated decline. As a consequence, the ones from the ACTH-treated pregnancies had less suppressor activity at both age points. This maturational decrease by 2 months of age coincides with the developmental appearance of other immune responses. Our studies on maternal stress during pregnancy have documented effects on many other immune functions (Coe et al., 1999). One difference immediately apparent in the neonatal period is an impact on the types of bacteria that will become established as the commensal microbiota of the gut. This research conducted by Michael Bailey was especially insightful because it revealed how a seemingly innocuous change could set the stage for later disease (Bailey et al., 2004). It also documented how rapidly bacteria invade the gut as soon as the baby disembarks from the sterile environs of the womb. Within 1–2 days of birth, millions of bacteria were in evidence as determined by coprocultures. The main assessment then focused on the establishment of two bacterial populations known to be of benefit to the host: Lactobacilli and Bifidobacter. These natural constituents of the gut microbiota can help to suppress the growth of enteric pathogens (Cebra, 1999). Briefly, monkeys were generated either from undisturbed, control pregnancies or from females that had Infant Suppressor Activity

% Suppression

FIGURE 6 Suppressor cell activity in infant rhesus monkeys at 2 and 6 weeks of age (Coe et al., 1996). Some mothers were administered ACTH for 2 weeks during pregnancy (Days 120–133 postconception), which resulted in infants with lower suppressor responses. All infants showed the typical maturational change from 2 to 6 weeks of age. Used with permission of Elsevier.

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experienced daily periods of acute stress for 6 weeks of their 24-week gestation. The stress periods were scheduled to occur either Early or Late in pregnancy (Days 50–92, or 105–147 postconception, respectively). From Day 2 postpartum through 6 months of age, rectal swabs were obtained to determine the concentrations of Lactobacilli and Bifidobacter. As shown in Figure 7, babies from both the Early and Late Stress pregnancy conditions had significantly lower levels of Lactobacilli. When Bifidobacter levels were enumerated, a prenatal effect was seen primarily in the infants from the Late Stress pregnancies, indicating that the timing of the gestational disturbance also had a selective effect on certain gut bacteria. The altered bacterial profiles were found to have disease ramifications. All of the babies were screened for the presence of Shigella, a gram-negative enteric pathogen, which is endemic in most monkey colonies. Whereas none of the control babies evinced any Shigella across the first 6 months of life, it was found to be present without symptoms in 43% of Early Stress babies. Similarly, Shigella occurred periodically in the cultures from 38% of Late Stress monkeys, and two developed diarrhea symptoms requiring treatment with antibiotics. In summary, the prior occurrence of maternal stress altered how the young baby would interact with bacteria present in the rearing environment. The prenatal conditions influenced how bacteria became established as the gut microbiota, which was associated with the capacity to prevent enteric infection.

Log (10) CFU/g

Control

Early Stress

Late Stress

FIGURE 7 Levels of Lactobacilli in 24 infants quantified across the first 6 months of life by coproculture (Bailey et al., 2004). Prenatal disturbance resulted in lower levels of protective microbiota in the gut, which was associated with an increased risk for infection with Shigella, an enteric pathogen. Infants from the Late Stress condition also had lower levels of the protective Bifidobacter. Copyright 2002, The Endocrine Society.

These conclusions are not restricted to non-human primates. It has long been known that mice and rats reared in gnotobiotic environments will grow up to exhibit abnormal immune responses because the normal bacterial invasion of the gut did not occur (Cebra, 1999; Hooper and Gordon, 2001). Moreover, it has been shown that the gut microbiota are critical for the normal maturation of the gut-associated lymphoid tissue (GALT). Changes in GALT immunity would then affect other mucosal and systemic responses (Barreau et al., 2004). While these microbiological processes are not typically studied within the context of PNI research, they offer important outcome measures, which are known to be associated with long-term health. To reinforce this point, it is worth noting that the infant’s most exposed surface is the gut. In an adult human, the gastrointestinal tract spans 400 square meters as compared to the typical 2 square meters of skin. Ultimately, over 500 species of bacteria will become established as the resident microbiota of the intestines (Mazmanian et al., 2005). The significance of this symbiotic relationship can be highlighted further by the fact that their numbers will reach 1014, while the developing human host will be composed of just 1013 cells. In our study of stressed monkeys, we were not able to determine exactly what factors mediated the long-lasting change in these important gut bacteria. But among the suspects were alterations in gut motility and acidity, both of which are known to influence bacterial concentrations. In addition, we speculated about the possible effect of gestational stress on the composition of the mother’s breast milk. Our suspicions about milk were based on many papers reporting that several of its constituents can promote the growth of both Lactobacilli and Bifidobacter. Nursing by the mother also has other benefits for enhancing the baby’s immune competence (Hasselbalch et al., 1999; Labbok et al., 2004). As mentioned earlier, it is the primary source of sIgA, which helps to protect the epithelial surface of the gastrointestinal tract. Thus, it can directly assist in neutralizing pathogens that utilize this route of entry. Both the sIgA and gut bacteria likely contribute to the known differences in illness frequency between breastfed and bottle-fed babies, especially with regard to the risk for diarrheic conditions. A specific influence of maternal care and nursing on immunity in monkeys is discussed at greater length in a later section describing immune differences found between mother-reared infants and ones raised on formula by humans. However, before addressing those findings, it is important to summarize a few of the more long-term immune effects that occur in monkeys generated from prenatally disturbed pregnancies.


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VI. SUSTAINED EFFECTS OF PRENATAL CONDITIONS ON LYMPHOCYTE FUNCTIONS The immune effects of prenatal stress and hormone treatments have been found to extend beyond the infancy period. In one project, lymphocyte proliferative and cytokine responses were examined in prepubescent animals (Coe et al., 2002). Blood samples were collected from 2–3-year-old monkeys that had been generated from either undisturbed control or psychologically stressed mothers. The whole-blood cultures were stimulated overnight with LPS (with either 10 or 100 ng/ml), and the supernatants harvested the next day for quantification of interleukin-6 (IL-6) and TNFα levels. In response to both low and high concentrations of LPS, the monkeys from stressed pregnancies produced less IL-6 and TNF-α (Figure 8). This finding of a lower cytokine response to protein derived from the cell wall of E. coli concurred with a previous paper reporting smaller in vivo inflammatory responses in a different cohort of monkeys (Reyes and Coe, 1997). In that study circulating IL-6 levels were determined 1 hour after administration of IL-1 to 1–2-year-old monkeys that had been generated from control and hormone-treated pregnancies. If mothers had been administered ACTH for 2 weeks during pregnancy, their offspring had a reduced IL-6 response in both blood and cerebrospinal fluid (CSF). Delineating all the possible processes that account for such long-term effects, and distinguishing which ones are primary, is extremely difficult. In some instances, the immune alterations have been associated with parallel changes in the endocrine system (Figure 9). We, and others, were inclined to look for changes in the pituitary-adrenal axis (Weinberg and Jerrells, 1991; Weinstock, 1997). The main hypothesis is usually that the offspring from disturbed pregnancies will either have higher adrenal hormone levels all the time or be more stress-reactive. While the studies in rodents have sometimes reported the maintenance of higher basal levels of corticosterone, prenatally stressed monkeys do not usually manifest such a pervasive upregulation of HPA activity. Instead, they can be distinguished more readily by having prolonged cortisol responses when provoked, such as after being moved to an unfamiliar cage. In addition to a slower recovery, they may exhibit a differential response to a test of corticosteroid negative feedback, the dexamethasone suppression test (DST). Overnight probes of HPA suppression in monkeys from disturbed pregnancies indicate that they more likely to “break through” the Dex-induced inhibition by the next morning (Coe et al., 2003).

FIGURE 8 Levels of cytokine (tumor necrosis factor-alpha and interleukin-6) released after overnight stimulation of whole blood cultures with a low or high concentration of lipopolysaccharide (LPS) (Coe et al., 2002). A trait-like effect of Early and Late gestational stress on these pro-inflammatory responses was still evident in these 2-year-old juvenile monkeys. Used with permission of LW&W.

This type of linkage between hormone and immune alterations in the developing monkey is not restricted to the effects of gestational stress. In one experiment, the proliferative responses of MNC were examined in 1-year-old monkeys that had been exposed to Dex in utero a month before term (Coe and Lubach, 2005). Antenatal corticosteroid exposure on Days 143–144 postconception resulted in yearling animals with cells that proliferated less to mitogen. Their cells also reacted differentially when a gradation of cortisol was added to the cultures (10−5 to 10−9 M). The MNC seemed more insensitive to cortisol, with an overall shift in the doseresponse inhibition curve. We hypothesized that the corticosteroid receptors on the lymphocytes were downregulated, because of the frequent occurrence of higher in vivo hormone levels. The possibility that variation in the amount of adrenal hormone secretion had contributed to the long-term immune differences led us to examine a brain region critically involved in the regulation of the HPA axis. The hippocampus is the primary feedback center in the cortex for corticosteroids, and it exerts a tonic inhibition over the hypothalamic control of adrenal secretion. As found in studies of the brains of prenatally stressed rodents, our pregnancy manipulations had impacted the hippocampus (Coe et al., 2003; Lemaire et al., 2000; Takahashi, 1998). The monkeys generated from stressed pregnancies showed a substantial reduction in hippocampal volume when measured at 3 years of age (approximately 10% across its length). Smaller size was also associated with a lower growth rate of new neurons (neurogenesis), as measured by cells that had been labeled with bromodeoxyuridine (BrdU). While this did not definitively prove that these hippocampal changes accounted for

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FIGURE 9 Overview of physiological alterations induced by prenatal disturbance and some of the pathways likely to be involved in mediating the immune effects. In addition to changes in the neuroendocrine axis, an interference with the normal placental transfer of nutrients and an abnormal profile of gut bacteria may affect the development of immune responses.

the co-occurring hormone and immune changes, it seems reasonable to conclude that they were a contributing factor. Figure 9 summarizes the broad conceptual model that emerged from these primate studies. Given the range and duration of the immune changes, it is likely that they reflect a multi-factorial causation. Long-term alterations in neuroendocrine activity postnatally would presumably add to the immediate effects of prenatal stress on lymphoid tissue and cells that had occurred during gestation. Disturbances in the gut bacteria could increase the chance of infection in infancy, and recurrent illness would have its own effect on the development of immunity. Finally, we, and others, have some additional evidence that maternal stress during gestation may act through nutritional pathways (Beach et al., 1982; Caulfield et al., 1998). When similar studies were conducted on the offspring of stressed females fed a diet with just adequate iron but not the extra supplemented amounts recommended for pregnancy, larger effects on infant immunity were found. Iron stores and utilization in these infants were affected, and the prenatally stressed ones

developed an iron deficiency anemia as they grew after birth. This developmental impairment in red cell functioning provided a second hit to the immune system at 4–6 months postpartum. Even innate immune responses were now impacted. NK responses that had been solidly established earlier at 2 months of age deteriorated in the increasingly anemic infants from stressed pregnancies. The possible significance of stress-related changes in the prenatal transfer of micronutrients from mother-to-infant is not restricted to monkeys. Because mice and rats are litter-bearing species, the dams are even more taxed by the demands of providing iron to multiple offspring (Conner, 1994). Iron deficiency is also a serious concern in humans, occurring in between 60% and 80% of pregnant women in developing countries (Allen, 1997). As recently as 1980, 30% of infants in the United States became anemic before the advent of more widespread iron fortification in baby formulas and cereals (Beard, 1994; Lonnerdal and Dewey, 1995). So a possible link between stress, nutritional deficiencies, and the development of immune responses is an issue that may apply to both animals and humans (Lozoff et al., 1998). Even today,


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up to 10% of babies in low SES families of industrialized countries are destined to become anemic.

VII. INFLUENCES OF THE EARLY REARING ENVIRONMENT ON IMMUNITY In the introduction we stated that an influence of the postnatal rearing environment on the development of immune responses is at least equal to the effect of prenatal conditions. Adverse and stressful rearing may also further compound any vulnerability created by prior undernutrition or disturbance during gestation. Not too surprisingly, much of this research has focused on the influence of maternal care given that a period of nursing is a sine qua non for all mammals and essential for infant survival. Perhaps because it is such a predictable feature of the postnatal environment, the normal development of many biological systems including immunity appears to rely upon the infant’s receipt of maternal behavior. Developmental researchers have known for many decades that monkeys will show marked behavioral abnormalities if reared away from the mother. Even brief separations of rat pups from the mother can have deleterious effects on brain development. Thus, it may not be too surprising that the absence of maternal care will cause some immune responses to go awry. However, at the time our research was conducted two decades ago, no one had previously investigated the immune consequences of different types of rearing in monkeys (Coe et al., 1989). Specifically, the lymphocyte responses of rhesus monkeys normally reared by their mothers were compared to bottle-fed animals raised by humans in a nursery setting. Our first study took advantage of an opportunity to collect blood from yearling rhesus monkeys that were still with their mothers (MR) and to compare them to age-matched monkeys initially fed by humans and then raised in small peer groups (NR). Samples were collected from eight of each type, and their MNC were separated and stimulated in culture with mitogens (Con A, PHA, or PWM). To our surprise, the lymphocytes of the NR monkeys proliferated at dramatically higher levels. Because the findings differed from our expectation that the absence of maternal care would cause an immune inhibition, a second study was designed to assess other monkeys at a younger age (Coe et al., 1992). Again, it was found that the proliferative responses were exaggerated by a history of nursery rearing. But inclusion of some additional assays provided preliminary evidence that higher proliferation was not necessarily better. These NR infants

FIGURE 10 Monkey infants that are bottle-fed and reared by humans have different immune responses than do motherreared animals (Lubach et al., 1995). In addition to altered lymphocyte proliferation, nursery-reared infants (NR) have a skewed ratio of CD4+ and CD8+ T lymphocytes, due primarily to low CD8+ cells in circulation. This influence of early rearing continued to persist after an attempt to ameliorate the immune effects by housing all monkeys in similar social groups at 1 year of age (n = 8 and 8). Used by permission of Oxford University Press.

tended to have lower NK activity and smaller antibody responses to vaccinations. To explore the reason for the increased proliferation, a final study was conducted to prospectively examine a third set of monkeys at 6-month intervals from birth through 2 years of age (Lubach et al., 1995). This time the mitogen assays were accompanied by an enumeration of lymphocyte subsets. As can be seen in Figure 10, the higher proliferation proved to be associated with a different profile of CD4+ and CD8+ cells in circulation. Specifically, the NR infants had a relative abundance of CD4+ cells, due in large part to lower numbers of CD8+ cells. There is one other aspect of these results that is worthy of note because it highlighted the enduring nature of these rearing effects. When the MR and NR monkeys reached one year of age, all were re-housed so that they would be living in identical conditions. Eight monkeys from each background were combined into a small, stable social group that also included an elderly female monkey to serve as a supportive adult. Despite the comparable housing, the immune differences between MR and NR monkeys persisted for another year (see Figure 10). Later assessments of the NR monkeys at 3 years of age indicated that their cells still exhibited high proliferative activity.

VIII. IMMUNE RESPONSES TO SOCIAL STRESSORS AND DISTURBANCE IN OLDER INFANTS The long-lasting nature of the effects seen in nurseryreared monkeys suggested that the immune differ-

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ences would be perpetuated into adulthood. In those experiments, however, the disruption of normal rearing commenced when the infant’s immune system was still at a more vulnerable, formative stage. Conclusions about the duration of immune changes are quite different when the consequences of early life events are evaluated in older infants. Immune responses may still be affected by challenge and stress, but the effects are more transient if the events first occur in 6– 12-month-old infants, and they usually resolve within a few days to weeks. The experimental paradigm used most frequently by researchers for this type of study in older monkeys involved a several-day to 2-week period of separation from either the mother or familiar social group (Coe, 1993; Gust et al., 1992; Laudenslager et al., 1982). While the immune effects do subside over time, these pioneering papers on the acute dysregulation of immunity in young animals provided important insights into how stressful events can affect the immune system. Immediately after the social separation, a marked lymphocytopenia was evident for several days, which included a significant decline in T- and NK cell numbers. Studies of their cellular responses in vitro indicated that proliferative responses and cytolytic activity were also inhibited at this time (Figure 11). Depending upon the monkey species used in the study and how the separated infant was housed—either alone or in a familiar group after removal of the mother—the inhibition of lymphocyte functions might be sustained for several days to a few weeks (Laudenslager et al., 1990). If the lymphocyte responses of the stressed monkey were probed further with antigen, it was clear that the changes were sufficient to impair antibody responses. Levels of antibody produced in vivo in response to keyhole limpet hemocyanin (KLH) or to a bacteriophage (φX174) were reduced in the separated monkeys (Coe et al., 1988b). Nevertheless, most papers that followed these separated infants over a longer period of time reported that

the period of inhibition was delimited. Even if the mother and infant were not reunited, or the monkey was not returned to its original social group, there would be a re-establishment of immune responses similar to those seen before the stressful event. This recovery suggested that the older infant had already attained a better and more mature capacity to regulate its immune functions. Presumably, the resiliency also indicated that there were some regulatory set points established to guide the cellular return back to the normal numbers and reactions of the undisturbed state. When these social separation studies were conducted nearly 2–3 decades ago, they also made another novel contribution to the PNI literature. In addition to showing that stress could inhibit lymphocyte functions, the research revealed that some arms of the immune system might be activated simultaneously. A pronounced neutrophilia was frequently seen in the infant monkeys during the first few days after weaning from the mother or separation from the social group. In fact, the neutrophil numbers often increased to such a degree that the rise exceeded the decline in lymphocytes and resulted in higher overall leukocyte counts. Neutrophils and Mφ were also found to be in an activated state and would release more superoxides when stimulated with phorbol myrisate acetate (PMA) in an in vitro chemiluminescence assay (Coe et al., 1988b). This turning on of innate immunity by social stressors also extended to the complement system. Increases in

TABLE 3 Alterations in Immune Responses Observed Acutely after Young Monkeys Are Either Separated from Their Mothers or Removed from the Social Group • • • • •

Lymphocytopenia in association with marked neutrophilia Decreased T and NK cells in circulation Reduced in vitro proliferative and cytolytic activity Lower antibody responses to antigen challenge Enhanced innate immunity (complement activity and Mφ superoxide production) • Increased inflammatory responses (Delayed Type Hypersensitivity, DTH)

FIGURE 11 Cytolytic activity in infant squirrel monkeys (n = 11) before, and at 1 and 7 days after separation from the mother (Coe and Erickson, 1997). Note both the significant inhibition on Day 1, as well as the rapid recovery back to baseline in these older infants that were between 7 and 12 months of age. Assays were run at 5 Effector-to-Target cell ratios (100 : 1, 50 : 1, 25 : 1, 12.5 : 1, 6.25 : 1).


hemolytic complement activity lasted for more than 2 weeks in the separated monkeys (Coe et al., 1988c). While the observation that stress can stimulate some innate and inflammatory processes is not surprising today, this formative research on young animals contributed to our awareness about the bi-directional nature of immune changes in the disturbed individual. Stressed infant monkeys also showed an enhancement of delayed hypersensitivity responses. When recall responses to dinitrochlorobenzene (DNCB) were elicited during the separation period, the erythema and induration were aggravated. This stress-related enhancement of inflammatory processes in young animals concurs with the more extensive and detailed investigations of cutaneous hypersensitivity in adult rats (Dhabhar and McEwen, 1999).

IX. CONCLUSIONS We now know that immune responses can be affected by environmental challenges and psychological disturbance at any point in the life span. However, the research on infant animals and children indicates that the effects may be more pronounced and long lasting in the immature individual. Disturbance of social relationships in monkeys of 6–12 months of age, which is developmentally equivalent to the 21-day-old rat and the 3–4-year-old child, has been found to impact a wide range of innate and adaptive immune responses (Coe, 1993). Nevertheless, there is already both a quantitative and qualitative difference in the reactions at this age when compared to immune alterations after perturbations that took place earlier during the fetal and neonatal stages. Immune changes in the older infant are more short-lived and usually resolve in a few weeks as the young animal adapts to the new situation. Thus, the recovery and resolution are more like that seen in the adult host. Any consequences of these transient immune changes for health would depend upon there being opportunistic infections or experimental probes at the maximal point of lymphocyte inhibition or enhanced inflammatory potential. The conclusions are quite different after disturbances of fetal or neonatal development. Permanent changes in immunity may then be observed if the perturbations were sufficiently salient. Even an abrupt, early weaning of the infant from the mother may cause long-lasting effects. This type of observation was first reported in a seminal paper 40 years ago when adult rats were found to respond poorly to a transplanted tumor if they had been removed from the mother on Day 14 rather than Day 21 (Ader and Friedman, 1965). The critical importance of maturational age at the time

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of insult generalizes from the rodent to the primate. For example, a retrospective examination of the reason for variation in how adult monkeys succumbed to infection with simian immunodeficiency virus (SIV) showed that it could be attributed to a history of disturbed early rearing and housing conditions (Capitanio and Lerche, 1991). Both maternal stress and hormone treatments during pregnancy can result in pervasive and lingering effects on the offspring. These developmental alterations in immunity are often associated with more widespread effects that span brain and neuroendocrine functions. As a consequence, it is difficult to designate just one pathway mediating the immune changes. Many investigators have pointed to the significance of increased adrenal activity during pregnancy and subsequently to the abnormal regulation of HPA activity in the offspring postpartum. Given the potent effects of adrenal hormones on the thymus and lymphoid cells, corticosteroids could undoubtedly play a role. However, in order to fully understand prenatal influences on the development of immunity, it is equally important to consider other mediators. This chapter highlighted a few other candidates, including prenatal and postnatal nutrition, the microbiota of the gut, and the infant’s early responses to viruses and bacterial infection. Relatively modest changes in the gut bacteria of prenatally stressed monkeys were found to set the stage for a vulnerability to enteric pathogens. We know that diarrheic disease is still the second largest killer of human babies worldwide. Adverse events postpartum can continue to disturb the gut microbiota and may even facilitate bacterial translocation through the gut wall into systemic circulation (Barreau et al., 2004; Wenzyl et al., 2003). This idea of accumulating risk, or a two-hit model of vulnerability after developmental insults, would concur with the views emerging in other disciplines. There is compelling evidence that a period of undernutrition or stunted growth in utero can alter the regulation of metabolism in such a way as to increase the risk for diabetes or cardiovascular disease in adulthood (Barker, 1994; Caulfield et al., 1998). Allergists have also started to take more seriously the idea that the origins of allergies and asthma may be found in fetal life (Herz et al., 2001; Warner et al., 2000). Some babies may already be born with an atopy-prone phenotype, which is then aggravated by allergens in the rearing environment, frequent upper respiratory infections, or inadequate medical and parental responses (Mrazek et al., 1991; Wright et al., 2005). Figure 12 illustrates how the developmental perspective on asthma has broadened to include more antecedent events. At the moment of birth, there may already be

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FIGURE 12 Developmental model of allergies and asthma highlighting the important influence of both prenatal and early rearing conditions. Some infants at birth may already show an immune predisposition toward atopic conditions, which can then be aggravated by exposure to allergens and recurrent upper respiratory infections.

elevated IgE levels in cord blood, cells activated against certain allergens, and lymphocytes more prone to produce Th2 cytokines (Karmaus et al., 2001). A premature birth presents a particular challenge for the immature immune system. Both prematurity and an extended gestation that goes past 40 weeks have been associated with an increased occurrence of allergies and asthma (Ferguson et al., 1997; Pekkanen et al., 2001). Given that an early activation of immunity is a risk factor, it may seem paradoxical that some bacterial infections and parasite infestations in young infants have been associated with a reduced risk for asthma. The latter observation has led to the so-called “hygiene hypothesis” (Yazdanbakhsh et al., 2002). This initially counterintuitive idea is in keeping with some of the findings discussed in this chapter. Aspects of the developing immune system are pre-programmed in anticipation of the infant’s needs to respond to antigens in the postnatal environment. This typical development and the “learning trajectory” of immunity could then become derailed in our modern, clean world, especially when common childhood infections are prevented by immunizations. The hygiene hypothesis proposes that the normal immunobiology could then be deflected and result in inappropriate reactions to innocuous antigens like pollen. Having highlighted how readily immune responses in young animals may be affected by disturbances of pregnancy or early rearing, a few caveats should also be acknowledged. For both scientific and ethical reasons, the experimental conditions in our primate studies were designed to ensure the viability of the

offspring. Thus, we did not study any extreme or noxious manipulations that caused fetal loss. In the real world, however, one clinical consequence of a maternal infection could be pregnancy failure or premature delivery. Similarly, our tests of antenatal corticosteroid treatments were limited to a 2-day course to emulate clinical practice. More prolonged dexamethasone regimens, at least in animals, can have devastating effects on fetal and infant immunity, as well as on brain development (for a review of primate studies, see Coe and Lubach, 2005). The elicitation of stress in the gravid females was also designed to approximate a level of disturbance that could occur naturally. Pregnant monkeys may experience periods of limited food availability, climatic challenges of drought and heat, a periodic threat of predation, and may be subjected to aggression from a more dominant animal. Thus, the natural demands and levels of arousal could exceed that evoked by the acute acoustical startle protocol pre-housing stress utilized in the laboratory. As a consequence, the immune alterations described for our monkey infants should be viewed as within the normal range of physiological variation. The offspring were not obviously sick or impaired to the same degree as a child with an immunodeficiency disorder or an autoimmune condition. Rather this research was designed to model how early life events can create developmental vulnerabilities in the infant. Our review also began with the important role that immune cells play in reproductive biology. This expanded function for the immune system goes beyond its traditional role in the response to infection. While the endocrine events associated with pregnancy usually receive more attention, immune processes are critical for the successful implantation and the maintenance of pregnancy. These early immune interactions between the mother and her developing baby foreshadow the equally vital functions that her maternal care will serve postpartum. Symbolically and concretely, the maternal facilitation of immune development begins prenatally via the placenta and then continues postnatally through breast milk and solicitous care giving. These contributions are critical for survival and for guiding the infant on the right trajectory to become an immune competent adult.

Acknowledgments This research was supported by grants from the National Institutes of Allergy and Infectious Disease as well as Child Health and Human Development (AI46521, HD39386). CLC has received partial salary support from MH61083 and AG20166. Special appreciation is due M. Bailey, H. Crispen, E. Fuchs, M. Kraemer, T. Reyes, M. Schneider, and A. Slukvina for invaluable help on specific studies reviewed in this chapter.


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C H A P T E R

22 Social Dominance and Immunity in Animals MARK L. LAUDENSLAGER AND SARAH KENNEDY

I. INTRODUCTION 475 II. ANIMAL MODELS 476 III. AN OVERVIEW OF AGGRESSION AND DOMINANCE RELATIONSHIPS 477 IV. DOMINANCE RELATIONSHIPS IN RODENTS 479 V. DOMINANCE RELATIONSHIPS IN NON-HUMAN PRIMATES 482 VI. DOES DOMINANCE MATTER? 491

tive social interaction in humans is observed as well among the non-human primates and can mitigate the negative consequences of social and environmental challenges. It is no longer possible to take simplistic approaches to behavior-immune relationships. A comprehensive view needs to include the underlying factors that contribute to observed error variance. The best victory is when the opponent surrenders of its own accord before there are any actual hostilities. . . . It is best to win without fighting. Sun-tzu “The Art of War” Chinese general and military strategist (∼400 B.C.)

ABSTRACT Similar stressors do not produce similar responses in an organism. The social organization of many mammalian species creates dominance hierarchies, which may or may not affect immunoregulation and neuroendocrine regulation, particularly for the glucocorticoids in response to environmental challenge. Not only can relative rank within a group affect these relationships, but individual differences in behavioral styles or personality also enter heavily into the equation. These relationships have been studied extensively in rodents and non-human primates for over a decade and are the focus of the present chapter. This chapter will review the nature of dominance relationships and aggressive behavior(s) across species and their relationships to glucocorticoids and immune measures. Although there is advantage to high rank such as priority of access to preferred or restricted resources (e.g., food and mates), it has the potential to carry with it occasional consequences. The essential role for posiPSYCHONEUROIMMUNOLOGY, 4E VOLUME I

I. INTRODUCTION Over a decade ago we noted that the reduction in specific antibody of an intruder rat was directly related to its behavior; e.g., did it fight back or show the residents a submissive response such as exposing highly vulnerable surfaces (Fleshner, Laudenslager, Simons, and Maier, 1989)? Signaling submission or defeat was associated with reduced specific antibody responses, whereas those intruders that fought back had antibody responses that were similar to controls exposed to the residents with a Plexiglas barrier between them, but without physical contact. The wisdom of Sun-tzu may have been lost among rodents as far as immune regulation of the defeated is concerned. If a number of individuals experience the same stressor or challenge under a common set of condi-

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tions, the observed impact of that event on the individual’s behavior and physiology is not necessarily consistent across individuals. The average response across a number of individuals is encompassed by a large confidence interval that most investigators would like to minimize. However, it is this variability in behavioral and physiological response patterns that will provide clues to the underlying organization of the system once the factors contributing to the variance are identified. There will always be factors out of our grasp, yet small steps in identifying any contribution to experimental variance are critical to the advancement of psychoneuroimmunology (PNI). In the present chapter, we will consider the influences of aggression and dominance or hierarchical relationships in rodents and non-human primates on endocrine and immune systems that are associated with individual and species survival. Although this chapter is not to be considered as comprehensive in coverage, we have attempted for the most part to focus on developments since the previous edition of Psychoneuroimmunology (Ader, Felten, and Cohen, 2001). The present chapter attempts to cover several areas. For example, is the dominant member of a dyad of a social group always in a more advantageous position compared to the lower-ranking member? High rank carries with it certain privileges. However noblesse oblige and higher social rank may be burdened with untoward side effects. The contributions of individual differences preclude simple descriptions of critical factors and their role in immunomodulation. We will focus on one of many potential endocrine mechanisms, the glucocorticoids, which may be related to immune modulation in association with dominance-related stressors that occur both acutely and chronically. Can animal models inform our understanding of brain-behaviorphysiology relationships in general and PNI more specifically?

II. ANIMAL MODELS A detailed empirical study of biological mechanisms underlying immune dysregulation in association with psychosocial stress, aging, affective disorders, cancer, autoimmune disorders, and many other medical conditions may not always be achievable in humans. The social situation of humans cannot be easily controlled; that is, we cannot experimentally introduce acute stressors (e.g., loss of a job) or set up situations of long-term stress in humans (e.g., poverty) for the study of underlying biological changes associated with immune dysregulation. Hence, animal models have an important place in biomedical research

when they are conducted in a careful and humane manner. One issue to consider in regard to an animal model is related to the concept of analogous and homologous phenotypes (Laudenslager and Worlein, 1999). Two analogous phenotypes may serve the same purpose but evolve via distinctly different paths such as flight in birds, bats, and flying squirrels. Consequently, the underlying structures, physiology, and regulation may differ substantially. Homologous phenotypes have similar etiologies from an evolutionary perspective and thus possess more common underlying mechanisms. Human and non-human primates share common evolutionary origins, and, as such, should reflect many homologous processes with regard to behavior-immune relationships. There are 320 identified primate species and perhaps as many as 600 subspecies. With regard to cross-species comparisons, it has been stated “. . . every living primate is literally more like a human than it is like any other living nonprimate animal. While humans share many conserved characteristics with the nonprimates, human characteristics that were derived during their evolution are homologous only with those of other primates . . .” (Erwin and Hof, 2002, p. viii). However, animal studies among the lower species do in fact inform the higher species by providing mechanistic clues when homologous processes might be involved. Some ethologists, notably Robert Hinde (1987), are quick to caution us that there are many examples wherein the non-human primate model may differ from the human condition and one must use “. . . humility . . .” in comparing non-human primates to humans, and to be careful not to focus on too narrow a perspective (e.g., a single species). According to Hinde, human/non-human primate comparisons must be drawn carefully with a full appreciation for the complexities of human emotions. When we speak of aggression in humans, we must address complex human emotions like jealousy, vengeance, and so on. Regardless, principles that generalize across several species are most likely to inform theory regarding their underlying mechanisms. The study of non-human primate social behavior began less than a century ago with studies of baboons in captivity (Zuckerman, 1932). Observations in captivity do not always mirror responses that might be noted under free-ranging situations. The semi–freeranging rhesus monkeys of Cayo Santiago, Puerto Rico, were introduced to this 17 hectare island as a research colony for the specific study of primate behavior by C.R. Carpenter in 1938 (Rawlins and Kessler, 1986). We will return to our studies on the Cayo Santiago population later in this chapter. The develop-

22. Dominance and Immunity

ment of computer-assisted behavioral data collection techniques (Noldus, 1991) has not only improved the quality of data that can be collected, but it has also increased observational reliability. However, nonhuman primate models also have some limitations with regard to sample size and accessibility in naturalistic settings. Animal models permit a level of control in research not always possible in studies involving humans. The life span of many animals used as model systems is far shorter than that of humans. In non-human primates, this allows for longitudinal studies encompassing the prenatal period, early development, adolescence, adulthood, and old age within the same cohort. If one is focused on rodent models, their life span is much shorter, ranging from 24–36 months, compared to nonhuman primates that might span as much as two to three decades for macaque species maintained in captivity (Erwin and Hof, 2002). In contrast, the great apes practically match humans in terms of longevity. Four criteria specify the essentials of a valid animal model with regard to psychopathology (McKinney and Bunney, 1969). These criteria include similarities in etiology, phenomenology, pathophysiology, and treatment. Specific animal models serve different purposes. For example, rodent models allow for larger sample sizes and permit microanalysis of relevant neurochemical (CNS localization studies, receptor-binding studies, endocrine control) and immunological (trafficking patterns within immune compartments or challenge with infectious agents) systems generally not available for study in human or non-human primates. However, there are exceptions that we will discuss later. Animal models actually form bi-directional relationships with the human condition (McGuire, Brammer, and Raleigh, 1983) in that human observations can inform the animal model. Homology implies that one is studying similar processes that have a shared phylogenetic basis (McGuire et al., 1983). This will be reflected in similar underlying anatomy and physiology. As behavioral scientists, we ask if seemingly similar behaviors serve the same underlying function in rodents and non-human primates. Does subordinance for a rodent have the same implications as subordinance for a non-human primate? Is subordinance in laboratory settings a feature of rodent behavior that is commonly seen in nature? Rodents do not typically have repeated social interactions with the same dominant rodent (a typical laboratory model of social dominance); instead, they take appropriate measures to escape the situation and move to a safer but less appealing nesting location. These features should be kept in mind as we describe the impact of social interactions in both rodent and non-

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human primates. However, observations of speciesspecific social signals in rodents associated with dominance and submission suggest that these interactions do occur, and behavior plays an important role in modulating interactions between the members of the dyad. In the present chapter we attempt to draw parallels between social dominance hierarchies and dominancerelated behaviors across two orders, rodents and primates. The attention herein is not to focus specifically on models of psychiatric disorders but rather indicate the extent to which low social rank or subordinance contributes to long-term stress and increased risk for medical illness as mediated by immune dysregulation.

III. AN OVERVIEW OF AGGRESSION AND DOMINANCE RELATIONSHIPS We will begin by carefully specifying what we are referring to when we refer to aggression, dominance, and dominance hierarchies. Aggression is a term that is frequently used in the discussion of dominance and dominance relationships. Aggression represents a wide range of overlapping behaviors that serve one of two purposes: resource/territorial competition or defense. Aggression can be induced by a variety of conditions but generally by threat from an intruder/ aggressor. Examples of laboratory conditions that can elicit aggression include foot shock (painful stimulus) or schedule-induced aggression (frustration). Archer (1988) distinguishes three types of aggression: (1) Protective Aggression (attack by conspecific, attack by a predator, sudden pain, territorial intrusion), (2) Parental Aggression (protection of the offspring and a more neglected form of aggression as far as research is concerned), and (3) Competitive Aggression (to gain a territory, reproduction, resources; most studied aspect). Protective and competitive aggression are involved in situations reflecting dominance interactions and are the focus of this chapter. There are significant variations in aggressive displays. For example, across the primate order: lemurs “stink fight” by rubbing pheromonal secretions to their forearms; male squirrel monkeys display an erect penis and urinate on their forearms; baboons flash eyelids showing a white streak; macaques bear their teeth; and gorillas stand upright, beating their chest while charging (Walters and Seyfarth, 1987). None of these visual displays involve actual contact, and the subsequent behavior of the engaged parties is a function of each member’s response to the other’s display. Across species when contact actually occurs, behaviors

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are more similar in characteristics and include chasing, grabbing, and biting. Infants are relatively protected from aggressive interactions since infants of many primate species possess different coloration that distinguishes them from adolescents of the species. The age at which this “shield” ends varies across species. As the gender makeup of the group varies, the frequency of male–male interactions is affected; e.g., unimale group males will have more frequent aggressive encounters since the stakes are higher. For some species such as the squirrel money, males are outcasts from the social group until breeding season when the females “allow” them back in the troop to mate. Rodents differ from non-human primates in that physical contact is often a component of aggressive encounters. Rodent displays of aggression include offensive behaviors such as chasing (following a fleeing animal), sideways attacks, mounting attempts (distinguished from mating behavior by the absence of pelvic thrusts), and biting. In contrast, defensive behaviors include displaying a defensive upright posture, fleeing, boxing, and a decrease in time spent in open or vulnerable locations (for a review, see Blanchard, Wall, and Blanchard, 2003). Aggressivity in male rodents can be encouraged by housing with female rats, which increases territorial aggression, whereas male rats housed only with male rats appear to form more equitable social structures. Female rats and mice tend to be less aggressive toward other females, with some exceptions. Species differences are considerable. Female golden hamsters, which may be aggressive towards cohort members during adolescence, will subjugate younger females in adulthood (Taravosh-Lahn and Delville, 2004). In the common voles, adult females maintain dominance over their daughters (Heise and Van Acker, 2000). In many respects, dominance structures of rodents are limited by the number of possible interactions that occur in a short period of time. Dominance is a learned relationship (Bernstein, 1981), not an absolute attribute of an individual. It is always in reference to another individual. Thus, dominance is referred to as regularities in winning or losing fights (Archer, 1988; Bernstein, 1981). The history of the relationship is not the only factor that will determine dominance relationships among members of a social group. Factors such as size, age, matrilines (many primate species are organized along maternal or matrilineal ancestry), experience, and gender are all critical in the resolution of dominance interactions. A dominance relationship may change with time (e.g., age) or repeated encounters. Dominance is a dynamic process between individuals. Once a dominance relationship is established, there will be an energetic savings. Injuries are avoided, and the energy

required for a fight is minimized as well as a stress response. A dominance relationship develops between two animals during their first encounter, when both animals begin by evincing similar agonist behaviors, but ensuing responses of one animal may result in the termination of further agonistic behaviors by the other (see Bernstein, 1981). The termination might be signified by species-specific indications of subordinance or defeat. There are clear visual signals sent by dominant and subordinate animals. These signals are particularly important for non-human primates that live in social groups. These serve to reduce the number of actual fights in which an injury might occur. For example in male rhesus monkeys, an erect tail is a sign of a dominant male, as indicated in Figure 1. In a social group in which there was a previously established dominance hierarchy and a lower-ranking animal is threatening a rank reversal, escalated aggression might occur. In macaque monkeys, a dyadic interaction might begin as a facial gesture where the lower-ranking animal may simply lip smack and move away. A subsequent threat follows if the first gesture is not effective. Baring the teeth might indicate this, or one animal might lunge at the other without making contact. If neither animal backs down, the next lunge might be accompanied by actual contact and grabbing. If both contestants continue to hold firm, the grabbing will escalate to holding the opponent down and ultimately biting. Departures from the area or relinquish-

FIGURE 1 A high-ranking adult male rhesus monkey on Cayo Santiago, with its rank distinguished clearly by an erect vertical tail.


ing the valued resource will ultimately terminate the agonistic interaction of the dyad. There are well-established neurochemical correlates (e.g., CNS serotonergic activity) associated with these aggressive challenges and how they are expressed (Mehlman et al., 1994). The animal remaining in the territory, the “winner,” is considered to be in control of a highly valued or preferred resource and is dominant to the departing, or “loser,” animal. Dominance relationships can be linear or transitive. In a linear relationship, A defeats B and B defeats C and C is always subordinate to A. A non-linear relationship occurs if C defeats A. Among primates, these contests are not always straightforward. Rank reversals frequently occur within the middle ranks where the distinctions are much less clear. Coalitions may form between two members of a troop that result in displacing a higher-ranking animal. Highly preferred resources are typically monopolized by the higherranking animals, particularly in times of scarcity. An interesting observation of displacement of a highranking animal from a preferred food resource was described by John Capitanio (personal communication) in which a lower-ranking macaque began a fight with two other monkeys while the high-ranking monkey was monopolizing a preferred resource in their field cage. The high-ranking animal left the resource to join in the fight at which point the lowerranking animal that started the fight moved to the preferred resource, while the higher-ranking animal continued to fight with the two animals remaining in the dispute. What starts an agonistic interaction has been intensively studied, although recent attention has turned to what terminates these interactions (De Waal, 2000). De Waal has argued that it is equally important to understand what modulates the intensity of these interactions and controls the end of these encounters. What happens between these two primates when they encounter each other again (which is highly probable in socially living species)? The subsequent encounters are often described in terms of conflict resolution and reconciliation. Other dimensions are added when the alpha male in a social group breaks up a fight between two females in the group. Socially grouped non-human primates provide a wealth of interactions not available to individually or even pair-housed monkeys. This was clearly recognized by researchers in the 1960s who formed social groups of macaque monkeys for the study of mother-infant relationships (Kaufman and Rosenblum, 1966), going beyond the earlier work of Harlow (Harlow, Harlow, Dodsworth, and Arling, 1966; Harlow and Zimmermann, 1958). The rich and complex connections existing in social species have

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been used as a means of understanding relationships between dominance status and various indications of immunoregulation. How do dominance relationships influence immune and health outcomes in rodents and non-human primates?

IV. DOMINANCE RELATIONSHIPS IN RODENTS Several models have been used to assess rodent social interactions and influence dominance interactions. One ethologically relevant model is the visible burrow system (Blanchard et al., 1995; Blanchard, McKittrick, and Blanchard, 2001; Tamashiro et al., 2004). The visible burrow apparatus consists of an open field connected to several smaller chambers by a series of tunnels. Typically, four male and two female rats are housed in the visible burrow; eventually, one male rat will become dominant over the other males. Dominant rats are distinguished behaviorally by increased time in open spaces, more aggressive behaviors, fewer wounds, and maintenance of body weight despite social stress (Blanchard et al., 1995; Blanchard, McKittrick et al., 2001). Dominant rodents are considered “proactive” in that they are more likely to attack and wound another rodent in an unstable social situation (Ebner, Wotjak, Landgraf, and Engelmann, 2005), and they will be the “winner” of these agonistic interactions (Stefanski, 1998; Stefanski, Knopf, and Schulz, 2001). The nature of this proactive activity can be used to measure the aggressive tendencies of an animal. For example, a proactive animal displays a shorter attack latency (SAL) (Koolhaas et al., 1999). Conversely, subordinate animals are “losers” of agonistic interactions and display a “reactive” coping style (Blanchard, Yudko, Dulloog, and Blanchard, 2001; Ebner et al., 2005; Koolhaas et al., 1999). Instead of instigating fights, subordinate animals will display more submissive/defensive upright postures, flee from aggressive animals, and show a lack of exploratory behavior. Defeat behaviors in rodents often include exposure of vulnerable surfaces (e.g., the dorsum) to the attacker, which signals defeat and often ends attack by the dominant animal. When they flee, bites and wounds occur predominantly on the back and neck. A delay in attack behavior denotes animals as having a long attack latency (LAL) (Koolhaas et al., 1999). Subordinates will have wound patterns targeted to their back and tail, and a rapid and sustained weight loss of up to 15% body weight over a 2-week period during establishment of a social hierarchy (Tamashiro et al., 2004). Subordinate rats will have basal corticosterone levels elevated in comparison to dominant rats.

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However, as has been noted in non-human primates, not all subordinate rodents are equivalent with regard to glucocorticoid modulation. Following an acute stressor, a smaller proportion of the subordinate animals will actually develop glucorticoid resistance, and fail to secrete a higher amount of corticosterone in response to an acute stressor, as seen in the dominant or control animals (Blanchard, Yudko et al., 2001; Tamashiro et al., 2004). Additional hormonal differences can be found after subdividing subordinate rodents into subdominant or submissive based on their behavior during the interaction. Using the social disruption model of social stress, an aggressive intruder rodent is introduced into a stable social environment; that is, a group that has a long history of living together. The animals are allowed to interact for a fixed period of time, during which time one animal is reliably defeated. The animal’s response to repeated defeat can either be passive (submissive) or active (subdominant). Submissive animals appear to have given up; they are more likely to hide or lie on their ventral side exposing their vulnerable surfaces and are generally more passive. Subdominant animals still fight, but they are repeatedly defeated by dominant and some subdominant animals. These behavioral differences affect the physiology of these animals depending on their initial status, e.g., resident or intruder. Subordinate animals that were original inhabitants of the housing show an increase in body weight, but the weight of intruder subordinate animals declines (Bartolomucci et al., 2004). Since food intake was equivalent, the difference in body weight was likely secondary to differences in activity levels and other factors. This divergence between reactions to subordinance may be due to stress inducing an increase in hypothalamic-pituitary-adrenal (HPA) activity or responding via activation of the sympathetic nervous system (Bartolomucci et al., 2005; Koolhaas et al., 1999). Submissive animals have an increased concentration of glucocorticoids in their blood, whereas subdominant animals have an increase in adrenal tyrosine hydroxylase (Koolhaas et al., 1999). This corresponds to greater weight loss in intruder submissive animals, secondary to increased corticosterone, but a continued readiness for fight in the subdominant animals, as suggested by an increase in epinephrine. Mice with the lowest levels of social investigation (passive behavior) were most likely to be submissive and develop corticosterone resistance following social disruption (Avitsur, Stark, and Sheridan, 2001). Physical contact appears to be a necessary component of this, as mice protected by a mesh screen did not develop corticosterone resistance (Bailey, Avitsur, Engler, Padgett, and

Sheridan, 2004). It has yet to be established if this state of resistance is advantageous, preventing the suppressive effects of corticosterone on wound healing, or a result of reaching a failure level in adaptation or allostatic load (McEwen and Wingfield, 2003). Considering the “learned helplessness” or passive component of human depression and its association with elevations in glucocorticoids (Patten, 1999), this phenomenon requires further study. Learned helplessness in rodents can be defined as the state wherein an animal fails to learn to escape from a controllable stressful situation (typically foot shock) after prior exposure to an uncontrollable stressor. This behavioral pattern has a U-shaped relationship to corticosterone levels, whereby intermediate corticosterone levels are actually protective against learned helplessness, but high or low levels of corticosterone levels lead to deleterious consequences (Kademian, Bignante, Lardone, McEwen, and Volosin, 2005). From an evolutionary perspective, depression, or this passive approach, may be adaptive in that suppression of environmental processing may prevent processing of further negative events (Patten, 1999). For rodents raised in a laboratory setting, the social challenge of establishing a dominance hierarchy or facing an aggressive intruder can be used as either acute or chronic stressors. Housing conditions affect reactions to challenges, which will vary according to strain of rodent (social or not) (Glasper and Devries, 2005) and the conditions under which the animals were raised (singly housed, pair or sibling group, or a stable social hierarchy) (Avitsur, Stark, Dhabhar, Kramer, and Sheridan, 2003; de Jong, van der Vegt, Buwalda, and Koolhaas, 2005). The hormonal profile and immune outcome are therefore somewhat dependent upon timing of measurement and challenge. Mice in stable social groups (3 weeks without disturbance) have differences in basal corticosterone, with lowestranking mice showing the lowest corticosterone levels (Bartolomucci et al., 2005). Social disruption induces the greatest increase in corticosterone after the first session compared to the seventh in animals of all social rank (Avitsur et al., 2001; Merlot, Moze, Dantzer, and Neveu, 2004a). The increase in corticosterone is greatest on the first day of disruption because attack intensity dissipates over 7 days of social disruption, perhaps reflecting an adaptation to the challenge and establishment of respective dominance relationships (Avitsur et al., 2001). It appears that subdominant animals have no change in corticosterone but a decrease in corticosteronebinding globulin (CBG), after 2 days of social conflict (Stefanski, 2001). A decrease in CBG is associated with an increase in free corticosterone, thus leading


to a functional increase in bioactive corticosterone (Fleshner et al., 1995). Circulating epinephrine and norepinephrine are also elevated at this time-point. Adrenocorticotropic hormone (ACTH) increases in subordinate intruder rats and non-aggressive resident/dominant rats by 15 minutes after the initial social confrontation, but not in aggressive dominant residents (Ebner et al., 2005). The duration of time freezing was positively correlated with circulating ACTH. However, after 7 days of social disruption, with all animals losing to an aggressive intruder, regardless of rank, animals showed equivalent increase in corticosterone in BALB/c mice (Merlot, Moze et al., 2004a). Conversely, in Long-Evans rats, dominant males have a decrease in total corticosterone in comparison to control animals, with equivalent CBG, whereas subordinate animals in one study had equivalent corticosterone to controls, but a decrease in CBG (Stefanski, 2000). Seven days of social disruption also appeared insufficient to cause glucocorticoid resistance in all ranks of rats (Merlot, Moze et al., 2004a), as was seen in a subgroup of subordinate rats after 13 days in the visible burrow system (Blanchard et al., 1995; Tamashiro et al., 2004). Furthermore, housing for 2 weeks in a visible burrow system is insufficient to alter corticosterone receptor levels in the spleen (Spencer et al., 1996), hippocampus, hypothalamus, or pituitary (Blanchard et al., 1995), despite circulating corticosterone concentrations elevated in subordinate animals to twice those seen in dominant animals and 10 times higher than in control animals. The foregoing hormonal changes are likely to be involved in immunoregulation and immune balance, since increases in glucocorticoids are associated with a shift towards Th2 immunity (Iwakabe et al., 1998; Miyaura and Iwata, 2002). Lymphocyte profiles change in response to intruder challenges differentially based on social rank. Intruder mice that become subordinate have a decrease in white blood cell count and lymphocyte numbers, whereas granulocyte number increased in the blood by 2 hours after conflict onset. By 48 hours, only the increase in granulocytes persisted (Stefanski and Engler, 1998). When two malefemale rat pairs are housed together, a chronic, but stable, social stress situation develops with clear dominant and clear subordinate behavior patterns among the rats (Stefanski, 2000). CD4+ cells are decreased after 7 days in non-biting winners and both bitten and non-bitten losers, with no change in CD4+ count in biting winners, whereas only non-bitten losers showed a reliable decrease in CD8+ cells (Stefanski, 2000). Leukocyte numbers increased in losers that were bitten over both non-biting winners and control animals, whereas all groups except non-biting winners showed

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an increase in granulocytes. This difference in leukocyte counts may be related to hormonal changes, as wounded animals secrete higher levels of corticosterone and interleukin-6 than non-physically wounded animals (Merlot, Moze, Dantzer, and Neveu, 2003). The patterns of hormonal secretion varying with stressor type may be adaptive, as animals subjected to repeated social disruption stress, including wounding, are able to heal wounds faster than animals subjected to a similar number of the sessions of psychological stressor of restraint (Sheridan, Padgett, Avitsur, and Marucha, 2004). The nature of the stressor is quite important in the overall adaptive response of the organism. As these investigators have noted, burrow collapse (restraint) is likely a typical component of their evolutionary experience and for which adaptation might be quite different from wounding due to aggressive encounters. In vitro immune responses are affected by social interactions in rodents. Seven days of social disruption stress are associated with increases in both basal splenocyte proliferation and proliferative response to lipopolysaccharide (LPS), but proliferation in response to concanavalin A (Con A) is reduced (Merlot, Moze et al., 2004a). When subdivided by rank, only submissive animals show a decreased proliferative response to Con A, whereas subdominant animals’ response remains intact (Stefanski, 1998). This is supported by in vivo data showing that the amount of time in submissive posture correlates with suppression of antibody response to keyhole limpet hemocyanin (KLH) (Fleshner, Laudenslager et al., 1989). Following repeated social intrusion in the home environments, there were two characteristic behavior responses: fighting back and/or evincing defeat postures. Antibody titers to the KLH were lowest in the defeated animals, whereas those that fought back were similar to handled controls. A reduction in anti-KLH antibody is also evident in Syrian hamsters exposed to one or five sessions of social defeat (Jasnow, Drazen, Huhman, Nelson, and Demas, 2001). In contrast, 7 days of social disruption cause no difference in anti-KLH IgG response (Merlot, Moze et al., 2004a) or response to infection with BCG (Merlot, Moze, Dantzer, and Neveu, 2004b) compared to controls. The outcome was not divided by social rank within the stable housing groups; thus, it is unclear if mixing of social rank obscured a rank effect. Timing of social stress and immune challenge is also important, as submissive mice immunized after 2 weeks of daily 30-minute agonistic interactions show a decrease in primary antibody response to sheep red blood cells (SRBC), whereas after 3 weeks of daily antagonistic interactions, dominant mice showed an enhanced

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primary antibody response to SRBCs (Gasparotto, Ignacio, Lin, and Goncalves, 2002). Dominance in rodents may be seen as advantageous. For example, when a food competition task is used, the dominant animal repeatedly succeeds in securing the food, whereas low-ranking rats obtain the remnants or nothing at all (Merlot, Moze, Bartolomucci, Dantzer, and Neveu, 2004). However, the cost of aggressiveness may then limit resources available for other tasks such as reproduction. When animals were categorized as biting winners (most aggressive) versus non-biting winners or losers, the biting winners sexually approached females three times less often than non-biting winners, although still with greater frequency than the losing rats (Stefanski, 2000; see Sapolsky [1993] for similar observations in baboons). The role of female scent triggering aggressive behavior is well established, and exposure to female scent alone can overcome the decrease in aggressive behavior usually exhibited by mice treated with anti-thymocyte serum (Barnard, Behnke, Gage, Brown, and Smithurst, 1997). It is apparent that exposure to female scent modulates the stress response as well, as it both potentiates the heat-shock protein response to acute stress and prevents the acute-stress induced depletion in splenic norepinephrine (Kennedy and Fleshner, unpublished observations). The stimulus for becoming a hyper-aggressive animal, such as the biting winner, versus non-biting winners is yet unknown, but reinforces the idea that individual differences in personality, even in an inbred rodent model, influence reaction to stress and therefore immune outcome. Similar to observations among the rodent genera (Fleshner et al., 1989), other mammalian species show immunoregulatory changes after losing a social confrontation that are affected by individual responses to the challenge regardless of their outcome. For example, among the insectivores, when two male tree shrews (Tapaia belangeri) are housed together in a large cage for the first time, they will begin to fight immediately until one is clearly a winner of this initial fight (vonHolst, 1997). Submission after the fight may be characterized in one of two distinct patterns: (1) cowering in a corner of the cage or (2) active but alert to the location and actions of the winning tree shrew. HPA activation of the winning tree shrew returned to basal levels, while the cowering losing animal continued to show evidence of HPA activation for several weeks. In contrast, the active/alert losing subordinate showed a return to baseline within a few days. If following this initial social interaction, the two participants are housed in the same cage but with a screen between

them, the loser shows altered immune parameters including reduced lymphocyte activation by mitogens and lower interleukin-1 and interferon in the absence of further physical contact between the two animals. Across the primate order, there are clear indications of immune modulation in association with social group reorganization, but these in vitro measures may not provide the best insights into immune regulation of the intact organism (Maier and Laudenslager, 1988). In the following sections we will examine several hormonal and immune effects of social interactions driven by dominance rank that in many respects differ little from the preceding examples.

V. DOMINANCE RELATIONSHIPS IN NON-HUMAN PRIMATES The impact of dominance status for non-human primates has been investigated via several approaches in laboratory and naturalistic conditions. We will begin first with laboratory studies and move to the more difficult setting of free ranging non-human primate populations in natural or semi-naturalistic settings. These varied approaches may permit disentangling the relationship between dominance status and immune regulation and health in general. Social dominance status has been linked to reproductive maturation and regulation and HPA regulation. Focusing specifically on the HPA system, there are wide species variations in plasma cortisol levels under non-stressed basal conditions (Coe, Savage, and Bromley, 1992; Laudenslager, Bettinger, and Sackett, 2006). This makes cross-species comparisons of absolute levels a bit complicated. For example, New World primates tend to have higher steroid levels in general when compared to Old World species for total plasma cortisol levels. However, this is not always the case in that there are exceptions to this general observation: some New World species, like the titi monkey, have lower cortisol levels comparable to Old World species (Klosterman, Murai, and Siiteri, 1986; Mendoza and Moberg, 1985). One aspect affecting bio-available cortisol is the binding capacity and affinity of corticosteroid-binding globulin (CBG), both of which are lower in New World species, resulting in free levels higher than present in Old World species (Klosterman et al., 1986). In spite of these enormous differences, New World species show no physiological evidence of glucocorticoid excess (Chrousos et al., 1982; Chrousos et al., 1984). It turns out that the New World primates have a cytosolic-binding protein that inactivates the excess free cortisol (Fuller, Smith, and Rogerson, 2004; Scammell, Denny, Valentine, and Smith, 2001). In


general, comparisons of glucocorticoid levels must remain within a species to avoid erroneous conclusions drawn across species with regard to absolute glucocorticoid levels. Another characteristic to be considered when making cross-species comparisons is the rather large differences in plasma/serum cortisol levels associated with body size. Smaller primates have higher cortisol levels than the larger apes (Coe et al., 1992). This variation associated with body mass extends to other non-primate species as well (Laudenslager et al., 2006). Stressful or challenging events have been associated with both up- and downregulation of in vitro immune measures (see Fleshner and Laudenslager, 2004). These opposite reactions are related to a variety of factors, including the duration of the stressor, its timing, and the exact immune parameter assessed. When one is considering dominance relationships, there are several levels to consider: (1) the immediate effects of brief dominance encounters that occur on a daily basis in social groups, (2) the impact of dominance status in modulating an individual’s response to an acute challenge that is unrelated to dominance status per se, and (3) the longer-term effects of high or low rank within a social structure. In the following sections, we will focus on both acute effects (social reorganization paradigms in the laboratory) and longer-term effects (in stable social groups where dominance status has been established). Immune and endocrine changes associated with the brief daily events in the context of typical dominance interactions in free-ranging non-human primates are difficult to capture (no pun intended). One might assume that the measures collected represent the summation of events occurring over the past hour or days. However, this is only a snapshot of the complicated interactions of these highly social organisms. The different players in each social situation will influence the nature of the changes noted in any biomarker or behavior, and thus generalities are difficult to make.

A. Primate Social Group Instability in Captive Settings Instability in primate social groups has been demonstrated to produce a number of pathophysiological changes that lead to immunomodulation and other medical risks. Instability in free-ranging conditions might be due to changes in the dominance structure of a group associated with attempts by a previously lower-ranking member to attain higher social status or the emigration of a new male into a troop and consequent rank changes. Social rank instability can be pro-

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duced in captive settings by rearranging membership in the group or by the introduction of an intruder to an established group as used in laboratory studies of rodents. Social group rearrangement of primates is associated with significant increases in aggressive behaviors. The social group rearrangement paradigm is created by taking four unfamiliar monkeys and placing them together in a common living area. Periodic reorganization may take place every 4–6 weeks, with monkeys encountering unfamiliar monkeys each time. This instability is associated with profound neuroendocrine changes, atherosclerotic disease, and ovarian dysfunction in cynomologous macaque monkeys (Macaca fascicularis) (Kaplan and Manuck, 1999; Kaplan and Manuck, 2004; Manuck, Kaplan, Adams, and Clarkson, 1988a; Manuck, Kaplan, Adams, and Clarkson, 1988b; Manuck, Marsland, Kaplan, and Williams, 1995; Mccabe et al., 2000). Social reorganization has been useful in the study of social challenges on immunoregulation including in vitro and in vivo markers and inoculation with benign protein antigens or replicating viruses. If cynomologous monkeys experiencing the reorganization paradigm were characterized as high or low with regard to behavioral measures of receiving and initiating aggression and affiliation during the reorganization, monkeys spending the greatest time performing social affiliation behaviors (grooming others, being groomed, or in passive contact or proximity to other monkeys) had the greatest response to mitogen stimulation and natural cytotoxicity of tumor targets (Kaplan et al., 1991). Although one might expect that significant relationships would exist with aggression, affiliation was positively associated with these immune measures during reorganization, and aggression was unrelated to immune outcomes. This is a subtle but important difference. Interestingly, relative rank in the reorganized groups did not influence these particular immune markers. Moreover, in stable social groups, relative affiliation and aggression were unrelated to in vitro immune parameters (Cohen, Kaplan, Cunnick, Manuck, and Rabin, 1992). We have also observed that relative dominance status does not affect natural cytotoxicity in stable social groups of bonnet (Macaca radiata) or pigtail (Macaca nemestrina) macaques (Boccia, Laudenslager, and Broussard, 1992; Boccia, Laudenslager, Broussard, and Hijazi, 1992). Enhanced natural cytotoxicity was observed when pigtail monkeys were contesting for a limited resource, but activation of the cytotoxic response was noted across group members independent of rank. These changes returned to basal levels within 48 hours.

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During social group reorganization of adolescent bonnet macaques, the presence of a “friend” in the new group mitigates acute changes of in vitro immune measure (Boccia et al., 1997). Friendship was determined, as suggested by Smuts for free-ranging baboons (Smuts, 1985), based on relative rates of reciprocal grooming and proximity. In the absence of an established social relationship with members of the new group, changes in natural cytotoxicity were noted as quickly as 2 hours following the reorganization. However, these parameters returned to baseline within a week. Reorganized group members with a pre-existing “friend” showed no change in immune parameters during the reorganization. One week after reorganization, all monkeys in the newly reorganized group had established new friendships in the group coincident with the return of immune parameters to baseline levels. Rates of relative affiliation were directly related to both lymphocyte activation and natural cytotoxicity as noted by others. We conclude from these observations among non-human primates that positive social relationships (e.g., associated with affiliative behaviors) promote these in vitro immune markers. Affiliation and agonism do not necessarily have opposite effects on immune modulation. Assessing the organisms’ specific antibody response to challenge with a benign novel protein antigen such as KLH or tetanus toxoid has been a particularly profitable means for observing immune modulation in the intact organism. Not surprisingly, social group reorganization of male cynomologous macaques immunized with tetanus toxoid (TT) affects primary and secondary responses (Cunnick et al., 1991). Immunization either 1 or 4 weeks prior to a single reorganization or at the time of reorganization did not have a significant effect on the primary response immunization with TT. However, across all groups regardless of the timing of immunization, dominance rank affected specific IgG levels; that is, subordinate monkeys demonstrated a greater primary IgG response to TT. These monkeys were followed and either experienced repeated monthly social reorganizations or were placed in a stable group. A secondary immunization with TT occurred after a tenth reorganization for the challenge group and 9 months after the last reorganization for the stable group. There was no effect on the booster response of social rank, but there was a significant effect of reorganization; that is, a significantly higher antibody titer was measured in the chronically reorganized group. Social rank affected the primary response to TT, and the social challenge independent of rank affected the secondary response. Thus, effects were dependent on whether memory or naive cells were involved.

B. Individual Characteristics Contributing to Immunomodulation As complex social organisms, non-human primates provide a unique opportunity to look at individual differences. Personality is an important aspect of the individual that contributes to immune modulation. It is not something that can be measured directly, but it appears as a general pattern of behavior(s) reflective of how the individual responds to different situations. Personality concepts drawn from the human literature have been successfully applied to macaque monkeys (Capitanio, 2004; Capitanio, Mendoza, and Bentson, 2004; Capitanio and Widaman, 2005; Clarke and Boinski, 1995; Laudenslager et al., 1999; Stevenson-Hinde and Zunz, 1978) and other species ranging from fish to dogs (Gosling, 2001). Several decades ago a list of adjectives was drawn together for describing impressions about their behavior that an observer might have after spending many hours watching monkeys (Stevenson-Hinde and Zunz, 1978). The 23-item adjective list included such items as Assertive, Aggressive, Confident, Dominant, Fearful, and so on. These adjectives were scored on a seven-point scale from 1 (extreme opposite of the behavior) to 7 (extreme manifestation) for each individual monkey. These investigators and others (Capitanio et al., 2004; Laudenslager et al., 1999) applied factor analytic techniques for clustering these behaviors into categories that reflect “personality” structure for the macaque monkey in much the same manner that was found useful for humans many years ago (Allport, 1937). The initial factor structure of these adjectives for rhesus monkeys revealed three main dimensional components: Confident/Fearful, Active/ Slow, and Sociable/Slow (Stevenson-Hinde and Zunz, 1978). Across studies the number of components has varied between two and four major factors. Variations in the number of these principal factors may be related to specific situations of the monkeys such as housing and the total amount of time the moneys were observed, to changes in the exact items included in the list to improve inter-observer reliability. There are, however, some consistencies that warrant attention as they are related to immune parameters, endocrine regulation, and viral illness following challenge in monkeys. The most recent application of the personality approach to individual differences comes from the work of Capitanio and colleagues (Capitanio, 2004; Capitanio and Widaman 2005). According to Capitanio, personality is mirrored in a number of traits or dispositions to respond in a given situation; that is, “. . . personality is manifested in a context . . .” (Capitanio, 2004, p. 16) much like dominance rank. It is difficult to discuss the concept of personality without


commenting on the construct of “temperament,” which has been used frequently by both human (Kagan, 1994; Kagan and Snidman, 1991) and primate (Champoux, Suomi, and Schneider, 1994; Clarke and Boinski, 1995; Fairbanks, 2001; Fairbanks et al., 2004) researchers. Temperament may differ from the concept of personality somewhat in that it has been described as a biological predisposition to behave in a particular manner, whereas personality characteristics may be shaped more by experience. This is an arguable point (Capitanio, 2004), and it will not be addressed here. Temperament is personality and may simply be a reflection of personality at an early age (J. Capitanio and S. Suomi, personal communications, 2005). Neither temperament nor personality derives exclusively from genetic or environmental factors. For the purposes of this chapter, we will consider the two terms interchangeable, with both reflecting similar underlying processes derived from both genetic and environmental factors. Thus, temperamental dispositions, central neurochemical organization, and HPA reactivity have heritable (Williamson et al., 2003), physiological (Fairbanks et al., 1999; Fairbanks, Melega, Jorgensen, Kaplan, and McGuire, 2001; Manuck et al., 1995; Manuck, Kaplan, Adams, and Clarkson, 1989; Manuck, Kaplan, Rymeski, Fairbanks, and Wilson, 2003), and identified genetic aspects (Bethea et al., 2004; Bethea et al., 2005) which further interact with environmental events (Barr et al., 2003; Barr et al., 2004a; Barr et al., 2004b; Barr et al., 2004c) and gender (Barr et al., 2004b). The temperament of macaque monkeys may modulate responses to social and challenging situations. For example, cardiovascular responses to acute stressors vary in a striking manner across species in spite of close genetic relationships such as those that exist for the Macaque genus (Clarke and Lindburg, 1993; Clarke, Mason, and Mendoza, 1994). Rhesus (Macaca mulatta), bonnet, and long-tailed (Macaca silenus) macaques exposed to differing situations (their home cage, a novel environment, and restraint), while heart rate was collected via telemetry, showed species-specific heart rate responses. Average heart rate across all conditions indicated that heart rate of long-tailed > bonnet > rhesus macaques. Large behavioral/temperamental/personality differences exist both across species (Clarke and Boinski, 1995; Clarke and Lindburg, 1993) and within species (Capitanio, 2004). There appear to be temperamental differences in captive rhesus monkey infants dependent on their region of origin (Indian versus Chinese) and which influence enumerative blood parameters (Champoux, Kriete, Higley, and Suomi, 1996). Similar differences exist for regulation of the HPA axis as well.

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The study of species differences in bonnet and pigtail macaques has been the focus of our laboratory for many years (Laudenslager, 1988; Laudenslager et al., 1995; Laudenslager, Berger, Boccia, and Reite, 1996; Laudenslager, Held, Boccia, Reite, and Cohen, 1990). As a part of a longitudinal developmental study beginning shortly after birth until the monkeys reached 4 years, we collected blood samples every 6 months from a cohort of bonnet (n = 40) and pigtail (n = 34) monkeys housed in conspecific social groups. At approximately 42 months of age, we removed these monkeys from their peer groups and placed them in individual housing for a brief period of 72 hours, after which they were returned to their social group (Laudenslager, in preparation). Un-anesthetized blood samples were collected before and at the end of the individual housing experience. For both species, cortisol levels rose from baseline to post housing (p < .001). There were both species and gender differences in cortisol level (p < .001). Since female pigtail monkeys had higher cortisol levels than pigtail males at all timepoints (species by sex interaction, p = .04), they may be partially responsible for the overall effect of gender. Furthermore, CSF levels of corticotrophin-releasing hormone (CRH) are lower in bonnet relative to pigtail macaques (Rosenblum et al., 2002). These observations are consistent with established temperamental differences between bonnet and pigtailed monkeys (Boccia, Laudenslager, and Reite, 1994; Boccia, Laudenslager, and Reite, 1995; Kaufman and Rosenblum, 1966; Laudenslager and Boccia, 1996; Laudenslager, Boccia, and Reite, 1993). Compared to pigtail monkeys, bonnet monkeys are known to be more affiliative in social situations with group members, and care of the young is allomaternal (e.g., many members of the social group freely interact with the infants). Overall, these differences are consistent with differences noted between these species with regard to physiological responses to social challenge (Laudenslager and Boccia, 1996; Laudenslager, Boccia, and Reite, 1993; Reite, Kaemingk, and Boccia, 1989; Rosenblum et al., 2002). Yet intruder bonnet monkeys received far greater agonistic behavior from unfamiliar conspecific residents in a new social group (Figure 2, Laudenslager, unpublished observations). Thus, the expected pattern for greater affiliative behaviors among bonnets does not fit all circumstances, e.g., to conspecific intruders. Does personality variation within a species modulate the HPA axis along with immune responses? The answer is, not surprisingly, yes. Using the personality dimensions described earlier for the rhesus monkey, it was noted that adult male rhesus monkeys rated high on an excitability dimension (excitable, active, and

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monkeys. All monkeys were mid ranking prior to the move, and thus rank was not a variable in these studies. However, one might be concerned when drawing conclusions based on a grouping variable associated with subjective ratings of only three adjectives. Yet these general dimensions appear to be consistent across animal cohorts, varying only slightly with regard to the specific adjectives loading on a factor (Capitanio, 2004; Capitanio, 2005).

C. Social Reorganization and Response to Viral Challenges

FIGURE 2 Rates of agonistic behaviors during social group introductions in bonnet and pigtail monkeys. Although the bonnet monkey is typically considered to be more affiliative toward conspecifics, this does not hold for unfamiliar monkeys.

subordinate) and had lower plasma cortisol levels than monkeys rated as low on the same dimension during un-anesthetized blood draws (Capitanio, Mendoza, and Bentson, 2004). High scores on confidence (confident and aggressive) were associated with higher cortisol levels. The authors concluded that enhanced negative feedback among the excitable and low confidence monkeys was associated with lower plasma cortisol levels. These differences are not unlike what is noted in individuals diagnosed with post-traumatic stress disorder, that is, a lower cortisol level in the patient population compared to controls (Yehuda, 2002; Yehuda, 2003). Regardless of whether lower cortisol and/or disrupted diurnal rhythms are associated with a specific personality phenotype or environmental traumas (Nemeroff, 2004), identifying sources of these individual differences is central to a more complete understanding of brain behavior and immune relationships. Personality in the rhesus monkey affects responses to immunization that occur during relocation experiences in which monkeys were removed from large social groups to individual housing (Maninger, Capitanio, Mendoza, and Mason, 2003). The dimension of sociability, a trait that positively loaded on the adjectives of affiliative and warm and negatively on solitary, divided by a median split into high and low sociable monkeys was associated with greater secondary responses to TT boosters in the high sociable

What is the impact of social reorganization on response to a replicating viral challenge such as the common cold virus? Cohen has provided indications of the role of social factors in modulating viral susceptibility among primates (Cohen et al., 1997). These were crucial experiments for PNI as they challenged monkeys with an infectious agent, adenovirus SV17, not just a benign protein in a paradigm where the subjects could be behaviorally manipulated. Cohen had previously indicated in a series of elegant studies that susceptibility to the common cold was affected by self-reported psychological distress in humans (Cohen, Tyrrell, and Smith, 1991). Furthermore, the social environment had a significant impact to experimentally inoculated common cold viruses in humans (Cohen, Doyle, Skoner, Rabin, and Gwaltney, 1997). These studies in human populations were landmark experiments in PNI as they identified psychosocial risk factors for a medical illness which previous studies assessing immune parameters alone could not conclusively demonstrate. The social reorganization studies in primates (Cohen et al., 1997) showed that the social stress did not affect viral expression; however, social rank did. Lower social rank was associated with increased risk for infectious illness after viral inoculation. Risk was not associated with aggressive encounters among new social members, but rather where in the social dominance hierarchy a monkey fell. Although lowerranking animals had elevated cortisol levels, this did not account for risk for infection, nor did body weight or alterations in lymphocyte populations. In point of fact, more aggressive animals were at reduced risk for viral infection. These observations are consistent with observations of rodents who fought back in association with territorial intrusion and who had a more robust response to antigenic challenge (Fleshner et al., 1989) and similarly for the tree shrew (vonHolst, 1997). Important experiments have been carried out that followed the progression of simian immunodeficiency


virus (SIV) after inoculation of rhesus monkeys. The viral response was studied as a function of prior stressor exposure, social reorganizations, or the personality dimension described previously. In an early pilot study based on a retrospective investigation of colony records (n = 22), it was noted that monkeys who experienced more frequent cage moves had shorter survival times (Capitanio and Lerche, 1991). Furthermore, post-inoculation weight loss was associated with the number of pre-inoculation cage moves. These effects were found after statistically controlling for viral strain, dose of virus, age at inoculation, and so on. Applying hazard analysis to a larger sample (n = 298), it was found that any social relocation occurring within 90 days prior to inoculation with SIV and up to 30 days following inoculation was associated with shorter survival (Capitanio and Lerche, 1998). Social housing (pair or group housing) after inoculation was associated with greater survival hazard. One must be cautious with regard to these observations, as the number of subjects was quite small (n = 17) and the housing was not part of a specific experimental design to investigate housing, but to look at the effects of horizontal and vertical transmission. The impact of unstable social grouping has been investigated systematically in SIV-inoculated rhesus monkeys (Capitanio, Mendoza, Lerche, and Mason, 1998). At 6 years of age, a group of 36 healthy male rhesus monkeys was removed from their natal social groups and transferred to individual housing. Three to 5 days a week, the monkeys were placed in either stable or unstable social groups for socialization for 100 minutes/day. In the stable groups, the individual animals were always the same. In the unstable groups, the group members during the socialization period varied for each exposure. After 3 weeks of these experiences, half of these monkeys in each group were inoculated with SIV, and half received control saline injections. Socialization continued following inoculation, but controls and SIV-inoculated animals remained separate. Animals in the unstable condition had a shorter median survival (420 days) compared to the stable group (589 days). Although the stable animals showed lower viral RNA levels and higher IgG response to SIV, these effects were not significant. However, aggression (receiving threats), independent of social stability, was positively associated with viral RNA levels and negatively associated with antibody titers to SIV. Animals in unstable compared to stable groups had a greater cortisol response to restraint stress and enhanced suppression by dexamethasone. The preceding results indicate that social stressors affect survival in monkeys inoculated with SIV. However, results indicated suppressed HPA activity

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following inoculation. In fact, this could be due to neurotrophic effects of the virus affecting CNS feedback mechanism although the authors suggest post-traumatic stress disorder–like phenomena. The well-known impact of pro-inflammatory cytokines on HPA regulation (Dunn, 1995) and the influence of SIV and HIV on HPA regulation (Clerici et al., 1997; Norbiato et al., 1992; Vago, Clerici, and Norbiato, 1994) together make it difficult to interpret HPA observations in SIV-infected monkeys. Not surprisingly, the aforementioned dimensions of personality affect responses to SIV inoculation in adult male rhesus monkeys as well (Capitanio, Mendoza, and Baroncelli, 1999). Low sociable (low subjective ratings on sociable, playful, and curious) monkeys had a higher viral load following inoculation. Viral load was directly related to survival in SIV (Watson et al., 1997) and in HIV as well (Mellors et al., 1995). These investigators have not determined the extent to which personality characteristics moderate social instability in disease progression. However, we will see below that personality characteristics moderate HPA activation in subordinate baboons in association with dominance status. Although laboratory settings allow control over many confounding variables, the field setting provides a far richer behavioral backdrop for the study of dominance, individual differences, and immune relationships.

D. Dominance Relationships in Naturalistic Settings When free-ranging animals are the subjects of study, a number of unforeseen factors can contribute to problems in research, as typified by a study that one of the authors (MLL) and Robert Sapolsky undertook. We were interested in individual differences in the response to antigenic challenge among a group of freeranging olive baboons that Sapolsky had studied extensively. We wanted to investigate the specific antibody response to a “novel” antigen, KLH, as it related to social dominance status. We first verified that we could in fact measure the IgG response to KLH in captive baboons. The captive baboon population showed a significant rise in specific IgG antibody titers following an intramuscular KLH challenge. Specific IgG levels rose from an absence of antibodies prior to immunization to significant levels measured from 7–28 days later. Sapolsky then spent a summer in Kenya, armed with KLH, tracking 20 male baboons, darting them for immunization, and re-darting them as close as possible 21 days later so we could assess their primary response to KLH. He succeeded in collecting plasma samples from the immunized baboons.

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Here, the realities of the field met the controlled conditions of the laboratory, or psychoneuroimmunology alfresco (paraphrased from Sapolsky, 1993). Every baboon we immunized had significant KLH-specific antibodies before immunization at the first darting. Antibody levels were higher for the second sample, but they were confounded by the differences that existed at baseline. We appeared to be measuring either background cross-reactivity or a booster response to KLH. It was highly unlikely that the animals had ever been exposed to keyhole limpets (a coastal tide pool mollusk) in their lifetime. We checked for crossreactivity to hemocyanin from the horseshoe crab in the baboons. We found antibodies to this protein as well at baseline in our baboon samples. We scratched our heads as to how they had acquired antibodies to this rather unique antigen until we realized that the free-ranging baboons fed on insects and land snails. Among invertebrates, the respiratory protein is represented by copper-based hemocyanin. The free-ranging baboon population appeared to have orally immunized themselves to hemocyanin by ingesting invertebrates as a part of their diet. Their immune system could not distinguish specifically between sources of hemocyanin. Thus, the study subjects did not have a controlled exposure to KLH. In seeking a novel antigen for the free-ranging baboons, we had been frustrated by their feeding habits. However, the study of free-ranging baboon populations can provide rich data with regard to endocrine regulation and its association with dominance status and personality characteristics in a variety of primate species. Sapolsky has focused much of his attention to dominance and personality/style influences on the male baboons free ranging in Kenya. Early studies indicated that male aggressiveness was associated with elevated testosterone but not mating success (Sapolsky, 1982; Sapolsky, 1991). Those successful in mating had lower initial cortisol levels but a greater rise in response to capture. In these studies dominance was based on the number of successful matings, assuming that mating success was reflective of dominance. During a period of social instability in the troop (dominance interactions were more frequent and less predictable), high-ranking males were more likely to participate in escalated aggression, but they showed endocrine levels quite different from lower-ranking males in the group (Sapolsky, 1983). During stable periods, there were no differences between high- and low-ranking male baboons, whereas when aggression was high and less predictable, low-ranking males had higher cortisol levels. During the period of instability, males’ testosterone levels were lower compared to the stable period, but most importantly, the greatest sup-

pression was among the low-ranking males. One of Sapolsky’s important contributions was the observation that aspects of social dominance in adult male baboons were modulated by individual characteristics of the baboons, e.g., their personality. Lower basal cortisol levels were most often noted among high-ranking males that recognized differences in social situations as either a threatening or neutral interaction with other males (Sapolsky, 2005a; Sapolsky, 2005b; Sapolsky, Alberts, and Altmann, 1997). Dominant males lacking this “skill” had cortisol levels as high as low-ranking members of the troop. This trait is quite similar to the adjective “understanding” in the Stevenson-Hinde and Zunz schema (1978). Overall, the hypercortisolism was related to disrupted feedback regulation at the CNS level in the subordinate baboons (Sapolsky, 2005a, 2005b). Another modifier of the impact of social instability is whether a baboon is rising or falling in rank. Ascending in dominance rank is associated with higher cortisol levels (Sapolsky, 1992). It is of note that rankrelated differences extend to cardiovascular reactivity as well (Sapolsky and Share, 1994), with high-ranking animals showing more rapid return to baseline following pharmacological challenge. The picture that emerges for the baboon is that rank is not the only determinant of hypercortisolism, but individual differences in personality contribute significantly to regulation of the HPA axis (Virgin and Sapolsky, 1997). Unfortunately, the nature and location of this field site have precluded in-depth assessment of immune regulation beyond changes in enumerative measures such as differential cell counts in this otherwise well-studied population. For this reason, we turned to a more accessible field setting. For over a decade, we have studied immune and neuroendocrine parameters in free-ranging rhesus monkeys (Macaca mulatta) on a provisioned island, Cayo Santiago, off the coast of Puerto Rico (Laudenslager, Rasmussen, Berman, Suomi, and Berger, 1993; Laudenslager et al., 1999). Macaque monkeys in large social troops are organized into smaller groups of related adult monkeys who are sisters, daughters, and granddaughters of a female, creating a matriline. Within the troop there are several matrilines, which have unique status relative to each other, that is, outranking others and having priority of access to valued resources. Recent analyses suggest that matrilineal dominance status affects hormones, in vitro immune parameters, and antibodies to a latent virus found in young macaque monkeys (Laudenslager, Rasmussen, and Suomi, in preparation). Unlike the aforementioned studies of the baboon, we have focused on earlier aspects of dominance status of the mother and its impact on her offspring. Among macaques,


dominance status can be either along matrilines or based on a nepotistic hierarchy (Chapais, 2004). In a nepotistic hierarchy, individual attributes of the females are irrelevant to status, as rank is dependent on alliances formed by the females in agonistic and dominance-related encounters. In a matrilineal dominance hierarchy, a female’s rank is based on her kinship. Thus, in the rhesus macaque, our study subject, maternal rank is assumed by her female offspring. A macaque matriline might consist of an older female, her daughters, and their offspring, and so on. The principle of youngest ascendancy confers higher rank to a daughter over her aunts and cousins in the matriline. As you can imagine, these relationships are complicated for the casual observer, but the monkeys are clear in their ranking. In contrast to the role of matriline in determining rank of the female rhesus monkey, males are somewhat different, as the males typically emigrate from their natal troop around puberty or later and immigrate into a new troop or remain outside a troop as a peripheral male. Note that peripheral male status is unique to provisioned situations such as Cayo Santiago where predation is lacking. Until the males emigrate from their natal troop, their dominance status is that of the matriline. However, the dominance status of the male in a new troop is not predicted by its kinship and prior rank. Blood samples were obtained from these freeranging rhesus monkeys between the ages of 3 and 6 during the annual census and health checks that take place each January and February. The monkeys were trapped in provisioned field cages where they typically obtain their daily monkey chow. Blood samples were collected under light ketamine anesthesia exactly 30 minutes after the doors to the field cage were closed (base sample). This permitted control over time from trapping to blood draw not always possible at field sites (see Sapolsky, 1993). Subjects were then transported to a field laboratory, where they were held in individual housing over the next 24 hours. Prior to release the following day, they were anesthetized and blood was drawn a second time (challenge sample, e.g., being overnight in individual housing). Maternal dominance status within a troop was categorized as either high or low. Plasma cortisol levels assessed at the time of trapping (base) and prior to release (challenge) rose more in monkeys from low-ranking matrilines (p < .001), as shown in Figure 3. Collapsed across base and challenge conditions and years, plasma cortisol levels were greatest in the young males from low-ranking matrilines (p < .001). There was no difference in age of emigration based on matrilineal dominance status. Thus, matrilineal dominance status has a significant effect on

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FIGURE 3 Mean plasma cortisol level (μg/dl) ± 1 SEM noted following capture (Base) and 24 hours later after overnight housing in an individual cage (Challenge) in rhesus monkeys as a function of matrilineal dominance status. Note levels are comparable across ranks at Base, but the low-ranking animals showed a greater rise in association with the challenge.

glucocorticoid regulation of young males prior to emigration. It is of interest to note that peripheral or solitary males in this island setting have a number of indictors of greater fitness as measured by greater body weight and skin fold thickness (Rasmussen, Berard, and Suomi, 1995). Although these solitary males had a history of movement from group to group and spending more time as solitary peripheral animals, these animals were devoid of rank, as they were not a part of an established social structure or group. Sapolsky (2005a, 2005b) suggested that one feature lacking in studies of subordinate relationships are assessments that reflect transient immune activation (see Fleshner and Laudenslager, 2004, for a review of this phenomenon) and subsequent recovery functions. To some extent, this is difficult to accomplish under free-ranging conditions. However, we have noted significant effects of low matrilineal dominance status with regard to natural cytotoxicity. If we assume that the closure of the gates to the field cage described above initiates a stress response in the trapped monkeys, differences in relative levels of lysis of tumor targets may shed light on Sapolsky’s question. We noted that natural cytotoxicity of these tumor targets was higher on the day of trapping (base) for the young monkeys from subordinate matrilines, as indicated in Figure 4. That is, on the day of trapping (base), lysis of the tumor targets was significantly higher (p < .001) in the low-ranking group. This relationship was noted

III. Behavior and Immunity Estimated Marginal Mean B Virus Antibody Titer

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FIGURE 4 Percent lysis of K562 targets by isolated peripheral blood lymphocytes (30 : 1 effector-to-target ratio) of rhesus monkeys from high-and low-ranking matrilines. Overall, low-ranking animals displayed significantly higher lysis of targets, but both high- and low-ranking animals showed a decline between capture (Base) and release (Challenge) the following day.

across 3 consecutive years (from 3 to 6 years of age), during which we obtained blood samples for in vitro immune measures. Lysis declined from the first sample to the sample collected prior to release the following day in both groups. Lysis was consistently higher on both days in the low-ranking group. Gender did not influence these relationships, nor were these measures related to plasma cortisol at the time of blood sampling. We have noted increased natural cytotoxicity in previously challenged human populations such as combat veterans with PTSD (Laudenslager et al., 1998) and in monkeys throughout adolescence into adulthood after experiencing early social challenges (Laudenslager, Berger, et al., 1996). Increased numbers of activated NK cells were observed in a traumatized population among individuals who report no distress following the trauma but showed elevated cardiovascular activity, e.g., repressors (Benight et al., 2004). Since the NK cell is involved in viral defense (Shieh et al., 2001) and its relative activity might reflect immune activation in response to environmental challenges, these results suggest that there might be potential health implications for increased natural cytotoxicity associated with social subordination. Do the previous results suggest that the cells responsible for natural cytotoxicity in young monkeys from lower social ranks are actually activated? Does this in any way affect viral defense mechanisms in these free-ranging monkeys?

Age (Years)

FIGURE 5 Rhesus monkeys from low-ranking matrilines show elevated antibody titers to Simian B virus, a herpes virus found in macaque monkeys.

Simian B virus (Cercopithecine Herpesvirus 1) is a naturally occurring herpes virus found predominantly in macaque monkeys (Eberle and Hillard, 1995). B virus is passed vertically through mucosal exposure to oral and genital secretions from infected monkeys actively shedding virus. After an initial infection that includes mild symptoms, the B virus passes into a latent state (within sensory ganglia) as is typical of the herpes viruses (Favoreel, Nauwynck, and Pensaert, 2000). When the B virus is reactivated, it is observable as herpetic sores in the oral and genital regions. Following the same reasoning presented many years ago by Glaser and colleagues (2005), we screened our subject population between 2 and 5 years of age for specific antibodies to B virus. We observed significant effects of year (antibody titers rose, p < .001), gender (females greater than males, p < .001), and maternal dominance status (low-ranking titers exceeding highranking offspring, p < .001) on viral antibody titers to herpes B virus (Figure 5). If we assume that viral antibody level is a marker of the ability of cell-mediated responses to hold the virus in latency (Glaser, 2005), the low-ranking monkeys appear to be immune compromised, as reflected in the significantly higher antibody titers to B virus (Laudenslager, Rasmussen, and Suomi, in preparation). Across the years that samples were collected in this cohort, a number of changes occurred, including puberty, pregnancy for some females, and emigration from the natal troop for some of the males. However, the relationship of neuroendocrine and immune markers to matrilineal dominance status remained consistent. As has been suggested by other researchers


(Coe, 1993; Coe, Lubach, Schneider, Dierschke, and Ershler, 1992), early events set the stage for immune regulation and its course throughout the life span. Certainly, early events affect regulation of the HPA axis, as has been shown repeatedly for the rodent (Liu et al., 1997; Meaney et al., 1993; Meaney, Aitken, Bodnoff, Iny, and Sapolsky, 1985). In the non-human primate, the early effects of matrilineal dominance may plant their seeds early in development. For the rhesus monkey, a female is stuck with the lot provided her via her mother—their matrilineal status. One might ask to what extent this will continue to direct the trajectory of the males who leave the social structure of their natal group. At present, these questions have not been addressed.

VI. DOES DOMINANCE MATTER? It is often customary to make comparisons between dominance rank in non-human species and socioeconomic status, with the assumption that a similar pathophysiology underlies both concepts with regard to lower social status (Goymann and Wingfield, 2004). Caste systems exist across nature from the social insects to higher primates, including humans. Dominance relationships confer a set of rules for the members of the group to follow in a situation in which resources might be limited. Dominance rank and signals associated with rank also provide individuals with predictability in their social interactions, regardless of social status. Living in social groups has clear advantages to the extent that groups provide protection from predation for the group that would be absent for the individual animal. Social grooming among group members assists in removal of parasites from the integument and also facilitates restoration of cardiovascular function following agonistic encounters (Boccia, Reite, and Laudenslager, 1989). Grooming is a reciprocal process wherein both the recipient and the groomer benefit, regardless of rank. However, for non-human primates, there are potential costs to low rank with regard to neuroendocrine regulation. For example, subordinates are frequently reproductively suppressed (Creel, 2001). One might pose the question, “Are the subordinate animals within a social group always stressed?” We indicated in stable social groups that immune differences are minimal between high- and low-ranking macaque monkeys (Boccia, Laudenslager, Broussard, and Hijazi, 1992; Boccia et al., 1992). Rank may not be stressful in stable groups. However, when the group is challenged as a whole, during a period of reduced resources, for example, then rank may have an impact. This relationship is far from simple.

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There are large species variations with regard to the impact of social status on physiology. Based on a metaanalytic study of cortisol in Old and New World monkeys, monkeys were ranked based on a relative cortisol level derived from basal cortisol levels of the subordinates expressed as a percentage of the dominants (Abbott et al., 2003). Surveyed investigators completed a series of 16 subjective questions regarding what it is like to be a male or female dominant or subordinate member of social groups corresponding to several New (n = 5) and Old (n = 5) World species. The social and breeding systems for each of these species were characterized. Group size and number of males in the group and cooperative versus polygamous breeding systems were considered. A number of interesting relationships were noted. Importantly, availability of social support to the subordinates was associated with lower relative cortisol among the subordinates. Our observations of higher cortisol levels in pigtail compared to bonnet macaques support this observation as well. Bonnet macaques have more affiliative relationships within their social group, and the presence of affiliative relationships after group introduction will significantly modulate immune responses during that transition (Boccia et al., 1997). Overall, the prior meta-analysis identified two factors that contribute to elevated cortisol levels in subordinate group members: the level of stress experience by the group and the availability of social support from other group members (Abbott et al., 2003). The presumed “stress” of low social rank has been compared to the concept of allostatic load (McEwen, 2003; McEwen, 2004). Briefly, allostatic load considers stress and challenges over a longer time frame than the original conceptualization of Hans Seyle. Allostatic load reflects a life span perspective (Seeman, Singer, Rowe, Horwitz, and McEwen, 1997). Allostasis and allostatic load are concepts that have elaborated on the old concepts of homeostasis, but extending them to life-long stressors and challenges on systems not immediately critical to survival such as pH or body temperature, which are prototypical homeostatic systems. The allostatic load model has been applied to non-human animal populations (McEwen and Wingfield, 2003), but the concept has been most frequently used for humans. It is beyond the scope of this chapter to discuss this concept in great detail but only to bring to the attention of the reader that variations in dominance status among social animals have been frequently used as models of allostatic load. Figure 6 summarizes the relationships observed between social factors such as rank or personality and in vivo and in vitro immune markers. This figure shows quite clearly that one cannot make broad-ranging

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FIGURE 6 The interrelationships of social aspects of behavior including sociability, aggression, and rank on various in vitro and in vivo measures of primate immunity.

summary statements regarding immune/social relationships based on single immune measures. Even simple in vivo responses such as response to antigenic challenge may provide different outcomes based on the observation of either a primary or secondary response. Responses to live viral challenge may not always reflect the same underlying mechanism(s) as responses to a simple non-replicating antigenic challenge. A life of challenges (e.g., low social rank under unstable conditions) is associated with increased risk for morbidity and mortality. However, even for nonhuman primates, social support moderates these effects in a significant manner, regardless of social status. Individual differences in behavior/personality contribute in a significant way to these relationships. We can no longer take simplistic approaches to behaviorimmune relationships but need to include these factors that contribute to larger error bars than we would often like to see in our data.

Acknowledgments Preparation of this manuscript and portions of the data present herein were supported in part by NIH grants MH37373 (MLL), AA13973 (MLL), and RR03640 (Caribbean Primate Research Center); funds from the Developmental Psychobiology Endowment Fund– University of Colorado Denver and Health Sciences Center; and intramural funding at NICHD (S. Suomi). We also acknowledge the expert assistance of Mark Goldstein and Anne Luckow in many aspects of this research and preparation of this manuscript.

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23 Social Context as an Individual Difference in Psychoneuroimmunology EDITH CHEN AND GREGORY E. MILLER

I. INTRODUCTION 497 II. SOCIOECONOMIC STATUS AND HEALTH 498 III. PATHWAYS LINKING SOCIOECONOMIC STATUS AND IMMUNE FUNCTIONS 498 IV. DEFINING AND MEASURING SOCIOECONOMIC STATUS 500 V. EVIDENCE OF SOCIOECONOMIC STATUS ASSOCIATIONS WITH IMMUNE FUNCTIONS 501 VI. CONCLUSIONS 505

Weitzman, Schoen, & Anton, 1999; Davidson, Coe, Dolski, & Donzella, 1999; Denollet et al., 2003; Marsland, Cohen, Rabin, & Manuck, 2001; Miller, Cohen, Rabin, Skoner, & Doyle, 1999; Miller, Dopp, Myers, Felten, & Fahey, 1999; Segerstrom, Taylor, Kemeny, & Fahey, 1998; Strauman, Lemieux, & Coe, 1993; Suarez, 2003; Suarez, Lewis, & Kuhn, 2003). Because several excellent reviews of this area of research have been published recently (Segerstrom, 2000; Segerstrom, 2003; Segerstrom, Kemeny, & Laudenslager, 2001), the goal of this chapter will be to introduce an alternative approach to conceptualizing individual differences, and discuss its implications for conducting and interpreting research in PNI. Though research on individual differences in PNI has been very fruitful, it has consistently overlooked questions about the origins of cognitive, affective, and behavioral characteristics. In this chapter we will argue that the larger social context is a critical factor in shaping individual differences and needs to be considered more thoroughly in PNI. The term social context refers to neighborhood, community, and family influences on an individual. In contrast, much of the individual differences literature isolates the individual and focuses on defining his/her characteristics, without much emphasis on how the larger social context may shape the development of these characteristics. As one representative indicator of this larger social context, we focus here on the role of socioeconomic status (SES). By SES, we refer to an individual’s position within a larger social hierarchy, as typically indicated

I. INTRODUCTION Individual differences refer to enduring characteristics that distinguish one organism from another and that are stable over time and across situations. Traditionally, these characteristics have included cognitive, affective, behavioral, and/or genetic traits ascribed to persons or animals. In humans, a large body of work has documented associations between individual differences and morbidity and mortality (Cohen, Doyle, Skoner, Rabin, & Gwaltney, Jr., 1997; Cohen et al., 1995; Cole, Kemeny, & Taylor, 1997; Cole, Kemeny, Taylor, Visscher, & Fahey, 1996; Cole et al., 2001; Kubzansky, Sparrow, Vokonas, & Kawachi, 2001; Miller, Smith, Turner, Guijarro, & Hallet, 1996; Reed, Kemeny, Taylor, & Visscher, 1999; Reed, Kemeny, Taylor, Wang, & Visscher, 1994; Scheier & Bridges, 1995; Scheier et al., 1999), and this work has given rise to mechanistic questions regarding the immunologic correlates of personality characteristics (Cole, Kemeny, PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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by social status (e.g., occupation, educational attainment) or material resources (e.g., income, savings). Focusing on the role of SES in PNI is important for several reasons. First, SES is a construct that simultaneously reflects neighborhood, community, and family influences. At the broadest level, markers of SES can be derived from community characteristics (e.g., the gap between the rich and the poor across a community). At a more proximal level, indicators of SES can be derived from features of a person’s neighborhood (e.g., the median home price or rates of violent crime), or the family that he/she is part of (e.g., annual family income or educational attainment). Second, SES is often considered to reflect a stable, enduring characteristic, a necessary quality for an individual difference variable. Although some SES characteristics can change from year to year (e.g., family income), many are fairly consistent across time (e.g., educational attainment). Finally, there is robust evidence that SES at one point in time can have long-lasting impacts on health. For example, early childhood SES has been found to predict health outcomes decades later in adult life, such as cardiovascular disease, stomach cancer, and hemorrhagic stroke (Barker, 1992; Galobardes, Lynch, & Smith, 2004). These observations suggest that SES is an important social context variable that contributes to the development of enduring individual differences across people.

II. SOCIOECONOMIC STATUS AND HEALTH SES has a profound influence on physical and mental health outcomes. Of all the social and psychological factors studied to date, SES exhibits the strongest and most consistent associations with morbidity and mortality. Individuals lower in SES are more likely to develop illnesses in the first place, to have difficulties managing them, and to die from them, compared with their higher-status peers. This “social gradient” exists for nearly all acute and chronic medical conditions. It also emerges in nearly all countries of the world, regardless of whether their citizens have universal access to care. Lastly, the social gradient persists across the life span, from early childhood to older adulthood (see Adler, Boyce, Chesney, Folkman, & Syme, 1993; Chen, Matthews, & Boyce, 2002; Marmot, Kogevinas, & Elston, 1987; Townsend & Davidson, 1982; Williams & Collins, 1995, for reviews). SES also is associated with a wide variety of psychological variables. These include traditional individual difference variables, such as hostility, optimism, depression, and anxiety (Adler et al., 1994; Barefoot et

al., 1991; Gallo & Matthews, 2003; Kubzansky, Sparrow, Vokonas, & Kawachi, 2001). Furthermore, low SES is associated with poor health behaviors, such as increased risk of smoking and decreased physical activity (Adler et al., 1994; Cohen, Kaplan, & Salonen, 1999; Lynch, Kaplan, & Salonen, 1997). Finally, evidence suggests that individuals from different SES backgrounds have different types of life experiences. For example, lower SES individuals are more likely to be exposed to stressful life events and are more likely to perceive stress in their lives (Brady & Matthews, 2002; Chen, Langer, Raphaelson, & Matthews, 2004; Cohen et al., 1999). Taken together, these findings suggest that SES is an individual difference variable that has profound effects on health and well-being. This highlights the importance of studying SES from a PNI perspective in order to understand mechanisms for how SES “gets under the skin” to influence health outcomes. Furthermore, SES clearly has psychosocial influences, underscoring the importance of considering SES as a psychologically relevant individual difference variable in PNI studies. In the next section, we briefly discuss possible pathways between SES and immune functions.

III. PATHWAYS LINKING SOCIOECONOMIC STATUS AND IMMUNE FUNCTIONS Why would a person’s SES influence the functions of his/her immune system? Although there are many potential answers to this question, they can usually be placed into one of two categories of explanation, depending on whether they emphasize SES’s role in fostering exposure versus vulnerability. Exposure hypotheses maintain that a person’s SES influences his/her chances of coming into contact with stimuli (exposures) that modify the immune response. These stimuli can range from micro-organisms that give rise to infectious disease, to environmental pollutants that set off inflammatory processes in the lung, to psychosocial stressors such as negative life events and community violence. Although these stimuli differ from one another in many respects, and influence immune functions through disparate mechanisms, what they share in common is a robust social gradient. For example, sanitation, environmental pollution, and violence vary by SES (Evans, 2004; Selner-O’Hagan, Kindlon, Buka, Raudenbush, & Earls, 1998). In addition, robust SES differences exist in the frequency of stressor exposure (Attar, Guerra, & Tolan, 1994; Brady et al., 2002; Garbarino, Kostelny, & Dubrow, 1991) and

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23. Social Context as an Individual Difference in Psychoneuroimmunology

in the way that stressors are appraised (Chen et al., 2004; Cohen et al., 1999). This cumulative stressor burden is expected to give rise to immune system dysregulation among low SES individuals, rendering them vulnerable to diseases that are immunologically resisted or mediated (Herbert & Cohen, 1993; Segerstrom & Miller, 2004). See Figure 1 for a graphical depiction of the exposure model. There are several mechanisms through which stressful experiences could “get inside the body” to modify immunity. They include activation of autonomic fibers descending from the brain to lymphoid organs, stressrelated secretion of hormones and neuropeptides that regulate leukocyte function, or changes in coping behaviors such as smoking or sleeping (Ader, Cohen, & Felten, 1995; Cohen & Williamson, 1991). Behaviors such as cigarette smoking, alcohol consumption, and a sedentary lifestyle also can be viewed as potential mechanisms of exposure. These behaviors are more frequently observed among low SES individuals (Adler et al., 1994; Cohen et al., 1999; Lynch et al., 1997), and they are known to modify various immune functions (Cohen, Miller, & Rabin, 2001; Kiecolt-Glaser & Glaser, 1988). However, social disparities in morbidity and mortality persist after statistical adjustment for health practices, suggesting that behavior is not likely to be the primary mechanism of action for SES (Lantz et al., 1998; Lantz et al., 2001). In contrast to exposure hypotheses, vulnerability hypotheses maintain that low SES makes individuals more vulnerable when an exposure occurs. That is, among low SES individuals, higher levels of exposure will relate to greater dysregulation of immune function. In contrast, high SES will buffer individuals from the effects of exposure, such that greater exposure will be only weakly or not associated with immune dysregulation. Thus, this hypothesis argues that low SES individuals have a more pronounced response to an equivalent exposure (stimulus) compared to high SES individuals. See Figure 2. Much of this work has been

conducted with testing stress as a type of exposure. In this context, the vulnerability hypothesis predicts that low SES individuals would exhibit heightened immunologic dysregulation during times of high stress compared to low stress; in contrast, high SES individuals will be buffered and show few immune differences during high stress versus low stress times. Why would low and high SES individuals differ in their response to an identical stressor? One hypothesis is that people continuously appraise ongoing stressful circumstances along dimensions of threat and manageability (Lazarus & Folkman, 1984). To the extent that circumstances are evaluated as posing significant threat and exceeding coping resources, they elicit a cascade of emotional, behavioral, and hormonal responses, which ultimately result in dysregulation of various immune system functions (Cohen & Williamson, 1991; Segerstrom & Miller, 2004). Moreover, a person’s tendency to appraise stressors as threatening is shaped by his/her SES (Chen & Matthews, 2001; Chen & Matthews, 2003). By virtue of living and working in settings that are unpredictable and sometime dangerous, lower SES persons develop a tendency to interpret situations as potentially threatening, even when the extent of danger is ambiguous. This vigilant cognitive style results in interpretations of an equivalent stressor as more threatening among lower SES individuals. This response strategy in turn may activate stress-response systems unnecessarily, and over time this may contribute to cumulative wearand-tear on the endocrine and immune systems ( Chen et al., 2004; McEwen, 1998).

SES

Exposure SES

Exposure

I mmune Dys reg ul ati on

Immu ne D y sr e g ul a t i o n

↓ SES: ↑ Exposure – ↑ Immune change ↓ SES

↑ Exposure -micro-organisms -pollutants -stress

↑ Immune Change

FIGURE 1 Exposure model. Low SES individuals receive higher levels of exposure to micro-organisms, pollutants, and stressors, and in turn, higher levels of exposure are associated with greater immune dysregulation.

↑ SES: 0 relation Exposure – Immune Change FIGURE 2 Vulnerability model. The relationship between exposure and immune function depends on SES. Among low SES individuals, high levels of exposure are related to greater immune dysregulation. In contrast, high SES buffers individuals from the effects of exposures, such that there is no relationship between exposure and immune dysregulation in this group.

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A second aspect of the vulnerability hypothesis suggests that a person’s SES governs the types of coping resources he/she has available and can utilize during stressful encounters. Persons of lower SES by definition have fewer economic and educational resources than their high-SES peers; these disparities are likely to accentuate the impact of life stressors like job loss or chronic illness in the family. In support of this hypothesis, previous research has documented that providing individuals with resources reduces cardiovascular reactivity during an acute laboratory stressor among low SES, but not high SES, individuals (Chen, 2005). Thus, these theories suggest that the reasons why low SES individuals may show heightened biological responses compared to high SES individuals to equivalent exposures have to do with psychological processes such as threat appraisals and coping.

IV. DEFINING AND MEASURING SOCIOECONOMIC STATUS SES is a multi-dimensional construct, and the measures one chooses of SES can reflect different underlying conceptualizations about what SES means (Winkleby, Jatulis, Frank, & Fortmann, 1992). One traditional approach to conceptualizing SES is as an indicator of a person’s status or standing within society. Researchers have termed these “prestige-based measures of SES,” as they indicate how a person is regarded within the community. Commonly used prestigebased indicators include education and occupation (Krieger, Williams, & Moss, 1997; Winkleby et al., 1992), with more advanced training and more prestigious jobs viewed as higher status. Others have argued for a resource-based model of SES, which suggests that the critical component of SES is the material resources a person possesses. These assets, including family income, assets, and wealth, are hypothesized to play a role in determining both health status and health trajectories over time in a family (Krieger et al., 1997; Lynch, Smith, Kaplan, & House, 2000). In contrast to these two more objective approaches, other researchers have argued for a more subjective, or relative, approach to measuring SES. This approach is based on the notion that most individuals do not understand SES in terms of absolute dollars, but rather in terms of where they stand relative to their peers, and that an individual’s perception of his/her SES or social standing will be more important to health outcomes than objective measures (Adler, Epel, Castellazzo, & Ickovics, 2000).

In addition, SES can be measured at multiple levels (Krieger et al., 1997). For example, one could measure characteristics of the community, neighborhood, family, or individual. Community measures include factors such as the level of income inequality in a society, or the level of social capital (community norms for cooperation and behavior) (Kawachi, Kennedy, Lochner, & Prothrow-Stith, 1997; Wilkinson, 1992). Neighborhood SES measures are narrower than community measures, and represent an aggregate measure of the group of individuals living in a neighborhood. These include indicators such as the percentage of adults with less than high school education in the neighborhood, median family income of the neighborhood, and percentage of people who own their own homes in the neighborhood. These characteristics describe the larger context that an individual lives in. At the family and individual levels, SES measures include direct assessments of the individual participant or the household that the participant lives in. Family measures include those described above, such as the income and savings of all family members living in a household. In contrast, individual SES measures focus on just the study participant, and could include either objective indicators, such as the individual’s occupation or educational attainment, or the subjective indicators described above, such as perceived social status. These various approaches highlight the importance of understanding the measurement approach when interpreting findings on SES and PNI. In addition, it also indicates the importance for future researchers in this area to consider the ways in which SES might exert effects on their immune outcomes of interest, and to choose theoretically meaningful approaches to measuring SES when designing PNI studies. For example, stronger associations with resource-based SES measures such as income and savings might suggest that more money allows families to afford higher quality medical care, which would presumably have effects on disease processes. In contrast, stronger associations with prestige-based SES measures such as educational attainment might suggest that higher SES families have better knowledge about healthy lifestyles, which in turn may lead to better health behaviors with immunological consequences. Finally, stronger associations with subjective SES measures might suggest that perceptions of social standing are related to the control one perceives over one’s life, or the degree to which one utilizes proactive coping strategies for dealing with stress, both of which may have implications for activation of stress-response systems.


V. EVIDENCE OF SOCIOECONOMIC STATUS ASSOCIATIONS WITH IMMUNE FUNCTIONS This section describes existing research on SES and immunity in humans. Following the conceptual distinction introduced earlier, studies are grouped according to whether they emphasize exposure versus vulnerability, and also according to kinds of immune system outcomes they include. Because this is a new area of inquiry, most research focuses on the simple question of whether SES and immune outcomes are related, without attempting to identify mechanisms that might be responsible for such an association. As we note, this will be an important direction for later research.

A. Exposure Hypothesis Studies guided by the exposure hypothesis examine associations between SES indicators and immune functions. The better research in this area goes a step further and seeks to identify potential underlying mechanisms, such as SES-related disparities in pathogen exposure, environmental pollutants, health practices, or stressful experience.

B. Studies in Community Populations A number of these studies have shown associations of SES with inflammatory molecules involved in the pathogenesis of coronary heart disease (CHD). For example, healthy adults from lower SES groups (as defined by a combination of education and income) had higher levels of CRP, IL-6, TNR-α, fibrinogen, and homocysteine (Panagiotakos et al., 2005). Adults from lower occupation groups had higher levels of CRP, IL-6, and serum amyloid A (though no differences were found for adhesion molecules that reflect endothelial activation) (Hemingway et al., 2003), and higher levels of fibrinogen (Brunner et al., 1993). Being unemployed also was associated with higher levels of CRP, although no differences in CRP were found for other SES markers such as education, car and home ownership (Danesh et al., 1999). Finally, adults from a low income group had higher levels of heat shock protein 60 compared to those from a high income group (Lewthwaite, Owen, Coates, Henderson, & Steptoe, 2002). Heat shock protein 60 is a protein released by cells that have faced trauma, such as heat, injury, or infection, and high levels have been related to CHD (Zhu et al., 2001). Other studies have examined immune markers different from those implicated in CHD. For example, one

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study measured the presence of the immunoglobulin secretory IgA (or sIgA) in maternal breast milk. Lower household income was associated with higher sIgA (Groer, Davis, & Steele, 2004). These findings suggest that low SES mothers may have been exposed to infectious micro-organisms to a greater extent than high SES mothers. At the same time, however, higher concentrations of sIgA may be beneficial for breastfeeding infants, who receive most of their mucosal immune protection through maternal transfer of antibody in milk.

C. Studies with Relative SES Measures Another study utilized a novel approach of measuring SES to investigate associations with immune variables. All of the above studies have relied on objective indicators of SES, such as education or occupation. However, families vary greatly in the extent to which they spend money on material possessions, regardless of their objective status. Dressler (Dressler, 1990; Dressler, Bindon, & Neggers, 1998) has argued that the discrepancy between outward displays of prestige (via material possessions) and actual objective circumstances (via education or occupation) is an indicator of “lifestyle incongruity,” and that higher levels of lifestyle incongruity are detrimental because they create stresses on the family. Consistent with this line of reasoning, one study found that in adolescents greater lifestyle incongruity (greater material possessions relative to objective circumstances) was associated with more antibody to the latent Epstein-Barr virus (EBV) (McDade, 2001). Higher levels of antibodies suggest that latent EBV may have been reactivated, either by stress hormones, poor immune control, or other mechanisms (Glaser & Gottlieb-Stematsky, 1982). Given that EBV plays a role in the pathogenesis of infectious mononucleosis, these findings could have implications for explaining some of the SES gradient in infectious disease.

D. Studies in Chronically Ill Populations Finally, one study examined associations of SES with immune markers in the context of a chronic inflammatory disease. Adolescents diagnosed with persistent asthma were recruited from either low SES or high SES neighborhoods (based on the percentage of people living below poverty in each neighborhood). Blood was drawn, and cells were stimulated with a combination of phorbol myristate acetate and ionomycin to induce the production of cytokines. Researchers have hypothesized that certain cytokines are impor-

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tant for the orchestration of cellular events related to airway inflammation and hyper-responsiveness (Chung & Barnes, 1999). For example, Th-2 cell secretion of IL-4 induces B-cells to produce IgE antibodies, which initiate an inflammatory cascade leading to airway constriction and mucus production (Bacharier & Geha, 2000). Th-2 cell secretion of the cytokine IL-5 has been found to increase eosinophil production, which also promotes airway inflammation and obstruction (Kamfar, Koshak, & Milaat, 1999; Ying et al., 1997). More recently, some researchers have argued that the cytokines that Th-1 cells produce (e.g., IFN-γ) also are elevated in patients with asthma, and can induce airway inflammation (Busse & Lemanske, 2001; Hansen, Berry, DeKruyff, & Umetsu, 1999; Holtzman, Sampath, Castro, Look, & Jayaraman, 1996; Marguet, Dean, & Warner, 2000). Thus, this study investigated stimulated production of IL-4, IL-5, and IFN-γ. Adolescents with asthma from low SES neighborhoods displayed significantly greater production of IL-5, IFN-γ, and marginally greater production of IL-4 compared to adolescents with asthma from high SES neighborhoods (Chen, Fisher, Jr., Bacharier, & Strunk, 2003). See Figure 3. These findings suggest that within the context of a chronic disease, low SES adolescents may exhibit heightened inflammatory responses to pathogens, and that the specific nature of these responses is consistent with pathways to more severe exacerbations of asthma.

E. Vulnerability Hypothesis

2000

20000

1600

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IFN-γ (pg/ml)

IL-5 (pg/ml)

Studies examining the vulnerability hypothesis often utilize the acute-stress paradigm. This entails bringing participants into the laboratory and collecting blood from them during a resting baseline. Participants then engage in a brief stressor, such as impromptu public speaking or pressured mental arithmetic, and additional blood is collected after the stressor. The vulnerability hypothesis is supported when low SES participants exhibit greater stressor-induced immune dysregulation compared with high SES participants.

1200 800 400 0

12000 8000 4000 0

Low SES

High SES

Low SES

High SES

FIGURE 3 Evidence for exposure model: Low SES adolescents with asthma have higher levels of stimulated production of IL-5 and IFN-γ compared to high SES adolescents with asthma.

Importantly, under this hypothesis SES differences are predicted to emerge during stressful circumstances, rather than during the baseline period.

F. Laboratory Stress Paradigms A number of studies have examined the vulnerability hypothesis in relation to inflammatory molecules implicated in CHD. These studies have been conducted by one research group and have all used a similar paradigm. Healthy adults underwent two acute stress tasks. One involved tracing an image that could only be seen in a mirror, as quickly and accurately as possible. The second involved identifying as quickly as possible the colors of color words that were printed in incongruent colors (e.g., the word red printed in blue. This is also known as the Stroop task). These tasks typically lasted 5 minutes each. Blood was drawn at baseline, in some studies immediately after the acute stressors, and then during a recovery period (ranging from 30–120 minutes post-stressor across the various studies). SES was defined by participants’ occupational status, for example, higher status jobs being managerial positions, and lower status jobs being clerical positions. One of these studies found partial support for the vulnerability hypothesis. Lower SES participants had higher circulating concentrations of the inflammatory cytokine interleukin-6 (IL-6) 120 minutes after the stressor ended compared with higher SES participants (Brydon, Edwards, Mohamed-Ali, & Steptoe, 2004). No SES differences in IL-6 were evident at baseline, or at 30 or 75 minutes after the stressor. Given that protein synthesis requires a minimum of 1–2 hours to occur, 30–75 minutes may have been too soon to detect stressrelated boosts in IL-6 expression. Regardless, if the findings at 120 minutes prove to be robust, they could help to explain the SES gradient in cardiovascular disease, as inflammatory processes have a key role in the development, progression, and clinical expression of atherosclerosis (Libby, Ridker, & Maseri, 2002; Ross, 1999). In contrast, several of this group’s other studies have found SES differences in inflammatory molecules during resting periods, but this difference did not change during or after participants were exposed to a stressor. For example, adults who are low in SES by virtue of having a low-prestige occupation (or lower education or income) showed elevated concentrations of the inflammatory molecule C-reactive protein during a resting baseline (Owen, Poulton, Hay, Mohamed-Ali, & Steptoe, 2003). CRP is a marker of systemic inflammation, and high levels are associated with CHD risk (Ridker, 2003; Ridker, Hennekens,

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G. Life Experiences with Stress It is important to remember that these studies focused on acute stressors in the laboratory, and the artificial nature of these tasks raises questions about their generalizability to the real world. A project conducted by a different research group found support for the vulnerability hypothesis when low and high SES persons were compared in the midst of a real-life chronic stressor. In this study, healthy adults were recruited who were either undergoing a major life chronic stressor (having a child being treated for pediatric cancer) or were under no chronic stress (having medically healthy children and no other major life

stressors). Parents underwent blood draw, and their cells were co-incubated in vitro with lipopolysaccharide (a bacterial product that stimulates cytokine production) and dexamethasone (a synthetic form of the hormone cortisol, which is a potent inhibitor of inflammation), and subsequent production of IL-6 was measured. This assay tests leukocyte sensitivity to the inhibitory properties of glucocorticoid hormones, which in vivo play a key role in regulating the magnitude and duration of inflammation. Interestingly, chronic stress-related alterations in the immune system were evident only among low SES participants. That is, parents of cancer patients showed reduced sensitivity to the anti-inflammatory properties of dexamethasone compared to parents of healthy children, but this was true only if they had lower levels of education (defined as a high school diploma or less). In parents of cancer patients who had some college education, there were no detectable alterations in glucocorticoid sensitivity relative to controls (Miller, 2002). See Figure 4. These findings suggest that low SES individuals facing chronic stressors may have disrupted mechanisms for regulating inflammation, which if sustained could place them at risk for conditions involving excessive inflammation, such as cardiac, allergic, and autoimmune diseases (Miller, Cohen, & Ritchey, 2002). More generally, these findings provide support for a vulnerability hypothesis of SES, at least in the context of real-world stressors that are severe and chronic.

H. Vulnerability to Viral Challenge A handful of studies have examined a modified version of the vulnerability hypothesis. In this view

IL-6 (percent resistance)

Buring, & Rifai, 2000). They also showed higher numbers of total lymphocytes, T lymphocytes, and natural killer cells at baseline compared with higher SES adults, but SES disparities did not increase in magnitude 1 or 45 minutes after the stressor ended, as the vulnerability hypothesis would predict (Owen et al., 2003). In another study by this group, women with lower occupational status had higher IL-6 at baseline, but again SES differences did not change in magnitude after the stressor. There also were no SES disparities for men (Steptoe, Owen, Kunz-Ebrecht, & MohamedAli, 2002). Other studies from this group have focused on coagulation processes involved in CHD progression. This work has yielded a similar pattern of findings. Participants with lower SES showed higher levels of coagulation markers such as fibrinogen (Steptoe et al., 2003a), leukocyte-platelet aggregate, and monocyte-platelet aggregate (Steptoe et al., 2003b) during resting-baseline periods. However, the SES disparities did not change in magnitude from the resting baseline to after the stressor. Finally, one study found quadratic effects of SES on inflammatory molecules linked to CHD. Adults in the intermediate SES group had the highest levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α and interleukin-1 receptor antagonist (IL-1ra) compared to those in the low or high SES groups at baseline. Again, these disparities did not diverge further during or after the stressor (Steptoe et al., 2002). Collectively, these findings suggest that SES differences primarily exist under basal conditions, supporting the exposure hypotheses (SES differences in immune markers evident across different conditions). These studies on the whole did not find evidence suggesting that among low SES individuals, there is a stronger relationship between exposure to stress and immune function compared to among high SES individuals (as the vulnerability hypothesis would suggest).

1

CANCER CONTROL

0.75 0.5 0.25 0 HIGH SCHOOL DIPLOMA

SOME COLLEGE

COLLEGE DEGREE

FIGURE 4 Evidence for vulnerability model: Among low SES individuals, high exposure to chronic stress (being a parent of a child with cancer) is associated with reduced sensitivity to the anti-inflammatory properties of dexamethasone compared to low exposure to chronic stress (being a parent of a healthy child). In contrast, among higher SES individuals, exposure to chronic stress is not related to sensitivity to dexamethasone.

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SES determines the efficacy of a person’s immune response to infectious challenge rather than (or perhaps in addition to) his/her response to stressful experience. The idea here is that SES influences the immune system’s capacity to eradicate invading pathogens, or modifies the nature and severity of clinical symptoms a person experiences once infected. This paradigm usually involves assessing healthy participants on a variety of SES variables and then exposing participants to a rhinovirus that causes upper respiratory infection. Participants are quarantined and monitored for signs of objective infection (viral shedding and antibody production) and clinical illness (mucus production and nasal congestion). Participants are considered to have a “cold” if they meet criteria for both infection and illness. One group of researchers has documented several SES-related findings using this paradigm. For example, adults who were unemployed (or underemployed) were three to four times more likely to develop a cold after rhinovirus exposure compared with adults who had sufficient work (Cohen et al., 1998). In another study, this group showed that SES during early periods of life has important consequences for disease susceptibility. Adults who had lower SES when they were children—as indicated by their parents not owning a home—were more likely to develop a cold following viral exposure (Cohen, Doyle, Turner, Alper, & Skoner, 2004). This was a dose-response association, such that the more years a family did not own a home, the more likely the participant was to develop a cold. Interestingly, owning a home later in life—either during adolescence or adulthood—did not attenuate these associations. This suggests that early childhood may be a critical period during which SES shapes the immune system in a fashion that cannot be “undone” by later improvements in a person’s social status. Finally, in addition to objective indicators of SES, these researchers also investigated the role of subjective indicators of status. Before exposure to the virus, participants were asked to rate where they stood in their own community relative to others. Adults who perceived themselves to be lower in social status were more likely to develop infection following exposure to a rhinovirus (Cohen, 1999).

I. Response to Vaccination Lastly, one study from another research group utilized a slightly different paradigm for examining SES differences in vulnerability to immune challenge. Rather than exposing participants to viruses that cause the common cold, these investigators tested the associations of SES with responses to vaccination for rubella

in adolescent girls. After vaccination, adolescents were classified as either infected with the rubella virus (being seronegative at the start of the study, then showing at least a four-fold increase in rubella antibody titers), or as showing no change in antibody titers (due to being seropositive at the start of the study). These researchers found that antibody response was associated with different behavioral effects, depending on the SES of the adolescents. Among low SES girls, those who showed an antibody response to vaccination reported more depression, attention problems, and delinquent behaviors 2–10 weeks after vaccination compared to those who did not respond to vaccination (Morag, Yirmiya, Lerer, & Morag, 1998). In contrast, among middle and high SES girls, there were no behavioral differences between those who did and did not show antibody response to vaccination. Research indicates that inflammation following pathogen exposure can produce a constellation of adjustments known as sickness behavior (Maier & Watkins, 1998; Yirmiya, 1996). These behaviors are thought to be similar to depression and can include negative mood, difficulty concentrating, and anhedonia. Thus, the findings from the above study suggest that low SES individuals may be particularly vulnerable to sickness behaviors that result from infectious challenges.

J. Potential Pathways between SES and the Immune System In addition to investigating associations of SES with immune markers, several of the above studies have also tested whether different mechanisms may account for relationships between SES and immune markers. These mechanisms include ones described earlier, such as stress and health behaviors, as well as other pathways such as social support and the endocrine system. Typically, these studies statistically control for possible mechanistic pathways, and compare the relationship of SES and immune markers prior to and after controlling for these variables. In their study on production of cytokines implicated in asthma, Chen et al. (2003) found that adolescents with asthma from low SES neighborhoods had heightened production of IL-5 and IFN-γ compared to adolescents with asthma from high SES neighborhoods. Stress was measured in this study both in terms of life stress exposure and perceptions of stress. When stress was included as a control variable, the relationship between SES and cytokine production decreased by 38–76%, and was no longer significant (Chen et al., 2003), suggesting that stress is a critical pathway linking SES and cytokine production in adolescents with asthma.


With respect to health behaviors, a number of studies have tested the effects of controlling for health behaviors. Some studies have found that controlling for health behaviors somewhat reduces the association between SES and immune markers. For example, controlling for smoking, alcohol, exercise, and diet reduced the relationship between low SES and high fibrinogen levels to non-significant in women (but not men) (Brunner et al., 1993). On the other hand, controlling only for smoking did not reduce the association between SES and fibrinogen to non-significant in another study (Steptoe et al., 2003a). This may be because the constellation of health behaviors as a whole best accounts for the SES-fibrinogen relationship, or because factors such as alcohol use or exercise are more important to the SES-fibrinogen relationship than smoking. In contrast, the association between SES and other inflammatory markers, including CRP, IL-6, TNF-α, fibrinogen, and homocysteine remained significant after controlling for smoking, diet, physical activity, and medication compliance (Panagiotakos et al., 2005). Similarly, in the viral cold studies described above, controlling for smoking, alcohol, exercise, sleep, and vitamin C consumption slightly reduced the magnitude of the relationship between unemployment and risk of cold; however, the risk of cold for those facing unemployment was still significant (Cohen et al., 1998). In addition, controlling for smoking, alcohol, exercise, and sleep did not diminish the relationship between perceived social status and likelihood of developing a cold (Cohen, 1999). Taken together, these findings suggest that as they have been assessed to date, there is not much evidence that health behaviors play a major role in relationships between SES and inflammatory processes. However future studies that conduct more thorough and objective assessments of factors such as sleep, exercise, and diet may reveal greater support for the role of health behaviors in SES-immune relationships. Other pathways that have been tested include social support and endocrine measures. No evidence has been found for either of these processes as pathways. For example, the relationship between lower income and higher heat shock protein 60 remained significant after controlling for social isolation (Lewthwaite et al., 2002). The relationship between unemployment and colds remained significant after controlling for the diversity of a person’s social network (Cohen et al., 1998). With respect to endocrine measures, the association between unemployment and colds, as well as between perceived social status and colds, remained significant after controlling for epinephrine and norepinephrine (Cohen et al., 1998; Cohen, 1999).

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Taken together, these findings suggest that additional research is needed to uncover the mechanisms by which SES exerts effects on the inflammatory processes. Perhaps the most promising mediator is stress; however, only one study has tested the role of stress on SES and immune markers (Chen et al., 2003). Thus far, health behaviors, social support, and endocrine measures do not have much evidence supporting their mediational role. Future studies should explore the role of stress in greater depth, as well as test other possible mediators psychosocially (e.g., trait variables such as locus of control) and biologically (e.g., cortisol).

VI. CONCLUSIONS Though it has received scant attention in the PNI literature thus far, SES is emerging as an important individual difference characteristic. Persons of low SES exhibit various indicators of immune dysregulation when they are at rest, including heightened expression of inflammatory and coagulation molecules involved in CHD, and greater production of cytokines that are responsible for the symptoms of asthma. Although low SES does not seem to render people especially vulnerable to immune dysregulation following acute lab stress, there is preliminary evidence that it amplifies the biological consequences of severe and chronic stressors in real-life settings. Perhaps most compelling are the influences of SES following infectious challenge; persons of lower status exhibit heightened susceptibility to respiratory infection and sickness behavior. The mechanisms underlying these findings have not yet been elucidated. However, with further efforts in this exciting new area of PNI research, we will soon have a better understanding of how social context “gets under the skin” to influence well-being and health outcomes in such a profound fashion.

Acknowledgments Support for this chapter was provided by National Institutes of Health grant HL073975, the Canadian Institutes of Health Research, and the Michael Smith Foundation for Health Research.

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24 Psychoneuroimmunology of Depressive Disorder: Mechanisms and Clinical Implications LUCILE CAPURON, ANDREW MILLER, AND MICHAEL R. IRWIN

I. INTRODUCTION 509 II. IMMUNOLOGICAL ALTERATIONS IN DEPRESSION 509 III. CLINICAL MODERATING FACTORS OF IMMUNE ALTERATIONS IN DEPRESSION 512 IV. BIOLOGICAL MEDIATORS OF IMMUNE ALTERATIONS IN DEPRESSION 515 V. BEHAVIORAL MECHANISMS OF IMMUNE ALTERATIONS IN DEPRESSION 516 VI. FROM CYTOKINES TO DEPRESSION 518 VII. PATHWAYS LINKING IMMUNITY TO DEPRESSION 520 VIII. CYTOKINE ABNORMALITIES IN DEPRESSION: TREATMENT IMPLICATIONS 523 IX. CONCLUSION 524

and innate immune responses that are associated with infectious disease susceptibility (Cohen and Miller, 2000; Leserman 2003), whereas other studies have found that depression is linked to immune activation in patients with inflammatory disorders such as rheumatoid arthritis (Zautra et al., 2004), cardiovascular disease (Lesperance et al., 2004; Miller et al., 2002c), or undergoing cytokine therapy (Capuron and Miller, 2004). In this chapter, we examine evidence linking major depressive disorder to reductions of innate and acquired immunity, as well as the associations between depression and alterations in the capacity of immune cell to express pro-inflammatory cytokines. Finally, we consider the hypothesis that the cytokine abnormalities found in depressed patients may have reciprocal influences on the central nervous system and contribute in part to the pathophysiology of the disorder.

I. INTRODUCTION Major depressive disorder, which exceeds a lifetime incidence of 10% (Irwin, 2002; Michaud et al., 2001), is a potent risk factor for disease morbidity, with depressed persons showing a mortality rate twice that found in non-depressed persons (Cuijpers and Smit, 2002; Penninx et al., 1999; Rudisch and Nemeroff, 2003; Wulsin et al., 1999). Altered functioning of the immune system is implicated as a mechanism that might contribute to medical morbidity of major depressive disorder, including risk of infectious disease (Evans et al., 2002) as well as inflammatory disorders (Zautra et al., 2004). Depressed persons show reductions of cellular PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

II. IMMUNOLOGICAL ALTERATIONS IN DEPRESSION Many immunological changes reliably occur in patients with major depressive disorder, as described in a comprehensive meta-analyses of over 180 studies with more than 40 immune measures (Zorrilla et al., 2001). This section provides a review of the psychoneuroimmunological research being conducted on the relationship between depression and immunity, with a detailed review of various immune findings that

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occur in major depression. In addition, the behavioral correlates and biological mechanisms that might contribute to immune changes in major depression are discussed.

A. Enumerative Measures Evidence for increases in the total number of white blood cells and in the numbers and percentages of neutrophils and lymphocytes was among the first immunological changes identified in depressed persons (Herbert and Cohen, 1993; Kronfol et al., 1984; Zorrilla et al., 2001). In contrast, changes in monocyte counts have not been consistently reported (Irwin et al., 1990c; Maes et al., 1992). Further evaluation of lymphocyte numbers in depression has used phenotype-specific cell surface markers to enumerate lymphocyte subsets, and found that depression is negatively related to the number and percentage of lymphocytes (B-cells, Tcells, T helper cells, and T suppressor/cytotoxic cells) (Herbert and Cohen, 1993). In addition, a decrease in the circulating number of cells that express the NK cell phenotype occurs, which appears to be moderated in part by gender, with a decline in NK cell numbers found in male but not female depressed subjects compared with gender-matched controls (Evans et al., 1992). However, in one of the largest study samples of depressed subjects, no difference in the number of peripheral blood lymphocytes or T-lymphocyte subsets was found between depressed patients and controls (Schleifer et al., 1989). Indeed, with the recent accumulation of studies and the results of comprehensive meta-analyses, it is questionable as to whether there are consistent changes in the number of circulating B-, T-, or NK cells in depression (Zorrilla et al., 2001).

B. Functional Measures For the evaluation of the function of the immune system in depressed patients, a majority of studies have relied on results from assays of non-specific mitogen-induced lymphocyte proliferation, mitogenstimulated cytokine production, and NK cytotoxicity. Among the first few studies, it was found that mitogen proliferation responses were reduced in depressed subjects compared with controls (Kronfol et al., 1983; Schleifer et al., 1984). Whereas subsequent studies failed to replicate these observations, raising questions about the reliability of this immune alteration in depression (Schleifer et al., 1989), more than a dozen studies have now been conducted on lymphocyte proliferation in depression, and a reliable association between depression and lower proliferative responses to the three non-specific mitogens including phytohem-

aglutinin (PHA), concanavalin-A (Con A), and pokeweed (PWM) has been found (Herbert and Cohen, 1993; Zorrilla et al., 2001). Attention has also been given to the assessment of natural killer (NK) cell activity in depressed patients. Following the initial observations by Irwin and colleagues (Irwin and Gillin, 1987), over a dozen subsequent independent samples have replicated the finding of reduced NK activity in major depression (Herbert and Cohen, 1993; Zorrilla et al., 2001). Nevertheless, several caveats must be considered when reviewing the associations between NK activity, NK numbers, and depression. Compared with controls, decreases in NK counts and NK activity have been primarily found in male but not female depressed subjects (Evans et al., 1992; Irwin, 2002), and other studies have found no difference of NK activity in depression (Schleifer et al., 1989). Additional factors that might moderate or mediate the effects of depression on NK activity are discussed later in this chapter. Importantly, the declines of NK activity in depression may be relevant for certain infectious disease outcomes. Evans and colleagues found that major depression in women with HIV infection was associated with lower NK activity, as well as increases in the numbers of activated CD8 lymphocytes and viral load, and suggested that declines of killer lymphocytes in association with depression may increase risk of HIV disease progression in women (Evans et al., 2002).

C. Stimulated Cytokine Production Studies of stimulated cytokine production have not yielded consistent findings. For example, in whole blood assays, Kronfol et al. (2003) found increased lipopolysaccharide-stimulated production of IL-1 and IL-6, but no change in the expression of tumor necrosis factor-α. Other studies have suggested a shift in the relative balance of T helper 1 versus T helper 2 cytokine production with increases in the capacity of lymphocytes to produce interferon in depression (Seidel et al., 1995), yet no difference in the stimulated production of IL-2 has been found (Irwin et al., 2003a; Seidel et al., 1995). These negative findings cannot be ascribed to differences in depressed samples, as depressed patients who show no difference in IL-2 production evidence declines of NK activity (Irwin et al., 2003a). Recent attention has focused on evaluating different patterns of cytokine activation in subtypes of depression. Whereas one study found no differences in the capacity of lymphocytes to produce IL-2 between melancholic and non-melancholic depression (Schlatter et al., 2004), another study suggested

24. Psychoneuroimmunology of Depressive Disorder: Mechanisms and Clinical Implications

that peripheral blood mononuclear cells of nonmelancholic depressed patients showed a greater stimulated capacity to produce interleukin-1β and interleukin-1 receptor antagonist as compared to responses from controls and melancholic depressed patients (Kaestner et al., 2005), although earlier work by this group of investigators did not identify such increases in IL-1 production (Rothermundt et al., 2001). Nevertheless, further observations suggest that the melancholic but not non-melancholic depressed patients showed evidence of HPA axis, which is thought to inhibit immune activation and the expression of inflammatory markers; this might account for the reported differences in these two groups of diagnostic depression (Kaestner et al., 2005).

D. Inflammation and Circulating Levels of Inflammatory Markers The presence of immune activation in major depression has also been evaluated by examining circulating levels of inflammatory markers. In contrast to the inconsistent findings regarding association between depression and production of inflammatory cytokines, meta-analyses indicate that depression is associated with increases in circulating levels of the proinflammatory cytokine, interleukin-6 (IL-6) (Zorrilla et al., 2001). Importantly, as compared to controls, elevated levels of IL-6 have been found in adults with major depression (Frommberger et al., 1997; Irwin and Pike, In press; Motivala et al., 2005), as well as in depressed elderly populations (Penninx et al., 2003) and in those with chronic medical disorders such as rheumatoid arthritis (Zautra et al., 2004), cancer (Musselman et al., 2001b), and cardiovascular disease (Empana et al., 2005). It is hypothesized that increases in circulating levels of pro-inflammatory cytokine are due to activation of monocyte populations, and increases in circulating levels of other pro-inflammatory cytokines such as tumor necrosis factor-α (TNF) and interleukin-1β (IL-1) have been reported in depressed patients (Anisman et al., 1999; Maes et al., 1993), including late-life depressive disorder (Thomas et al., 2005), although the numbers of studies that have examined these additional cytokines are too few to make firm conclusions. One study also reported increases of plasma levels of IL-12 in a large cohort of depressed patients (Kim et al., 2002); IL-12 is a hertodimeric cytokine that is produced primarily by monocytes and macrophages and plays a central role in the early phases of inflammation. Given evidence that depression is associated with immune activation with increases primarily of monocyte-derived cytokines, additional studies have

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extended these observations and assayed markers of systemic inflammation such as acute phase proteins and/or levels of sIL-2r. Although early reports suggested increases in haptoglobin and other acute phase proteins in depressed patients (Zorrilla et al., 2001), these data were primarily limited to reports from one laboratory, and recent efforts have failed to identify abnormal increases in acute phase proteins (Irwin and Pike, In press) consistent with the preliminary conclusions from meta-analyses (Zorrilla et al., 2001). Nevertheless, increases in c-reactive protein have been found in association with depression, with elevated levels in healthy-depressed adults (Miller et al., 2002c), as well as in those depressed patients with acute coronary syndrome (Lesperance et al., 2004; Miller et al., 2005). In turn, systemic immune activation is thought to lead to endothelial activation in depression with increases in the expression of soluble intercellular adhesion molecule (sICAM) (Lesperance et al., 2004; Motivala et al., 2005); elevated levels of c-reactive protein and sICAM are associated with cardiovascular disease and are prospectively linked to myocardial infarction risk (Ridker, 2001).

E. Dissociation between Declines of Innate Immunity and Inflammatory Markers As reviewed above, a number of studies have evaluated different cellular immunity and inflammation of immunity in depression, yet little attention has been given to the potential relationship between measures of innate immunity, such as NK activity, and levels of inflammatory markers in the context of major depression (Raison and Miller, 2003; Segerstrom and Miller, 2004). Rather, these immune differences have been generated in separate samples of depressed patients. In a recent study, Irwin and Pike measured levels of NK activity, circulating levels of interleukin-6 (IL-6), soluble interleukin-2 receptor (sIL-2r), and acute phase proteins in patients with current major depressive disorder (Irwin and Pike, In press). Consistent with prior observations, patients with major depressive disorder showed lower NK activity and higher circulating levels of IL-6 as compared to age-, gender-, and body-weight– matched controls, yet levels of NK activity were not correlated with IL-6 or with other markers of immune activation including acute phase proteins or IL-2r. The independent effect of depression on inflammatory markers as compared to NK responses has implications for understanding individual differences in the adverse health effects of major depressive disorder. Altered functioning of the immune system is thought to contribute to medical morbidity of major depressive

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disorder, including risk of infectious disease (Evans et al., 2002) as well as inflammatory disorders (Zautra et al., 2004). Some depressed persons show reductions of cellular and innate immune responses that are associated with infectious disease susceptibility (Cohen and Miller, 2000; Leserman, 2003). As noted above, other studies report that depression is linked to immune activation in patients with inflammatory disorders such as rheumatoid arthritis (Zautra et al., 2004) and cardiovascular disease (Lesperance et al., 2004; Miller et al., 2002c). It is not known what mechanisms might account for increases of IL-6 levels in some depressed patients and for decreases of NK activity in other patients with depression. Raison and Miller (2003) have proposed that depressed patients show inadequate glucocorticoid-mediated feedback inhibition of immune activation (Raison and Miller, 2003); for example, increased concentrations of plasma IL-6 positively correlate with post-dexamethasome suppression test (DST) cortisol levels in depression (Lowy et al., 1988). In contrast, several studies of patients with major depression have failed to show a relationship between altered NK activity and plasma or urinary concentrations of free cortisol (Irwin et al., 1988; Kronfol et al., 1986; Miller et al., 1991). Together, these data suggest that glucocorticoid resistance in depression is associated with immune activation (Miller et al., 2002b; Raison and Miller, 2003), yet not with declines of NK activity. Hence, it is speculated that insufficiency in glucocorticoid signaling of inflammation might identify a group of depressed patients at risk for exacerbations of inflammatory disorders such as rheumatoid arthritis (Raison and Miller, 2003). Genetic and metabolic variation in the expression of pro-inflammatory cytokines may also operate independently of processes that regulate NK activity. Whereas chronic psychological stress is found to induce increases in the expression of the proinflammatory cytokines (Kiecolt-Glaser et al., 2003) as well as decreases of NK activity (Pike, 1996), stressinduced increases of plasma C-reactive protein are reported to occur only in stressed persons who have the A allele of tumor necrosis factor-α −308 G/A polymorphism (Jeanmonod et al., 2004). Likewise, polymorphism of the 174 bp upstream of the transcription initiation site of the IL-6 gene, the −174 G/C allele, correlates with plasma IL-6 levels (Fishman et al., 1998), although the relationship of this polymorphism with major depression or with NK immune responses is not known. Finally, one-third of total IL-6 in the circulation is estimated to originate from adipose tissue (Mohamed-Ali et al., 1998). Even in depressed patients and controls who are similar in body weight and/or adiposity, metabolic alterations in adipose tissue sig-

naling might contribute to increases of IL-6 in depression independent of immune cell production of this pro-inflammatory cytokine.

F. Viral-specific Immune Measures Extension of these non-specific measures of immunity to viral-specific immune response has begun to yield promising findings relevant to the increased risk of infectious disease in depression and psychological stress. For example, major depression is associated with a functional decline in memory T-cells that respond to varicella zoster virus (Irwin et al., 1998), and this immune response is thought to be a surrogate marker for herpes zoster risk (Oxman et al., 2005). Such findings are consistent with a broader literature which has demonstrated that psychological stress is associated with decline in specific immune responses to immunization against viral infections (Miller et al., 2004; Vedhara et al., 1999), although extension of this work to major depression has not yet been conducted.

G. Assays of in vivo Responses Basic observations in animals have raised the possibility that depression can alter in vivo immune responses, as administration of chronic stress in animals suppresses the delayed type hypersensitivity (DTH) response (Dhabhar, 2000). Indeed, in depressed patients, suppression of the DTH response to a panel of antigenic challenges has been found (Hickie et al., 1993). In contrast, Shinkawa et al. (2002) found that older depressed patients were more likely to show positive tuberculin responses than non-depressed patients. To our knowledge, no study has investigated whether depression alters immunological response to vaccination, although several studies have revealed that psychological stress is associated with declines of hepatitis B antibody responses as well as antibody response to influenza immunization (Glaser et al., 1992; Miller et al., 2004; Vedhara et al., 1999).

III. CLINICAL MODERATING FACTORS OF IMMUNE ALTERATIONS IN DEPRESSION A. Demographic and Clinical Factors Alterations in the immune system during major depressive disorder may be influenced by the severity of depressive symptoms and other factors such as age, gender, ethnicity, body mass, and hospitalization stress (Stein et al., 1991) (see Figure 1). Whereas many


Depression

- Age - Gender - Ethnicity - Body mass

- Sleep disturbance - Physical activity - Tobacco smoking - Alcohol abuse

Corticotropinreleasing hormone

Adrenocortiocotropic hormone

Adrenal g land

Cortex

Autonomic innervation

Noradrenaline

Lymphoid organs

Medulla Immune cell activity Cortisol NK cell Adrenaline

Th2

Th1

Cytokines IFN IL-10 IL-6, TNF-α, IL-1

Monocyte Inhibitory

APC

Stimulatory Increase Decrease

FIGURE 1 Model of depression-associated modulation of immune cell activity. The effects of depression on immune cell activity are moderated by clinical variables such as age, gender, ethnicity, and body mass. Behavioral mechanisms such as sleep disturbance, physical inactivity, and substance dependence co-occur in depression, have independent effects on immune cell function, and alter the magnitude of depression-related immune changes. Depression results in the central release of corticotrophin-releasing hormone that activates the hypothalamic-pituitaryadrenal (HPA) axis and the sympathetic-adrenal-medullary axis. The production of adrenocorticotropic hormone by the pituitary gland results in the production of glucocorticoid hormones. Autonomic activation leads to the production of adrenal medulla adrenaline, and the sympathetic neural release of noradrenaline in lymphoid organs. Immune cells have receptors for cortisol and catecholamines; receptor binding of cortisol, adrenaline, or noradrenaline induces a downregulation of natural killer (NK) cell function; a shift in the stimulated production of cytokines with decreases of the Th1 cytokine interferon γ (IFN) and increases of the Th2 cytokine interleukin-10 (IL-10); and increases in the production of pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF), and interleukin-1 (IL-1). These cytokines can also have bi-directional effects and can modulate the activity of the hypothalamus and also induce behavioral symptoms.

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studies have controlled for the influence of these factors in the selection of controls who are comparable to the depressed patients on demographic and clinical characteristics, some evidence suggests that these factors may interact with depression status in altering immune variables. For example, older adults show declines in cellular immunity; the presence of comorbid depression may further exacerbate age-related immune alterations (Schleifer et al., 1989). Gender also exerts differential effects on pituitary, adrenal, and immune systems by modulating the sensitivity of target tissues, and one study, for example, showed that women have an exaggerated expression of pro-inflammatory cytokines in response to psychological stress (Rohleder et al., 2001), which is thought to place women at increased risk for autoimmune disorders. Other data indicate that declines of T-cell and NK cell response appear to be more prominent in depressed men than depressed women (Evans et al., 1992). In regards to ethnicity, African American ethnicity interacts with a history of alcohol consumption to exacerbate immune abnormalities (Irwin and Miller, 2000), but the influence of ethnicity on depression-related changes of immunity is not known. Increases in body mass index and the presence of obesity are associated with increases in markers of inflammation such as circulating levels of c-reactive protein and IL-6 (Yudkin et al., 2000). In healthy adult depressed patients, Miller and colleagues showed that adiposity and great body mass partially mediated the increase of these inflammatory markers (Miller et al., 2002c). However, other studies have found increases of IL-6 and of c-reactive protein in healthy non-obese depressed persons that is independent of the contribution of body mass (Lesperance et al., 2004; Motivala et al., 2005), suggesting that the effects of adiposity on depression-related increases of inflammatory markers may only occur above a threshold or in those with obesity. Psychological stress may also impact measures of cellular immunity (Kiecolt-Glaser et al., 2002), and depressed patients show elevated rates of life stress and adverse events as compared to nondepressed persons (Kendler et al., 2000). Indeed, depressed patients who evidence greater life stress show greater declines of NK activity than depressed patients who are not stressed, although depression alone is associated with significant declines of NK activity (Irwin et al., 1990c). Finally, specific diagnostic comorbidity such as anxiety disorder (Andreoli et al., 1992) and sleep disturbance (Motivala et al., 2005) might moderate immune system functioning in depression. As discussed below, a number of studies have identified sleep in the regulation of some aspects of immune function (Irwin et al., 1992b; Irwin et al., 1994; Irwin et al., 1996), and depressed individuals with

somatic symptoms of depression such as insomnia are more likely to show immune changes (Cover and Irwin, 1994), which for markers of inflammation may be mediated by increases in the latency for sleep onset and rapid eye movement (REM) sleep (Motivala et al., 2005).

B. Depression Treatment: Effects of Antidepressant Medications Only a limited number of studies have investigated the clinical course of depression and changes of cellular immunity in relation to antidepressant medication treatment and symptom resolution. In one longitudinal case-control study, Irwin and co-workers (1992a) found that depressed patients showed an increase in NK activity during a 6-month course of tricyclic antidepressant medication treatment and symptom resolution, although the improvements of NK activity were correlated declines of symptom severity and not treatment status at the time of follow-up. In another longitudinal follow-up study (Schleifer et al., 1999) of young adults with unipolar depression involving 6 weeks of treatment with nortriptyline and alprazolam, clinical improvements in the severity of depressive symptoms were associated with decreased numbers of circulating lymphocytes and decreased responses to PHA and Con A but not PWM. In addition, decreases in T-cells, CD4+, and CD29 were found although there were no changes in B-cell numbers or CD8+ cells. None of these changes were related to nortriptyline blood levels (Schleifer et al., 1999). In addition, Frank et al. (1999) found that in vivo and in vitro treatment with fluoxetine, a selective serotonin reuptake inhibitor, resulted in enhanced NK activity, along with changes in depressive symptoms, consistent with the finding of Ravidran et al. (1995) in which a number of different antidepressants were used, including nafazodone, paroxetine, sertraline, and venlafaxine. In regards to pro-inflammatory cytokine expression and inflammation, Castanon et al. (2002) recently reviewed animal research that focused on the immunological effects of antidepressant medications and concluded that antidepressants decrease proinflammatory cytokine (e.g., IL-β, TNF, and IL-6) expression, with similar findings generated using in vitro and in vivo experimental approaches. Although the specific mechanism or mechanisms involved have not yet been identified, antidepressants modify the expression of glucocorticoids and their receptors (Barden ,1999; Goujon et al., 1995); limit the synthesis of prostaglandin and nitric oxide, which contribute to


pro-inflammatory effects (Yaron et al., 1999; Yirmiya et al., 1999); and act on intracellular messenger pathways with downstream effects on pro-inflammatory cytokines (Hindmarch, 2001). A few studies have extended these pre-clinical data and examined the effects of antidepressants on pro-inflammatory cytokine levels and productin in depressed patients. Increased levels of IL-6 during acute depression were normalized after 8 weeks of fluoxetine treatment (Sluzewska et al., 1995). Similarly, production of IL-6 and TNF was decreased in depressed patients treated with amitriptyline, although this decrease was confined to those depressed patients who responded to treatment, suggesting that symptom resolution is an important predictor of cytokine changes (Sluzewska et al., 1995). In contrast, 4-week treatment with clomipramine was found to be associated with increases in the mononuclear cell production of IL-1 in depressed patients (Weizman et al., 1994). A few studies have also examined the effects of antidepressant treatment on Th1 versus Th2 cytokine production in depression. Treatments in vivo and in vitro with imipramine, venlafaxine, or fluoxetine increased stimulated cellular production of IL-10, with a decrease in the ratio of interferon (IFN) to IL-10 (Kubera et al., 1996). In hospitalized psychiatric patients (with either schizophrenia, depression, or bipolar disorder), one study found that treatment with psychotropic medications reduced IL-12 expression as compared to controls (Kim et al., 2002), consistent with the view held by Kenis and Maes (2002) that antidepressants decrease pro-inflammatory cytokine expression and induce a shift toward Th2 cytokine expression.

IV. BIOLOGICAL MEDIATORS OF IMMUNE ALTERATIONS IN DEPRESSION A. Central Modulation of Immunity: Effects of Corticotropinreleasing Hormone Depressed patients show elevated levels of corticotropin-releasing hormone (CRH) (Owens and Nemeroff, 1991), and this key peptide is involved in integrating neural neuroendocrine as well as immune responses to stress. Release of this peptide in the brain alters a variety of immune processes including aspects of innate immunity, cellular immunity, and in vivo measures of antibody production (Friedman and Irwin, 1995; Friedman and Irwin, 1997). Peripheral immune measures also change following lesioning of the brain (e.g., hypothalamus) or in response to the stimulation

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of certain brain regions which ultimately impact CRH systems. The brain controls immune cells in lymphoid tissue in the same manner it controls other visceral organs, namely by coordinating autonomic and neuroendocrine pathways; when these pathways are blocked by specific factors that bind to sympathetic or hormone receptors, the effects of CRH on immune function are also blocked (Irwin et al., 1990b; Irwin et al., 1990d).

B. Autonomic Nervous System (ANS) At rest and in response to acute physical and/or psychological challenge, depressed patients show elevated levels of circulating catecholamines and neuropeptide Y (Irwin et al., 1991), consistent with the notion that depression is associated with activation of the peripheral sympathetic nervous system (SNS). The SNS regulates multiple aspects of the immune system and is thought to mediate changes of immunity in depression. It is known, for example, that sympathetic nerve terminals are juxtaposed with immune cells in organs where the immune system cells develop and respond to pathogens (e.g., bone marrow, thymus, spleen, and lymph nodes) (Friedman and Irwin, 1997; Sanders and Straub, 2002). When sympathetic release of norepinephrine and neuropeptide Y occurs, receptor binding serves as a signal in this “hard-wire” connection between the brain and the immune system. In addition, sympathetic nerves penetrate into the adrenal gland and cause the release of epinephrine into the bloodstream, which circulates to immune cells as another sympathetic regulatory signal. In depressed patients, excretion of 3-methoxy-4-hydroxy-phenylglycol (MHPG), a metabolic breakdown product of catecholamines, has been used as an index of total body noradrenergic turnover, or sympathetic activity, and MHPG excretion was inversely related with lymphocyte proliferative responses in depressed patients (Maes et al., 1989). Under both laboratory and naturalistic conditions including depression, sympathetic activation has been shown to suppress the activity of diverse populations of immune cells, including NK cells and T lymphocytes (Friedman and Irwin, 1997; Sanders and Straub, 2002). In contrast, other aspects of the immune response can be enhanced. For example, catecholamines can increase the production of antibodies by B-cells and the ability of macrophages to release cytokines and thereby signal the presence of a pathogen. Additional studies indicate that sympathetic activation can also shunt some immune system cells out of circulating blood and into the lymphoid organs (e.g., spleen, lymph nodes, thymus) while recruiting other types of

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immune cells into circulation (e.g., NK cells). It is thought that sympathetic activation reduces the immune system’s ability to destroy pathogens that live inside cells (e.g., viruses) via decreases of the cellular immune response, while enhancing the humoral immune response to pathogens that live outside cells (e.g., bacteria) (Sanders and Straub, 2002).

C. Neuroendocrine Axis A hallmark of major depression is dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and the overexpression of cortisol. Cortisol exerts diverse effects on a wide variety of physiological systems and also coordinates the actions of various cells involved in an immune response by altering the production of cytokines or immune messengers (Moynihan and Stevens, 2001). Similar to sympathetic activation, cortisol can suppress the cellular immune response critical to defending the body against viral infections. Cortisol can also prompt some immune cells to move out from circulating blood into lymphoid organs or peripheral tissues such as the skin (Dhabhar et al., 1996). The relationship between HPA axis activation and immune alterations in depression has not been compellingly demonstrated. For example, in depressed patients, decreased lymphocyte responses to mitogens are not associated with dexamethasone nonsuppression (Kronfol and House, 1985) or with increased excretion rates of urinary-free cortisol (Kronfol et al., 1986). Dexamethasone non-suppression refers to the diminished feedback inhibition of the HPA axis and is associated with HPA overactivity. Furthermore, in bereavement which is complicated by depressive symptoms, changes of NK activity are not associated with plasma cortisol levels (Irwin et al., 1988). Nevertheless, disruption of one’s physiological homeostasis is implicated in depression, and neuroendocrine-immune interactions are thought to contribute to this dysregulation. As noted above, major depression is associated with the increased expression of pro-inflammatory cytokines, which in turn leads to the progression of the immune response and activation of the HPA axis (Besedovsky and del Rey, 1996; McEwen et al., 1997; Schobitz et al., 1994; Turnbull and Rivier, 1999). In a study of 28 inpatients with major depression, Maes and colleagues (Maes et al., 1993) found a significant positive correlation between mitogen-induced IL-1β production and postdexamethasone cortisol values, which suggested to the authors that IL-1β oversecretion may contribute to HPA axis overactivity in depression.

V. BEHAVIORAL MECHANISMS OF IMMUNE ALTERATIONS IN DEPRESSION In addition to the biological mediators of immune changes in depression, several behavioral factors associated with depression might contribute to immune dysfunction. Indeed, as reviewed by Cohen and Miller (Miller et al., 1999b), examination of health status and behavioral factors is needed in clinical psychoneuroimmunology. For example, alcohol and tobacco have well-recognized effects on immunity. Yet, there is limited empirical information on the processes by which these substances alter immune function in depressed subjects, despite their high rates of use in depressed patients. In the following sections, four of the more pertinent behavioral factors linking depression and immune dysfunction are discussed, namely, tobacco smoking, alcohol/substance abuse, activity/ exercise, and sleep disturbance.

A. Smoking: Prevalence and Immune Effects Cigarette smoking has long been considered a health risk, with effects on immunity via direct actions or possibly endocrine-mediated mechanisms (Sopori and Kozak, 1998). Nicotine is reported to affect the HPA axis (Rosecrans and Karin, 1998) and to alter measures of humoral and cellular immunity (McAllister-Sistilli et al., 1998) and markers of immune activation (Mendall et al., 1997). For example, specific immunological alterations occur in adult smokers affecting a variety of parameters, including increases of WBC counts, declines of NK activity, and increases of markers of immune activation such as IL-6. Hence, several studies of depressed patients have begun to evaluate the contribution of cigarette smoking to depression-related declines of NK activity and increases of IL-6 and other inflammatory markers (Andreoli et al., 1993; Irwin et al., 1990b; Lesperance et al., 2004; Miller et al., 2002c; Motivala et al., 2005). In a large study of 245 depressed and comparison controls stratified by smoking status, Jung and Irwin (1999) reported that depression and smoking status interact to produce greater declines of NK activity than those found in depressed or smoking groups alone. In addition, smoking status was associated with increases of IL-6 and sICAM in depression (Lesperance et al., 2004; Miller et al., 2002c; Motivala et al., 2005), although evidence of immune activation occurs in depression independent of smoking status (Irwin and Pike, In press; Lesperance et al., 2004; Miller et al., 2002c; Motivala et al., 2005). Nevertheless, epidemiologic evidence suggests that depression interacts


with cigarette smoking to impact health, rather than the presence of a unitary link between depression and cancer. In a 12-year follow-up of 2,264 adult men and women, depressed mood together with cigarette smoking were associated with a marked increase in the relative risk of cancer (Linkins and Comstock, 1990), as compared to the risk associated with smoking or depression status alone.

B. Alcohol Dependence: Prevalence and Immune Effects in Depression According to Cadoret (Cadoret, 1981), nearly 30% of individuals with depression also suffer from alcohol dependence, and over 30% of depressives escalate their drinking during the depressive episode. Whereas alcohol/substance dependence and depression each have a significant negative impact on the immune system, the interaction of alcohol/substance abuse with affective disorders may result in significantly more immune impairment than either condition alone. In a study by Irwin et al. (1990a), individuals with a dual diagnosis of alcoholism and depression had further decreases of NK activity compared with individuals diagnosed with only alcoholism or depression. Furthermore, these researchers found that depressed subjects with histories of alcoholism had lower NK activity compared with depressed subjects without such histories. Alcoholics with secondary depression showed a further decrease in cytotoxicity compared with alcoholics who were not clinically depressed (Irwin et al., 1990a). Strikingly, this result reflects the effects of past consumption of alcohol. Depressed and alcoholic subjects were free of alcohol for a minimum of 2 weeks, and thus the decline of NK activity was not due to a direct pharmacological effect of alcohol. More systematic assessment of current alcohol use along with dependence histories is needed.

C. Activity and Exercise: Immune Consequences in Depression Activity, or a lack thereof, can have negative consequences on the immune system, and some data suggest that older adults with depression may be especially vulnerable to the harmful effects of sedentary lifestyles. Conversely, exercise has been shown to have potent salutary effects on immune measures and has even been found to promote a remission of depressive symptoms in older adults. In the meta-analysis from Herbert and Cohen (Herbert and Cohen, 1993), melancholic depression correlated with greater impairments

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of cellular immunity, which may be due, at least in part, to an increased predominance of neurovegetative symptoms. Cover and Irwin (1994) found that severity of psychomotor retardation uniquely predicted declines of NK activity, similar to the effects of insomnia. Given that behavioral interventions that include a component of physical activity have been found to boost antibody response to a novel antigen (Smith et al., 2004), antibody production in response to influenza vaccination (Kohut et al., 2005), and viral-specific T-cell memory response to the varicella zoster virus (Irwin et al., 2003b), future studies are needed to determine whether interventions that increase physical activity and/or exercise capacity will yield improvements in immunity in depression. The administration of aerobic exercise has been found to alleviate depressive symptoms and to decrease the risk of relapse in major depressive disorder (Babyak et al., 2000; Blumenthal et al., 2005).

D. Disordered Sleep and Immunity: Relevance to Depression Disordered sleep and loss of sleep are thought to adversely affect resistance to infectious disease, increase cancer risk, and alter inflammatory disease progression. Animal studies show that sleep deprivation impairs influenza viral clearance and increases rates of bacteremia. In humans, normal sleep is associated with a redistribution of circulating lymphocyte subsets, increases of NK activity, increases of certain cytokines (e.g., IL-2, IL-6), and a relative shift toward Th1 cytokine expression that is independent of circadian processes. Conversely, sleep deprivation suppresses NK activity and IL-2 production, although prolonged sleep loss has been found to enhance measures of innate immunity and pro-inflammatory cytokine expression. Insomnia is one of the most common complaints of depressed subjects, but its role in moderating and/or mediating immune alterations in depression has been relatively unexplored. However, with evidence that subjective insomnia correlates with NK activity in depression, but not with other depressive symptoms, including somatization, weight loss, cognitive disturbance, or diurnal variation, the hypothesis has emerged that disordered sleep may be a distinct factor accounting for some of the observed immune alterations found in depression. In depressed samples who are at risk for disordered sleep, alterations of natural and cellular immune function among depressed patients correlate with disturbances of EEG sleep (Cover and Irwin, 1994; Irwin

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et al., 1992b). Recent studies in bereaved subjects have replicated this correlation, and studies have shown by way of causal statistical analyses that disordered sleep also mediates the relationship between severe life stress and a decline of NK responses (Hall et al., 1998). Further studies involving subjects with primary insomnia (e.g., no depression, other psychiatric disorder, and medical disorder) have found that prolonged sleep latency and fragmentation of sleep are associated with nocturnal elevations of sympathetic catecholamines and declines in daytime levels of NK cell responses (Irwin et al., 2003a; Savard et al., 2003). A more extensive line of research has focused on alcohol-dependent patient populations who have profound disturbances of sleep continuity and sleep architecture (Irwin et al., 2002; Irwin et al., 2000); decreases of total sleep time, declines of delta sleep, and increases of REM are associated with increases in the nocturnal and daytime expression of IL-6, possibly with consequences for daytime fatigue (Redwine et al., 2003). Finally, studies of depressed patients have found the prolonged sleep latency and increases of REM density are associated with elevated levels of IL-6 and sICAM in depression, and these EEG sleep measures fully account for the association between depression and IL-6 in healthy adults (Motivala et al., 2005). Findings linking sleep and IL-6 and sICAM may have important health implications. Recent epidemiological data show that self-reported difficulty initiating sleep is a predictor of cardiovascular disease mortality (Mallon et al., 2002). Similarly, objective measures of difficulty initiating sleep (i.e., prolonged sleep latency) yields a two-fold elevated risk of death in a healthy older adult population (Dew et al., 2003). Prospective data further indicate that sICAM and IL-6, directly implicated in atherosclerotic disease processes, predict risk for myocardial infarction independent of cholesterol levels, smoking status, and obesity (Ridker et al., 1998; Ridker et al., 2000). Likewise, in patients recovering from an acute coronary artery event, comorbidity for depression is associated with elevated levels of sICAM independent of smoking status, obesity, and other traditional risk markers (Lesperance et al., 2004). Thus, further examination of disordered sleep along with inflammation and endothelial activation may prove to be a key biobehavioral mechanism contributing to excess cardiovascular mortality in persons with depression.

VI. FROM CYTOKINES TO DEPRESSION There are many reasons to believe that the immunologic changes found in depressed patients described

above may in part contribute to the pathophysiology of the disorder. First, medically ill patients, who exhibit evidence of immune activation and/or inflammation secondary to tissue damage and destruction, infection, autoimmunity, or neoplastic disease, exhibit high rates of depression. Second, cytokine therapies for infectious diseases and cancer are notorious for causing behavioral alterations. Finally, there are many pathways known to be involved in the pathophysiology of depression that are influenced by cytokines, including neuroendocrine function, neurotransmitter function, and information processing.

A. Depression in the Medically Ill Major depression is frequent in the medically ill. Whereas the prevalence rate of depression in the general population ranges from 2–5%, patients with medical illnesses exhibit rates up to 50% and higher, depending on the type and severity of disease (Evans et al., 1999). Relevant illnesses include neurologic disorders (e.g., Parkinson’s disease), cancer, infectious diseases, autoimmune disorders, and cardiovascular disease (Evans et al., 1999). While there has been substantial focus on the psychosocial factors that may contribute to depression in medically ill patients, new developments in the biology of mood disorders have raised novel considerations regarding the pathophysiology and treatment of depression in the medically ill (Maier and Watkins, 1998). Specifically, recent theories have proposed that pro-inflammatory cytokines, released as a function of disease-related inflammatory processes, participate in the pathophysiology of depression, given their potent effect on neurotransmitter function, neuroendocrine function, and behavior (Besedovsky and del Rey, 1996; Dunn and Wang, 1995; Yirmiya, 1996). Behavioral changes induced by cytokines include the induction of a syndrome referred to as “sickness behavior” that has many overlapping features with major depression (Kent et al., 1992). (See Table 1.) Thus, cytokine-induced sickness behavior may account in part for the high rate of major depression found in the medically ill. Sickness behavior is typically associated with the behavioral changes seen in humans and laboratory animals suffering from microbial infections and includes symptoms of cognitive dysfunction, fatigue/anergia, psychomotor slowing, anorexia, anhedonia, sleep alterations, and increased sensitivity to pain (Kent et al., 1992). Relevant to its mediation by pro-inflammatory cytokines, sickness behavior can be reliably reproduced by administration of each of the pro-inflammatory cytokines in isolation or by administering agents (e.g., endotoxin or lipopolysaccharide


[LPS]) that induce the pro-inflammatory cytokine cascade (TNF-α to IL-1 to IL-6) (Yirmiya et al., 1999). Relevant to the role of cytokines in behavioral pathology in medically ill patients, depressed patients with cancer were found to exhibit significantly higher plasma IL-6 concentrations compared to non-depressed cancer patients and healthy controls (Musselman et al., 2001b). In addition, elevated plasma concentrations of cytokines have been found in association with specific symptoms of depression, including elevated plasma concentrations of IL-1ra and soluble TNF receptor type II in cancer patients with significant fatigue (Bower et al., 2002) and elevated IL-6 concentrations in cancer patients with impaired executive function (Meyers et al., 2005).

B. Model of Cytokine Therapy The model of cytokine therapy has been recently used to better investigate the physiopathology of cytokine-induced depression. Because of their immunomodulatory, anti-viral and anti-proliferative properties, cytokines—principally interferon (IFN)-alpha and interleukin (IL)-2—are currently used for the treatTABLE 1

Overlapping Symptoms of Sickness Behavior and Major Depression

Sickness behavior

Major depression

Anhedonia Anorexiaa Decreased libido Cognitive disturbance Psychomotor retardationa Fatiguea Weight lossa Sleep disturbance Hyperalgesia Social isolation Sad mood Worthlessness/guilt Suicidal ideation

Anhedonia Anorexia Decreased libido Cognitive disturbance Psychomotor retardation Fatigue Weight loss Sleep disturbance Increased pain complaints Social isolation Sad moodb Worthlessness/guiltb Suicidal ideationb

a

More frequent in sickness behavior than depression. More frequent in depression than sickness behavior. Reproduced with permission from Miller et al., Clinical Neuroscience Research, 2005. b

TABLE 2

Symptoms Time course Responsiveness to antidepressant

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ment of immune-mediated medical illnesses, including cancer and viral infections (e.g., chronic hepatitis C, AIDS). Despite their therapeutic efficacy, cytokine therapies are, however, notorious for causing neurobehavioral symptoms, including major depression, in a large number of patients. Indeed, data indicate that approximately 30–50% of patients undergoing treatment with the cytokine IFN-alpha develop major depression during the course of their treatment, depending on the dose, route, and duration of treatment (Capuron et al., 2002a; Miyaoka et al., 1999; Musselman et al., 2001a). Similar rates of depression have been reported in patients treated with the cytokine IL-2 for renal cell carcinoma (Buter et al., 1993; Capuron et al., 2004; Denicoff et al., 1987). It is important to note that in the context of cytokine administration in medically ill patients, major depression refers in the classification of the DSM-IV to a substanceinduced affective disorder (American Psychiatric Association, 1994). Recent advances made using the model of cytokine therapy have revealed that cytokine-induced neuropsychiatric symptoms in patients treated with IFNalpha therapy represent two distinct behavioral syndromes with different phenomenology and responsiveness to antidepressants (Capuron et al., 2002a). (See Table 2.) The mood and cognitive syndrome, characterized by the typical symptoms of depression such as depressed mood, anxiety, irritability, memory, and attentional disturbance, develops usually between the first and third month of IFN-alpha therapy in vulnerable patients (Capuron and Miller, 2004; Capuron et al., 2002a; Capuron et al., 2004; Musselman et al., 2001a). In contrast, the neurovegetative syndrome, characterized by symptoms of fatigue, psychomotor slowing, anorexia, and altered sleep patterns, develops earlier (within 2 weeks of IFN-alpha therapy) in a large proportion of patients and persists at later stages of therapy. For instance, fatigue, the symptom most common during cytokine therapy, has been shown to occur in up to 80% of patients undergoing IFN-alpha therapy (Capuron et al., 2002a). In terms of their responsiveness to antidepressants, the mood and cognitive syndrome was found to be highly responsive to pre-treatment with the antidepressant paroxetine (a

Neurobehavioral Effects of Interferon-Alpha Therapy Mood/Cognitive syndrome

Neurovegetative syndrome

Depressed mood, anxiety cognitive dysfunction Appears later (mean onset: week 8) Responsive

Psychomotor slowing Fatigue, sleep disturbance, anorexia Appears early and persists (onset: 1st 4 weeks of treatment) Minimally responsive

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selective serotonin inhibitor), whereas the neurovegetative syndrome was antidepressant non-responsive (Capuron et al., 2002a). This differentiation between mood versus neurovegetative symptoms has been also found in cancer patients undergoing chemotherapy, where it was also noted that depression was responsive to paroxetine treatment, whereas fatigue was treatment resistant (Morrow et al., 2003). Moreover, in a factor analysis of behavioral symptoms in a large number of cancer patients (N = 527) with a variety of different types of cancers in different stages of treatment, neurovegetative symptoms including weakness/fatigue, anorexia, and inability to get things done were found to be a distinct factor versus mood/cognitive symptoms, which clustered in a factor that included symptoms of worrying, irritability, sadness, nervousness, and problems with attention and memory (Cleeland, 2000). Taken together, these data suggest that differential pathophysiological pathways may be involved in the development of specific symptom dimensions including mood/cognitive versus neurovegetative symptoms in the context of cytokine system activation.

VII. PATHWAYS LINKING IMMUNITY TO DEPRESSION A. Do Cytokines Produced Peripherally Reach the Brain? As previously noted, evidence of increased inflammatory markers, including plasma concentrations of pro-inflammatory cytokines, has been found in patients with major depression. Given that pro-inflammatory cytokines are relatively large molecules that do not freely cross the blood-brain barrier, research has focused on how cytokine signals reach the brain. At least three pathways have been described (discussed in more detail in Chapter 13) including (1) passage through leaky regions in the blood-brain barrier; (2) active transport via cytokine-specific transport molecules; and (3) activation of vagal afferent nerves, which signal relevant brain nuclei such as the nucleus of the solitary tract. Once cytokine signals reach the brain, there is a cytokine network within the brain that can amplify and transpose relevant signals into those that interact with pathophysiologic pathways that are known to be involved in the development of depression.

B. Cytokines in the Brain: Pathophysiological Effects There are several potential mechanisms by which cytokines may contribute to the pathophysiology of

depression. These mechanisms, which are not mutually exclusive, involve (1) neuroendocrine effects, (2) alterations in neurotransmitter function, and (3) effects on neuronal activity/function. 1. Neuroendocrine Effects Cytokines have profound effects on neuroendocrine function. The HPA axis and immune system axis interact via a complicated circuitry composed of both feed-forward excitatory and feedback inhibitory loops. Under normal conditions, inhibitory elements such as glucocorticoids and anti-inflammatory cytokines limit the activation of the immune system to levels that are appropriate for clearing the initial antigenic stimulus (Maier and Watkins, 1998). However, it is becoming increasingly recognized that in certain conditions these inhibitory feedback loops become impaired, allowing for chronic immune activation and the persistence of sickness symptoms that follow from this activation. Depression may correspond to one of these conditions where inhibitory feedback loops are altered and both neuroendocrine and immune systems become persistently activated (Raison and Miller, 2003). As described in more detail elsewhere (see Chapter 13), a number of inflammatory and immunoregulatory cytokines have been found to have potent effects on the HPA axis (Besedovsky and del Rey, 1996). One consistent finding is the capacity of the cytokines that mediate innate immune responses (e.g., IFN-alpha, IL-1, IL-6, and TNF-alpha) to increase the release of corticotrophin-releasing factor (CRF) (Besedovsky and del Rey, 1996; Gisslinger et al., 1993; Raber et al., 1997). CRF is a key neuropeptide in the regulation of the response to stress, orchestrating stress-induced activation of the HPA axis (and the release of ACTH and cortisol) and the sympathetic nervous system (and the release of catecholamines) (Owens and Nemeroff, 1993). Activation of CRF pathways by inflammatory cytokines represents one of the primary mechanisms by which cytokines may contribute to the development of depression (Owens and Nemeroff, 1993). Indeed, hypersecretion of CRF is a reliable finding in patients with major depression as manifested by increased csf concentrations of CRF and increased CRF mRNA and protein in the hypothalamus of postmortem samples from depressed patients (Owens and Nemeroff, 1993; Pariante and Miller, 2001). In addition, administration of CRF to laboratory animals leads to behavioral symptoms that overlap with those seen in both major depression and sickness behavior, including alterations in food intake, locomotor activity, and sleep (Owens and Nemeroff, 1993).


Recent studies in humans have indicated that cytokine-induced activation of the HPA axis may represent a risk factor for the development of depression. Indeed, cancer patients who developed major depression while undergoing treatment with the cytokine IFN-alpha exhibited significantly higher ACTH and cortisol responses to the first injection of IFN-alpha compared to cancer patients who remained free of depression during IFN-alpha therapy (Capuron et al., 2003b). Moreover, a significant correlation was found between the degree of the initial ACTH and cortisol response to IFN-alpha and the development of depression, anxiety, and cognitive dysfunction after 8 weeks of IFN-alpha treatment (Capuron et al., 2003b). Interestingly, IL-6 responses to the initial injection of IFNalpha were no different in patients who became depressed versus those who did not, suggesting that the vulnerability to depression is a function of the interaction of cytokines with stress responsive pathways (Capuron et al., 2003b). Thus, individual differences in stress responsivity including hyperactive CRF pathways, such as has been described following early life stress (Heim et al., 2000), may lead to increased vulnerability to cytokine-induced behavioral alterations. Indeed, the oft-replicated finding of a correlation between baseline mood state and subsequent development of depression during cytokine therapy indicates that baseline mood status may be a behavioral marker of this neuroendocrine vulnerability (Capuron and Ravaud, 1999; Raison et al., 2005). Another pathway by which cytokines may influence the neuroendocrine system and thereby contribute to depression is through disruption of the functioning of the glucocorticoid receptor (GR) (Miller et al., 1999a; Pariante and Miller, 2001). Patients with major depression have reliably been shown to exhibit alterations in GR function as manifested both in vivo (as reflected by an abnormal dexamethasone suppression test and/or dexamethasone-CRF test) and in vitro (as reflected by reduced sensitivity of PBMCs to dexamethasone-induced inhibition of immune cell function) (Pariante and Miller, 2001). Of specific relevance to the GR, inflammatory and immunoregulatory cytokines have been shown to alter virtually every aspect of GR function, including GR expression, GR phosphorylation state, GR translocation, GR protein-protein interactions, and ultimately GR binding to DNA (Goleva et al., 2002; Miller et al., 1999a; Pariante et al., 1999; Smoak and Cidlowski, 2004). The signal transduction pathways by which cytokines affect GR function also have been described and include NFκB, p38 MAPK, JNK, and STAT5 (Goleva et al., 2002; Smoak and Cidlowski, 2004; Wang et al., 2004; Wang et al., 2005). Finally, cytokines (e.g., IL-2 and IL-4) have been

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shown to induce the beta isoform of the GR, which is inactive and thereby serves to compete for ligand and reduce activation of the GR alpha isoform, the primary mediator of GR effects (Leung et al., 1998). Given the role of glucocorticoids in regulating immune activation and inflammation, cytokine-induced disruption of GR function may lead to a feed-forward cascade in which increasing inflammation leads to increased glucocorticoid resistance, which in turn leads to reduced glucocorticoid-mediated feedback inhibition of inflammatory responses (Raison and Miller, 2003). 2. Effects on Neurotransmitter Function Both in animals and humans, cytokines have been shown to have profound effects on the metabolism of brain neurotransmitters. Special emphasis will be placed on the effects of cytokines on serotonin and dopamine neurotransmission. Serotonin There is abundant evidence for abnormalities of serotonin (5HT) function in depression (Delgado et al., 1994; Owens and Nemeroff, 1994; Price et al., 1991; Ressler and Nemeroff, 2000). In many studies, cytokines have been shown to interfere with 5HT metabolism and activity, a mechanism that may account for the depressogenic effects of cytokines. Indeed, significant and consistent decreases in serum/plasma concentrations of tryptophan, the primary precursor of 5HT, have been reported in patients undergoing IL-2 and/or IFN-alpha therapy (Bonaccorso et al., 2002; Brown et al., 1989; Brown et al., 1991; Capuron et al., 2003a; Capuron et al., 2002c). Interestingly, in some of these studies, decreases in blood tryptophan concentrations were found to correlate with depressive symptoms (Bonaccorso et al., 2002; Capuron et al., 2003a; Capuron et al., 2002c). More specifically, it has been recently shown that tryptophan degradation during IFN-alpha therapy was associated with the development of mood and cognitive symptoms (e.g., depressed mood, anxiety, memory/attention disturbance) but not with neurovegetative and somatic symptoms (e.g., fatigue, psychomotor slowing, pain) (Capuron et al., 2003a). At the experimental level, treatments with IFNalpha and IL-1-beta were found to significantly upregulate the expression of the 5HT transporter mRNA, both in human cells, an effect which is opposite the activity of certain antidepressants (Morikawa et al., 1998; Ramamoorthy et al., 1995). In contrast to findings obtained in humans, several data in animals have shown stimulating effects of acute treatment with proinflammatory cytokines on 5HT metabolism (Dunn

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et al., 1999b; Linthorst et al., 1994). Nevertheless, recent data have indicated that cytokine effects on 5HT release in the nucleus accumbens were cytokine-dependent (Song et al., 1999). For instance, IL-1 and IL-6 were found to modestly increase extracellular 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5HT, from the nucleus accumbens in the rat, whereas IL-2 did not. Other data have shown that IFN-alpha reduced concentrations of 5HT and 5-HIAA in the midbrain and striatum of rats and in the frontal cortex in a dosedependent manner (Kamata et al., 2000). In addition, TNF-alpha has been found to enhance 5HT uptake, whereas IL-4, which acts as an anti-inflammatory cytokine within the central nervous system, induced a dose-dependent reduction of 5HT uptake (Mossner et al., 2001; Mossner et al., 1998). Finally, chronic administration of IFN-alpha was found to affect the low-affinity 5-HT1A receptors in the rat brain (Abe et al., 1999) and to increase the levels of 5HT transporter mRNA both in the midbrain and adrenal gland in rats (Morikawa et al., 1998). Strong support for a role of 5HT alterations in cytokine-induced depression is the involvement of cytokines in the induction of the enzyme indoleamine 2,3 dioxygenase (IDO). IDO is induced by cytokines, especially IFN-gamma, in a variety of immune cells including monocyte-derived macrophage and microglia, upon the immune system activation (Konan and Taylor, 1996; Mellor and Munn, 1999; Taylor and Feng, 1991; Widner et al., 2000). (See Figure 2.) IDO catalyzes the rate-limiting step of tryptophan conversion into kynurenine and then quinolinic acid (Byrne et al., 1986), thereby reducing the availability of tryptophan for conversion into 5-HT, the primary mediator of serotonin effects in the brain. In vivo, the activity of IDO is reflected by the ratio of kynurenine/tryptophan (Widner et al., 2000). Findings in experimental animals have shown that IDO is induced in the brain in response to peripheral immune stimulation by LPS and Bacille Calmette-Guerin (BCG) inoculation in mice (Lestage et al., 2002; Moreau et al., 2005). In addition, it has been recently shown in vitro, using IFN-gamma–stimulated purified cultures of neurons, astrocytes, microglia, and macrophages, that all these cellular types expressed IDO, but only microglia were able to produce detectable amounts of quinolinic acid. However, astrocytes and neurons were found to have the ability to catabolize quinolinic acid (Guillemin et al., 2005). Of relevance to depressed patients, TRP depletion in patients who became depressed during IFN-alpha therapy was associated with marked increases in kynurenine, suggesting that increased IDO activity contributed to the TRP decreases observed in depressed patients (Capuron et al., 2003a). Taken together, these data support the

Immune Stimulus

Cytokines (e.g., IFN-°)

Tryptophan

IDO

Kynurenine

Qu in olinic Acid

TPH

5-Hydroxytryptophan

Serotonin

FIGURE 2 Effects of immune activation on tryptophan metabolism. The induction by cytokines (e.g., interferongamma) of the enzyme, indoleamine-2, 3-dioxygenase (IDO), upon immune activation leads to tryptophan catabolism into kynurenine and quinolinic acid, thereby reducing the availability of tryptophan for serotonin synthesis via the enzyme tryptophan hydroxylase (TPH).

notion that IDO may represent a key player in the pathophysiology of cytokine-induced depression. Dopamine Dopamine is a neurotransmitter that is involved in multiple brain functions, including motor activity regulation, addictive behavior, and certain psychiatric disorders, including schizophrenia and depression (Bedard, 1995; Koob, 1996; van Praag et al., 1990). Consistent with the effects of cytokines on motor/movement activity, hedonic tone, and fatigue, several clinical and pre-clinical studies have shown that cytokines are able to modulate dopamine function. In clinical studies, symptoms characteristic of dopamine dysfunction (e.g., Parkinson-like symptoms) have been reported in patients treated with the cytokine IFN-alpha (Horikawa et al., 1999; Mizoi et al., 1997). In that regard, intravenous administration of levodopa significantly ameliorated a refractory case of akathisia induced by


IFN-alpha (Sunami et al., 2000). Consistent with these clinical observations, alterations in basal ganglia activity, suggestive of decreased/impaired dopamine function, have been reported using positron emission tomography (PET) in medically ill patients undergoing treatment with IFN-alpha and exhibiting the neurovegetative symptoms of psychomotor slowing, fatigue, and anhedonia (Capuron et al., 2002b). Interestingly, dopamine uptake, as assessed by PET with 18 18 [ F]fluorodopa ([ F]DOPA), has also been found to be significantly lower in the left caudate of depressed patients with marked psychomotor retardation and affective flattening compared with depressed patients with high impulsivity and healthy controls (Martinot et al., 2001). These findings support the hypothesis that psychomotor retardation as well as anhedonia, which constitute two core neurovegetative symptoms induced by IFN-alpha, may be caused by diminished presynaptic striatal dopamine function. Regarding the symptom of fatigue, alterations in dopamine pathways may also participate to fatigue induction in patients undergoing treatment with cytokines. In support of this notion, treatments with levodopa or other pharmacologic agents increasing dopamine release (e.g. stimulants) have been shown to improve physical fatigue in patients with Parkinson’s disease as well as IFN-alpha–treated patients (Lou et al., 2003; Schwartz et al., 2002). In animals, chronic treatment with IFN-alpha was found to induce a dosing-schedule–dependent alteration of rhythmicity in locomotor activity (Ohdo et al., 2001) and a robust depression of motor activity (De Sarro et al., 1990; Dunn and Crnic, 1993). In addition, this cytokine was shown to decrease dopamine activity in the mouse brain (Kamata et al., 2000; Shuto et al., 1997). More recently, treatment with chronic IFN-alpha in rats was found to potentiate latent inhibition, a phenomenon believed to rely mainly on mesolimbic dopamine system (Bethus et al., 2003). In addition, chronic infusion of lipopolysaccharide into rat brain leads to delayed and selective degeneration of nigral dopaminergic neurons through microglial activation (Gao et al., 2002). Finally, recent data have indicated that nitric oxide induced by intramuscular injection of IFNalpha in rats was able to cross the blood-brain barrier and suppress both tetrahydrobiopterin biosynthesis (a co-factor for monoaminergic neurotransmitter biosynthesis) and dopamine production in the amygdala and raphe areas (Kitagami et al., 2003). Other cytokines, such as IL-2, were found to reduce dopamine turnover in the caudate and substantia nigra in mice when administered repeatedly (Lacosta et al., 2000). Consistent with the effects of cytokines on dopamine function, receptors for pro-inflammatory cytokines are

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abundantly expressed in the basal ganglia (Haas and Schauenstein, 1997), and basal ganglia and relevant dopamine pathways during cytokine activation appear involved in infectious diseases associated with neuropsychiatric alterations similar to those seen during cytokine treatment, including human immunodeficiency virus (Berger and Arendt, 2000). Effects on Brain Functions Aside from effects on neurotransmitter and neuroendocrine function, recent data suggest that cytokines are able to alter higher cognitive brain functions including fundamental information processing. Indeed, in a recent brain functional magnetic resonance imaging (fMRI) study, patients with chronic hepatitis C (HCV) treated with IFN-alpha for 12 weeks were found to exhibit significant activation in the dorsal part of the anterior cingulate cortex (Capuron et al., 2005), a brain region that has been involved in conflict monitoring and cognitive control during cognitive tasks with high demand (Botvinick et al., 2001; Bush et al., 2000; Carter et al., 1998; Carter et al., 1999; Paus 2001). Interestingly, in IFN-alpha–treated HCV patients, activation of the cingulate cortex highly correlated with errors made in a visuo-spatial attention task administered during the fMRI session (Capuron et al., 2005). This activation of the anterior cingulate cortex was not apparent in untreated HCV patients, although these patients exhibited a similar error rate and similar task-related performance compared to IFN-alpha–treated subjects. Similar to IFN-alpha–treated patients, activation of the dorsal anterior cingulate cortex in the context of a lowerror rate has been found in individuals with personality characteristics (e.g., high-trait anxiety, neuroticism) known to be risk factors for the development of mood and anxiety disorders (Paulus et al., 2004). These data suggest that cytokines (IFN-alpha) induce alterations in information processing (as revealed by increased activity in the anterior cingulate cortex) that may manifest by an increased sensitivity to processing conflicts and events perceived as potentially threatening (Capuron et al., 2005), possibly reflecting a so-called “danger” signal in the brain. Such changes in brain activity and relevant cognitive processes may in turn impart an increased vulnerability to negative effects and more generally to psychopathology (e.g., mood disorders).

VIII. CYTOKINE ABNORMALITIES IN DEPRESSION: TREATMENT IMPLICATIONS Based on the potential role of cytokines in the pathophysiology of depression, opportunities exist for

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“translational” research strategies that focus on the management of sickness/depressive symptoms using novel therapies targeted at the pathways by which cytokines may contribute to depression. Probably the most obvious targets are the pro-inflammatory cytokines themselves, including the use of interleukin (IL)1 receptor antagonist (e.g., anakinra) to target IL-1; the soluble TNF receptor (e.g., etanercept) or antibodies to TNF (e.g., infliximab) to target TNF; and the antiinflammatory cytokine, IL-10, to target multiple inflammatory cytokines. Such cytokine “antagonists” may find both systemic usefulness as well as local applications, such as the treatment of peripheral nerve pathological pain syndromes (Watkins et al., 2003). Of note, etanercept, infliximab, and anakinra are all available commercially for the treatment of rheumatoid arthritis, and therefore are available for preliminary analyses of efficacy in patients with altered mood status. Other promising approaches include the targeting of corticotropin-releasing factor (CRF), which, as noted above, is induced by pro-inflammatory cytokines and has been shown to cause many of the same symptoms as sickness behavior when administered to laboratory animals (Owens and Nemeroff, 1993). Antidepressants targeting CRH and novel CRH antagonists are being developed by several pharmaceutical companies (Stout et al., 2002). Other targets include inflammatory mediators such as the prostaglandins as well as the CNS monoamines such as serotonin, norepinephrine, and dopamine. Given the capacity of cytokine exposure to influence brain monoamines (Dunn et al., 1999a), therapies targeted at influencing monoamine neurotransmission (including selected antidepressants as well inhibitors of IDO) might be especially useful. Finally, because glucocorticoids serve to potently inhibit inflammatory signaling pathways, pharmacologic agents that enhance glucocorticoid signaling and/or inhibit inflammatory signaling, including type 4 phospodiesterase inhibitors, may be worthy of consideration (McKay and Cidlowski, 1999; Miller et al., 1999a; Miller et al., 2002a).

IX. CONCLUSION In conclusion, there is strong evidence that depression involves alterations in multiple aspects of immunity that may not only contribute to the development or exacerbation of a number of medical disorders, but also may contribute to the pathophysiology of the disease itself. Accordingly, aggressive management of depressive disorders in medically ill populations or individuals at risk for disease may improve disease outcome or prevent disease development. On the other

hand, in light of data suggesting that immune processes may interact with the pathophysiologic pathways known to contribute to depression, novel approaches to the treatment of depression may target relevant aspects of the immune response. Taken together, the data provide compelling evidence that a psychoimmunologic frame of reference may have profound implications regarding the consequences and treatment of depression.

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C H A P T E R

25 Immune and Neuroendocrine Alterations in Post-traumatic Stress Disorder GAIL IRONSON, DEAN CRUESS, AND MAHENDRA KUMAR

I. PSYCHOLOGICAL TRAUMA, PTSD, AND IMMUNE FUNCTION 531 II. PTSD: ENDOCRINE 539 III. ENDOCRINE-IMMUNE INTERACTIONS IN PTSD 542 IV. CONCLUSION 543

diabetes, and thyroid disease (Boscarino, 2004); musculoskeletal problems (Boscarino, 1997); and asthma and other respiratory problems (Dobie et al., 2004; Green and Kimmerling, 2004; McFarlane et al., 1994; Walker et al., 1999). However, the mechanisms linking PTSD to these conditions have yet to be elucidated. One possibility is the immune system.

I. PSYCHOLOGICAL TRAUMA, PTSD, AND IMMUNE FUNCTION

2. Overview The purpose of this chapter is to review the immune findings in trauma and PTSD. Since it is impossible to interpret the immune findings without reference to the endocrine system, that literature is covered as well. Thus, the chapter is divided into three primary sections. After the introduction and review of symptoms comprising the PTSD diagnosis, the first primary section deals with immune alterations in PTSD. Next, the endocrine findings in PTSD are covered, followed by a discussion of immune endocrine interactions in the third major section. A concluding section summarizing the main points completes the chapter.

A. Introduction and Overview 1. Interest in PTSD: PTSD and Health Interest in studying post-traumatic stress disorder (PTSD) and its associated physiological states has risen dramatically in recent years. One reason for this is the association of PTSD and severe trauma with increased morbidity and mortality (Friedman and McEwen, 2004; Green and Kimmerling, 2004). For example, a 50-year longitudinal study found those with combat exposure had a greater chance of early death and more chronic illnesses by age 65 (Lee et al., 1995). In another study exposure to severe stress was associated with more diseases over a 20-year follow-up (Boscarino, 1997). More specifically, studies have found severe trauma/PTSD to be associated with increased rates of coronary heart disease (Boscarino and Chang, 1999b); gastrointestinal disorders (Schnurr et al., 2000), including irritable bowel syndrome (Dobie et al., 2004; Irwin et al., 1996); autoimmune diseases such as rheumatoid arthritis (Boscarino, 2004; Grady et al., 1991), psoriasis, PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

3. Types of Trauma Studies Trauma studies can be differentiated in many ways. A few obvious ones are the type of stressor (natural disaster vs. war), the duration of the trauma (time limited vs. an extended period of time), age at which the trauma occurred (i.e., as an adult vs. as a child), the time between the trauma and assessment, and the severity of the trauma (did people think they might die, damage/loss). Tables 1, 2, and 3 summarize these

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studies. Table 1 covers studies involving traumas in which a diagnosis of PTSD is not explicitly made, in which the traumatic event has usually been time limited, and assessments have primarily been done in closer proximity to the trauma than traumas such as combat or childhood abuse. Tables 2 and 3 cover studies where the diagnosis of PTSD has been explicitly made. In recognition of the potential importance of the time elapsed between the trauma and assessment, Table 2 covers studies in which the diagnosis of PTSD has been made, but the time frame between the trauma and assessment is shorter than in Table 3, or is not explicitly stated in the article. Table 3 covers studies in which the diagnosis of PTSD has been made, and the time frame between the trauma and assessment is long. The traumas in Table 3 include such traumas as being a combat veteran or a victim of childhood sexual abuse. These types of traumatic events usually (but not always) have occurred over an extended period of time, and assessment is usually, but not always, long after the trauma has occurred. For the traumas covered in Table 1, which are primarily studies of time-limited TABLE 1 Investigator McKinnon et al., 1989

Dekaris et al., 1993

traumas in which the diagnosis of PTSD is usually not explicitly made, many of the people may have PTSD or develop it. For example, in the Ironson et al. (1997) study after Hurricane Andrew, symptoms of PTSD were assessed, but the diagnosis of PTSD was not. Using high scores on the impact of event scale (measured 1 to 4 months after the event), 44% could be estimated to have PTSD. Subsequent assessment of 46 people from this sample, augmented with 15 people living in the same damaged areas, showed that 36% met the criteria for PTSD several months later (6 to 12 months after the storm) (David et al., 1996). Additionally the diagnosis of PTSD requires at least 1 month’s time to have elapsed since the trauma. These studies of trauma were included as they are relevant to PTSD even if they do not directly diagnose PTSD. 4. Diagnosis of PTSD The diagnosis of PTSD requires several criteria to be met. First and foremost, a person must have been exposed to a traumatic event that involved a threat

Time-Limited Traumas, No PTSD Diagnosis, Measurement Time Frame Mostly 3 months after their index trauma.

Baker et al., 2001

11 combat veterans with PTSD vs. 8 healthy control subjects. (time frame not explicit)

Tucker et al., 2004

58 PTSD and 21 controls. PTSD subjects participated in a 10-week double-blind treatment with citalopram (n = 19), sertraline (n = 18), or placebo (n = 7) (time frame not explicit)

of death or serious injury, and the person’s response involved intense fear, helplessness, or horror (American Psychiatric Association, 1994). Three symptom clusters must be present: re-experiencing, avoidance and numbing, and increased arousal. Symptoms described by people from the re-experiencing cluster include flashbacks, recurrent distressing dreams of the trauma, intense distress at exposure to things reminding one of the trauma, intrusive and recurrent thoughts of the event, and physiologic reactivity to reminders of the event. Examples from the avoidance/ numbing cluster include avoidance of thoughts of the event and avoidance of activities, places, or people that remind one of the event; an inability to recall significant aspects of the event; withdrawal from activities, feeling of detachment from others, and inability to

Positive relationship between sIL-6r and the RevisedIES(RIES) intrusion score. Positive relationship also between CRP and DTS intrusion scores, GHQ depression, and RIES intrusion in PTSDdiagnosed patients. Increased CSF levels of IL-6; no group differences in plasma IL-6. Plasma IL-6 and norepinephrine levels positively correlated in the PTSD but not control groups. At baseline, PTSD patients had higher levels of depression and IL-1β, and lower IL-2R levels than controls. Both treatment groups had lower PTSD, depression, and IL-1β levels and increased IL-2R similar to control levels post treatment.

have loving feelings; and lowered expectations about having a future. The third cluster, increased arousal, is exemplified by such symptoms as difficulty concentrating, hypervigilance, an exaggerated startle response, problems sleeping, and increased irritability. As noted above, the three symptom clusters must be present at least 1 month for PTSD to be diagnosed. In addition, the symptoms must have caused significant distress or impairment in ability to function in social or occupational roles.

B. Studies of PTSD and the Immune System There are several reasons why one might expect an association between PTSD and the immune system.

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III. Behavior and Immunity TABLE 3

Investigator Mosnaim et al., 1993

Burges-Watson et al., 1993, 1995

Diagnosed PTSD, Assessment Distant (>2 Years after Trauma) Patients/Design

Key immune findings

Vietnam combat veterans with chronic PTSD: 13 with comorbid alcoholism, 11 with drug use disorders, 8 with both alcohol and drug use disorder, 8 healthy control subjects with no psychiatric or substance abuse disorders. 25 Vietnam combat veterans with PTSD clinical treatment. 28 civilians (no military service) and 20 non-combat vets. Controls had no diagnosed psychiatric disorders. All males.

Low NKCC with MET challenge in PTSD subjects with comorbid drug use but not alcohol use disorders.

Spivak et al., 1997

19 male patients with combat-related PTSD and 19 age- and sex-matched controls. 3 had served in the Yom Kippur War, 6 in the war with Lebanon, and 10 during the Intifada.

Laudenslager et al., 1998

10 male veterans of the Vietnam War diagnosed with long-term PTSD and 9 controls (combat veterans without PTSD). Male Vietnam veterans were analyzed and divided into groups. Males with current partial posttraumatic stress = 286, anxiety = 274, depression disorders = 192. They were compared to 2,179– 2,272 veterans without these symptoms 20 years after military service.

Boscarino and Chang, 1999a

Wilson et al., 1999

10 female subjects with childhood sexual trauma, diagnosed with PTSD, and 10 controls.

Altemus et al., 2003

16 women with PTSD due to childhood sexual or physical abuse and 15 women with no history of abuse, trauma, or psychiatric disorder. Chronic PTSD in a sample of 2,490 Vietnam veterans

Boscarino, 2004

First, the description of PTSD above, which includes re-experiencing of a stressful traumatic event and the occurrence of autonomic arousal, suggests the involvement of biological stress response systems such as the sympathetic nervous system. In addition, established interconnections between the brain and immune systems have been made, both by showing the existence of hardwiring (Felten and Felten, 1994), and endocrine mediators (such as cortisol, epinephrine, and norepinephrine, which interact with receptors on immune cells). There are several different effector cells of the immune system as well as indicators of their functioning that may be measured. Thus, this review will cover both functional measures and enumerative measures, and will review each in turn. Functional measures are covered first, as studies of chronic stress have been found to have a stronger relationship with those (Segerstrom and Miller, 2004).

The arousal/hyper vigilance were more important than depression or anxiety correspondence. Need to control for medication, alcohol, or smoking. Veterans with PTSD had enhanced CMI compared with civilians. Serum IL-1β but not sIL-2R were higher in the PTSD patients. IL-1β did not correlate with cortisol levels, severity of PTSD, depressive symptoms, or alexithymia scores, but they did correlate with duration of the PTSD symptoms. NK activity was higher in the PTSD sample than in the controls. No difference hormone levels or CD4, CD8, or NK cells between groups. Veterans with PTSD had higher adjusted leukocyte, lymphocyte, and T-cell counts (including CD4’s) compared with vets without PTSD, but no difference in DTH response to CMI skin multi-test. Veterans with anxiety disorders had adjusted lymphocyte and T-cell counts above range and highly reactive CMI. Depressed veterans were less likely to have B-cell counts above reference range. Ratio of CD45R0-positive to CD45RA-positive lymphocytes (an index of lymphocyte activation) was higher in PTSD subjects. No differences in the number of PBL or any major T, B, or NK lymphocyte subsets. PTSD patients had higher DTH.

Those with PTSD had clinically higher T-cell counts, hyper-reactive DTH, clinically higher IgM.

1. Overview of Immune System There are two main arms of the immune system: innate or natural, and adaptive or specific. The innate arm does not require specific recognition of an invader and can function quickly after an invader appears. Some of the cell types in this arm of the immune system are neutrophils, macrophages (monocytes), and natural killer (NK) cells. Neutrophils and macrophages (monocytes) are phagocytic; that is, they digest the foreign invader. Macrophages (monocytes), particularly when activated by non-self substances, also secrete cytokines (IL-1, IL-6, TNF-alpha), which serve a communication function and are pro-inflammatory. Natural killer cells are particularly important in fighting off viruses and tumor cells, as they can recognize cells that are not self or that have become malignant. They kill by secreting toxic substances. The major cell types in the adap-

25. Immune and Neuroendocrine Alterations in Post-traumatic Stress Disorder

tive or specific arm are T helper, T cytotoxic, and B-cells. T helper cells secrete cytokines that are chemical messengers which direct and “help” increase the immune response. Two types of cytokines are produced: Th1 and Th2 (see paragraph below on cytokines). Th1 cytokines are involved in cellular immunity and activate T cytotoxic and NK cells. Th2 cytokines are involved in humoral immunity and activate Bcells. B-cells, in turn, produce antibodies that are particularly important in helping to fight off bacteria and parasites and in increasing the effectiveness of innate immunity. 2. PTSD and Functional Measures of Immunity The functioning of natural killer cells is one of the measures included most often in studies of stress and trauma. As noted, natural killer cells are part of the innate arm of the immune system and can thus fight against invaders without specific recognition of a particular invader. They are particularly important not only because they fight off viruses and tumors, but because they are effective before specific immunity engages. NKCC measures the effectiveness of NK cells at killing tumor cells (typically, the K562 target cell line). NKCC was decreased in people (n = 180) with post-traumatic stress symptoms 1 to 4 months after Hurricane Andrew (Ironson et al., 1997), in 155 male workers with PTSD symptoms 14 to 18 months after an earthquake (Inoue-Sakurai et al., 2000), in 29 men detained in a prisoner of war camp in Bosnia (Dekaris et al., 1993), and in 12 Japanese male workers with a past history of PTSD (Kawamura et al., 2001). Finally, NKCC response to stimulation with methionineenkephalin (MET) was decreased in Vietnam War veterans with PTSD and comorbid drug use (Mosnaim et al., 1993) 10 days or more into a drug detoxification program, although unstimulated NKCC showed no difference. In contrast, NKCC was elevated in workers exposed to bodies and body parts at a plane crash site 2 months after the crash (Delahanty et al., 1997) but normalized at the 6-month follow-up. Higher levels of NKCC were correlated with intrusive thoughts of the disaster. NKCC was also elevated in 68 people experiencing an earthquake but returned to normal within 4 months (Solomon et al., 1997) and in Israelis experiencing SCUD missile attacks during the Persian Gulf War (Weiss et al., 1996). Finally, NKCC was increased in a sample of 10 male veterans of the Vietnam War with PTSD and substance abuse problems compared to 9 controls who had only substance abuse problems (Laudenslager et al., 1998). Thus, the picture for NKCC is inconsistent, with some studies showing more of an acute stressor response (with initial increases), fol-

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lowed by decrements when the stressor persists over time, but other studies fitting more of a chronic stress pattern of decreased NKCC (Segerstrom and Miller, 2004). The delayed-type hypersensitivity (DTH) response measured by the cell-mediated immunity (CMI) multitest was among the first immune measures studied in PTSD and is thought to involve primarily T-cell responses. The earliest study (Burges-Watson et al., 1993) found enhanced cutaneous CMI multi-test compound scores in 25 Vietnam combat veterans with PTSD versus 28 civilians (and although the compound score on the CMI multi-test was greater for the combat veterans vs. non-combat servicemen, the number of positive reactions on the CMI was not). Consistent with this, Altemus et al. (2003) compared 16 women with PTSD due to childhood sexual or physical abuse to 15 women without any history of trauma or psychiatric disorders and found the PTSD patients had higher DTH. However, in the largest study to date, Boscarino and Chang (1999a) found no differences on the DTH response to the CMI multi-test between 286 Vietnam combat veterans with PTSD and 2,179 veteran control subjects with no PTSD (see Table 3). However, the same author (Boscarino, 2004) found hyper-reactive immune responses to standardized cutaneous hypersensitivity tests in a national sample of 2,490 Vietnam veterans. Thus, findings suggest either an enhancement or no difference. A few studies have examined lymphocyte proliferative responses to mitogens. Proliferation to the mitogen PHA measures primarily T-cell proliferation. Sabioncello et al. (2000) found decreased proliferative capacity (ratio of cells in stimulated to non-stimulated cultures) to PHA in 20 female civilians displaced by the Croatian War, which is consistent with both the acute stress literature and the chronic stress literature (Segerstrom and Miller, 2004). Solomon et al. (1997) found that people who had recently experienced an earthquake and reported less distress had higher proliferation to PHA, suggesting a psychologyimmune connection. However, Weiss et al., (1996) found no difference between war and post-war periods in thymidine uptake of PBMCs under PHA stimulation. 3. PTSD and Cytokines The next set of immune-related measures that have been included in studies of trauma and PTSD are cytokines. Cytokines, mediators of the immune response produced by immune cells, fall into a few major classes: Pro-inflammatory cytokines (produced predominantly by macrophages), monocytes, and T-cells have been

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examined in the largest number of studies (IL-1, IL-6, TNF-alpha). Th1 cytokines such as IL-2, IL-12, and IFN-gamma are thought to be involved in cellular immunity. T cytotoxic cells and natural killer cells are activated by these cytokines. They are particularly useful in fighting off viruses and tumors. Th2 cytokines include IL-4, IL-5, IL-6, and IL-10. These cytokines help to direct and coordinate humoral immunity which involves antibodies, specific recognition of extracellular pathogens, and fighting against bacteria and parasites. Cytokines are most commonly measured in plasma/serum, or in culture supernatants before and after stimulation with an appropriate antigen or with a more general stimulant such as the T-cell stimulant PHA. Pro-inflammatory cytokines: Higher salivary IL-6 was found in 20 combat veterans with PTSD and rapidly progressive periodontitis (RPP) compared with non-combat controls with adult periodontitis (Aurer et al., 1999). Maes et al. (1999) found elevated serum IL-6 and soluble interleukin-6 receptor (sIL-6R) to be significantly higher in 13 PTSD victims of a fire or car crash. Interestingly, Miller et al. (2001) found that higher serum levels of the receptor to IL-6 (sIL-6R) were positively correlated with revised IES intrusion scores. Baker et al. (2001) found increased levels of IL-6 in cerebrospinal fluid, although they did not find any differences in plasma IL-6 between 11 combat veterans to 8 healthy controls. Interestingly, plasma IL-6 correlated with NE levels in the PTSD group. Another study finding no differences in basal plasma levels of IL-6 between PTSD and control groups was Wong et al. (2000), who compared 15 mixed trauma survivors with chronic PTSD to 8 control subjects without PTSD. Interestingly, Wong et al. (2000) found IL-6 response to LPS was lower in 15 people with PTSD from mixed traumas than 8 control subjects without PTSD. While IL-6 is the most studied pro-inflammatory cytokine, other pro-inflammatory cytokines have been studied. Tucker et al. (2004) found higher levels of another proinflammatory cytokine, IL-1β, in 58 PTSD patients versus 21 controls. Serum interleukin-1β was also higher in 19 Israeli soldiers with combat-related PTSD (Spivak et al., 1997) than healthy controls, and it correlated with duration of PTSD symptoms (but not severity). This suggests higher activity of the mononuclear phagocytes that produce this cytokine. A related cytokine, IL-1 receptor antagonist, was no different between trauma victims (fire, car crash) as compared with healthy controls. Furthermore, IL-1β produced by PBMC after stimulation by PHA was no different between Israeli civilians undergoing SCUD missile attacks pre- to post-war (Weiss and Hirt, 1996). Thus, IL-6 has the most studies, and several of these

studies have shown elevations of basal levels in PTSD. This result is consistent with the significant increase in IL-6 production seen in acute stress (Segerstrom and Miller, 2004), although there are not many PTSD studies of IL-6 production in cultured supernatants. IL-6 has also not been studied enough in chronic stress to compare it to the stress literature. (The only cytokine whose production has been studied enough in chronic stress is IL-2, whose production has been found to decrease [Segerstrom and Miller, 2004.]) It may further be pointed out that among these inflammatory cytokines, TNF-alpha appears first and is followed by tandem secretion of IL-1 and IL-6. Also, all three of these cytokines stimulate their own secretion. In addition, TNF-alpha and IL-1 stimulate secretion of IL-6, and conversely IL-6 inhibits secretion of TNF-alpha and IL-1 (Chrousos, 2000). It may be therefore inferred that pro-inflammatory cytokines should be investigated as a group, and there is a scarcity of such studies in PTSD. Moreover, in addition to studying the secretion of cytokines, it is highly desirable to rule out changes in the rate of catabolism of these cytokines, and their specific mRNA activity should also be investigated to understand the mechanisms involved. The next cytokines are those referred to as Th1, which are involved with cell-mediated immunity (CMI). Tucker et al. (2004) found lower levels of IL-2 receptors in 44 PTSD patients as compared with 21 controls. Wong et al. (2000) found decreased soluble interleukin-2 receptor (sIL-2R) in 15 people with mixed traumas with chronic PTSD in comparison with 8 control subjects without PTSD. In contrast, Spivak et al. (1997) found no difference in sIL-2R between 19 male PTSD combat-related PTSD subjects and 19 ageand sex-matched controls. Tucker et al. (2004) showed that these differences in results may be due to gender differences in the sample, as their study had predominantly females with abuse histories, and Wong’s study had predominantly females with mixed traumas. This is consistent with Dekaris et al. (1993), who found no difference in IL-2 in detainees from a war camp who were primarily male civilians. One study that found marginally higher levels of IL-2 (in supernatants of PHA-stimulated cells) was that of Weiss et al. (1996), who measured Israeli civilians during and after the SCUD missile attacks of the Persian Gulf War. Of additional interest, in Tucker’s study (2004) after treatment of PTSD and depression, the IL-2R levels increased for the PTSD patients. Thus, the most evidence is available for IL-2 receptors, and they appear to be decreased or unchanged. Only a few studies have been done on IFN. Kawamura et al. (2001) found lower IFN in 12 Japanese volunteers with PTSD. Weiss et al. (1996) found no differences between those experiencing


SCUD missile attacks pre- and post-war in IFN-gamma in supernatants of CML cultures. Only two studies have looked at Th2 cytokines. Wong et al. (2001) found higher levels of basal plasma and whole blood IL-10 in 15 mixed trauma survivors with chronic PTSD versus 8 control subjects without PTSD. In contrast, Kawamura et al. (2001) found lower levels of IL-4 in 12 Japanese men compared to 48 men with similar life experiences but no PTSD. Weiss et al. (1996) found no differences in IL-4 production in 22 Israeli men experiencing missile attacks pre- to postwar. While there are gender differences in these samples, results are too preliminary and samples too small to reach any conclusions. Finally, one study looked at treatment effects on the cytokine levels in people with PTSD (Tucker et al., 2004). Patients with PTSD were randomized in a double-blind fashion (2 : 2 : 1) to treatment with citalopram (n = 19), sertraline (n = 18), or placebo (n = 7). Treatment was successful in lowering PTSD, depression, and IL-1β levels, and increased IL-2R compared to controls. 4. PTSD and Enumerative Immune Measures Studies have also investigated numbers of different phenotypes of immune system cells. Abnormally high leukocyte and lymphocyte counts (including higher T-cell/CD4 cells) were found by Boscarino and Chang (1999a) in a large study of 286 Vietnam combat vets with current partial chronic PTSD compared to 2,190 veteran controls without symptoms of PTSD, depression, or anxiety. Consistent with this, Ironson et al. (1997) found significantly higher white blood cell counts in people who had recently experienced Hurricane Andrew compared to laboratory controls. Boscarino also found that in a national sample of 2,490 Vietnam veterans, those with PTSD were more likely to have clinically higher T-cell counts. In contrast, Kawamura et al. (2001), who had much smaller samples (12 with PTSD and 48 without, matched on similar life experiences), found lower lymphocytes and T-cell counts in the group with PTSD, and Solomon et al. (1997) found a decrease in T-cell numbers over 4 months after an earthquake. Lower T-cells were associated with greater distress and disruption at the initial assessment. Ironson et al. (1997) found lower CD4 and CD8 cells 1 to 4 months after Hurricane Andrew compared to laboratory controls. McKinnon and Weisse (1989) also found lower CD8 T-cells in residents living in the vicinity of the Three Mile Island nuclear reactor disaster 6 years after the incident. In contrast, Maes et al. (1999) found no differences in CD8 cells between 13 people with PTSD from accidents (car crash or fire) and 32 healthy volunteers, as did a study by Wilson

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et al. (1999), which found that 10 women with PTSD and a history of childhood trauma did not show differences in numbers of peripheral blood lymphocytes or T, B, or NK subsets. However, it should be noted that the latter study was on women, and both Wilson et al. (1999) and Maes et al. (1999) had much smaller sample sizes than the three studies showing differences. Another interesting finding of the Wilson et al. (1999) study was that although there were no differences in the number of PBL or major subsets (T, B, or NK), people with PTSD had a higher ratio of CD45R0 cells to CD45RA cells, an indication of lymphocyte activation. This ratio of increased memory to naïve Tcells is not only an indication of T lymphocyte activation, but is increased with aging. In one of the few other studies investigating the naïve T-cell subset (CD4+/CD45RA), Dekaris et al. (1993) found the percentage of these cells was decreased in men just released from a prisoner of war camp. Dekaris et al. (1993) also reported high levels of activated T and B lymphocytes in 29 men liberated from a war prisoner camp in the Croatian War, as did Sabioncello et al. (2000) in 20 female civilians displaced by the Croatian War. The latter study also found a higher level of activated NK phenotypes in the displaced women versus 14 control female residents of Zagreb, Croatia, not directly exposed to trauma. Thus, weighing the studies by the sample sizes, it appears that trends are there for increased lymphocytes and leukocytes, alterations in cell phenotypes, especially T-cells, and with activation a distinct possibility. In a summary of the acute stress literature (Segerstrom and Miller, 2004), significant increases have been found for leukocytes, natural killer cells, lymphocytes, and T cytotoxic lymphocytes (CD8). A review of the chronic stress literature showed no significant changes in any of the subsets studied (Segerstrom and Miller, 2004). A few studies have also looked at NK cells. Wilson et al. (1999) found no differences; the large Boscarino and Chang study did not look at this subset. Two studies in trauma (without PTSD diagnosis) found increased NK cell numbers (Ironson et al., 1997; Sabioncello et al., 2000), but two others found lower NK cells (Solomon et al., 1997; McKinnon and Weisse, 1989). Solomon et al. (1997) found a decrease in NK cell numbers over 4 months after an earthquake. A re-examination of this study showed that those in a high worry group had lower NK cell numbers than those in the low worry group or laboratory controls (Segerstrom et al., 1998). McKinnon and Weisse (1989) found that residents who lived near the Three Mile Island nuclear reactor disaster had lower NK cells as compared to control people 80 miles away, even 6 years after the disaster.

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5. Other Measures A few other immune-related measures were included in other studies. Residents living near the Three Mile Island nuclear reactor had higher antibody titers to herpes simplex virus and cytomegalovirus (McKinnon and Weisse, 1989) compared to people living in control cities 80 miles away, suggesting worse control over these latent viruses. They further determined that the results were not just due to immune activation, as there were no differences of the TMI and control groups on antibodies to rubella virus, IgG, and IgM. Plasma antinuclear antibodies were examined in one study in which girls who had been sexually abused for an average of 23 months were compared to healthy adult women (DeBellis et al., 1996). The abused girls were found to have higher levels of plasma antinuclear antibodies, which is suggestive of B-cell dysfunction. One study (Miller et al., 2001) found a measure of inflammation (C-reactive protein or CRP) to be correlated with intrusion scores, thus lending support to the notion that PTSD symptoms may be associated with increases in inflammatory substances. Weiss et al. (1996) found higher cell-mediated lympholysis (CML) in 22 men who experienced the SCUD missile attacks in Israel during the 1991 Persian Gulf War versus postwar, thus indicating enhanced T-cell lytic capacity of PBMCs. Finally, Dekaris et al. (1993) investigated phagocytic functioning of polymorphonuclear neutrophilic leukocytes (ingestion, digestion, and antibodydependent cellular cytotoxic [ADCC] abilities toward oopsonized sheep red blood cells) and found that ingestion and digestion (but not ADCC) were significantly depressed in 29 liberated detainees for a prisoner of war camp compared to 15 healthy controls and pre-war historical controls. 6. Some Methodological Issues Before concluding this section, some important methodological issues need to be raised in interpreting PTSD immune interactions. First, many of these studies were done with very small sample sizes. The larger samples were only able to measure a limited number of variables. Second, as noted above, characteristics of the trauma need to be taken into account, such as duration of the trauma, age at which trauma occurred, time from trauma to measurement, etc. Third, there are many potential confounders that occur with PTSD and are known to affect the immune system. For example, psychiatric disorders are comorbid with PTSD. Prominent among these are major depression and generalized anxiety disorder (David et al., 1996; Kessler et al., 1995). These disorders have been known to affect the

immune systems in their own right (Irwin et al., 1990) and may impact the immune system above the effects of PTSD alone. For example, Maes et al. (1999) found that PTSD patients with concomitant major depression have a distinct immune inflammatory profile. Serum IL-6R was higher in patients with PTSD and depression than in those with PTSD alone. In one of the few other studies that tried to disentangle these effects, Boscarino and Chang (1999a) attempted to compare veterans with and without PTSD as well as with and without an anxiety disorder (presumably excluding PTSD), and with and without depression. Differences were found only between the PTSD and no PTSD groups after controlling for sociodemographic and health behaviors (drug use and alcohol consumption). As noted above, those veterans with PTSD had higher WBC’s lymphocytes and T-cell counts than those without PTSD but did not differ on the CMI multi-test. Another comorbid condition found in PTSD that affects the immune system is alcohol and drug use (Irwin et al., 1990). It is interesting to note that in a study by Mosnaim et al. (1993), which looked at NKCC (after 10 days or more of detox), there were no differences between a group of veterans with PTSD (all of whom had a history of substance abuse), several control groups from the hospital’s Substance Abuse Unit (a control group with drug use, another control with alcohol use, and a third with drug and alcohol use), and drug-free, healthy controls. Note, however, NKCC response to stimulation with methionine-enkephalin (MET) was decreased in Vietnam War veterans with PTSD and comorbid drug use (Mosnaim et al., 1993) 10 days or more into a drug detox program. Thus, confounds such as psychopathology and alcohol and substance use need to be accounted for before being able to interpret associations with PTSD as being due to the stress of PTSD. In addition to psychiatric conditions comorbid with PTSD, another variable that may have a confounding effect is sleep. Sleep has been found to be disrupted in trauma (Davidson et al., 1987) and is an important immunomodulator (Irwin et al., 1994). In fact, Ironson et al. (1997) found that sleep disturbance mediated the effect of post-traumatic stress symptoms on NKCC in a community sample of people experiencing Hurricane Andrew. Finally, nutritional status is another potential confounder, especially, for example, when comparing prisoners of war to non-combat veterans (Dekaris et al., 1993). In addition to nutritional status as an immune modulator and potential confounder, Kiecolt-Glaser and Glaser (1988) described other important confounders to consider when doing human PNI work.


II. PTSD: ENDOCRINE A. Hypothalamic-Pituitary-Adrenal (HPA) Axis and PTSD There is a substantial body of evidence demonstrating altered functioning of the hypothalamic-pituitaryadrenal (HPA) axis among individuals diagnosed with post-traumatic stress disorder. HPA axis functioning has been assessed within a variety of PTSD populations through various methods, such as determining the levels of specific HPA axis hormones (e.g., cortisol), examining the response of the negative feedback mechanism of the HPA axis to a number of challenge and stimulation tests, and also evaluating the number and sensitivity of glucocorticoid receptors. Following is a summary of some of the main findings in this area. It is important to note that the complex nature of PTSD, including a wide range of traumatic events and responses to trauma; a diversity of symptoms and possible co-occurrence of depression, substance use, and sleep disruption; and also a broad array of demographic and individual difference (e.g., personality, coping, support) variables among subjects and comparison groups, poses a challenge in fully discerning HPA axis functioning among individuals diagnosed with PTSD. 1. Cortisol Levels Because cortisol is one of the major stress hormones produced by the HPA axis, the vast majority of studies have assessed the cortisol levels of PTSD patients as a general indicator of HPA axis activity. In early studies of male Vietnam veterans, lower 24-hour urinary cortisol output was observed among combat veterans with PTSD compared to psychiatric patients without combat experience, combat veterans without PTSD, and also normal control subjects (Kosten et al., 1990; Mason et al., 1986; Yehuda et al., 1990; Yehuda et al., 1993b). Reductions in urinary cortisol output have also been reported among other PTSD populations as well, such as Holocaust survivors and the adult children of Holocaust survivors (Yehuda et al., 1995b; Yehuda et al., 2000). However, some studies have reported higher levels of urinary cortisol output among combat veterans with PTSD compared with combat veterans without PTSD (Pittman and Orr, 1990), among women with PTSD resulting from sexual abuse compared to women victims of sexual abuse without PTSD and normal control subjects (Lemieux and Coe, 1995), among people living in the areas damaged by Hurricane Andrew (Ironson et al., 1996), among people living in areas close to the Three Mile Island damaged

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nuclear plant compared to control sites (Schaeffer and Baum, 1984), and among victims of flash fires and multiple car crashes with PTSD compared to psychiatric patients and normal control subjects (Maes et al., 1998). In fact, in the Ironson et al. (1996) study, reductions in cortisol over the course of a year were significantly correlated with the decrease in psychological symptoms of trauma (re-experiencing, intrusive thoughts, and avoidant thoughts). It is important to note that the bulk of the studies conducted in this topic area, regardless of whether cortisol increases or decreases, report fluctuations in cortisol levels that remain within the normal range. Studies assessing the cortisol levels of PTSD patients assayed from either plasma or saliva samples have also shown similarly mixed results. Lower morning cortisol levels have been observed among Vietnam veterans with PTSD (Boscarino, 1996), adult women traumatized by childhood sexual abuse (Stein et al., 1997), and also adolescents with PTSD as a result of the 1988 Armenian earthquake (Goenjian et al., 1996). However, some other studies have shown elevated levels of cortisol among PTSD patients compared to various comparison groups (Hoffman et al., 1989; Liberzon et al., 1999). Some studies have also examined the circadian rhythms of cortisol secretion in PTSD patients as well. An increased dynamic range in plasma cortisol levels (primarily due to lower levels in the late evening and early morning hours) has been observed among combat veterans with PTSD as compared to combat veterans with depression or nonpsychiatric comparison subjects (Yehuda et al., 1996b). However, it was recently reported that salivary cortisol levels were significantly lower at awakening, at 8:00 a.m., and at 8:00 p.m. in Holocaust survivors with PTSD than in non-survivors, which resulted in a flattening of the circadian rhythm (Yehuda et al., 2005). These results run contrary to those reported in younger combat veterans and may be particular to HPA axis alterations seen in aging as opposed to PTSD. Although there are conflicting findings, the majority of studies conducted to date seem to point to reductions in cortisol output among PTSD patients. Rachel Yehuda, one of the prominent researchers in this area, has noted that there are a number of methodological issues that may have led to these differing results across studies regardless of whether urine, plasma, or saliva samples were studied, including variability in the selection of subjects and comparison groups, different methods of collecting and assaying biological samples, and in some cases inadequate sample sizes and dissimilar inclusion/exclusion criteria (Yehuda, 2002). In addition, PTSD patients often vary in the

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degree of severity, duration, proximity, and type of traumatic event, and also gender (i.e., many of the early studies were of male Vietnam veterans), as well as the degree of certain coping strategies (i.e., dissociation) employed and availability of social support to deal with the traumatic event and its aftermath. Furthermore, PTSD, by definition, is composed of multiple symptoms with many patients also often contending with depression, substance-related issues, and sleep problems that can substantially disrupt the HPA axis and add to the complexity of quantifying a distinct hormonal profile of PTSD. Thus, while the majority of controlled studies conducted over the past 20 years seem to show hypocortisolism among PTSD patients (which runs contrary to what might be expected in a stress-related condition), there are notable exceptions. The circumstances under which trauma is associated with low or high cortisol are not currently clear. In addition, the role of cortisol in PTSD is not clear. It is not understood whether cortisol has any causal relationship (to PTSD symptoms) or is an outcome. Studies suggest that people with PTSD may have smaller hippocampal regions (Gurvitis et al., 1993). A smaller hippocampus in PTSD may be a result of neuronal loss, as has been reported using magnetic resonance imaging (MRI) with high resolution that is capable of distinguishing the amygdala from the hippocampus (Gurvitis et al., 1993). Could the smaller hippocampal area leave people susceptible to PTSD, or does PTSD result in a smaller hippocampal area? Sapolsky (1996) observed that among individuals with PTSD, there is no information as to the extent of the glucocorticoid levels during the trauma. Glucocorticoids are known to cause neural destruction in the brain (Sapolsky, 1996, 1997), especially in the hippocampal area, leading to a decrease in the size of the hippocampus. Alternately, given the cognitive role of the hippocampus and the association of a smaller hippocampus with both PTSD and depression (Steffens et al., 2000), could a smaller hippocampus lead to a greater disposition towards depression as well as PTSD? One possibility is that a smaller hippocampus may be associated with lack of signaling to the HPAC system, leading to lower levels of glucocorticoids. At any rate, this is an intriguing area that awaits further study. 2. Challenge and Stimulation Tests Researchers have also examined the HPA axis functioning of PTSD patients through evaluating how the negative feedback mechanism of the HPA axis responds to various challenge or stimulation tests. Because dexamethasone mimics the biological activity of cortisol,

the dexamethasone suppression test (DST) is commonly employed to assess the relative strength of this negative feedback mechanism, with elevated cortisol levels following DST indicative of a weaker negative feedback mechanism and diminished levels indicative of a stronger negative feedback mechanism. A number of DST studies using low doses of dexamethasone have shown an enhanced suppression of cortisol (i.e., lower cortisol levels) among combat veterans with PTSD compared to combat veterans without PTSD (Yehuda et al., 1993b; Yehuda et al., 1995a) and among adult female survivors of childhood sexual abuse compared to non-victimized women (Stein et al., 1997). Another study reported similar results among activeduty Gulf War soldiers (Kellner et al., 2000). In addition to the DST, several other challenge and stimulation tests have also shown an increase in the negative feedback mechanism among PTSD patients. For example, an exaggerated ACTH response to the metyrapone stimulation test (Yehuda et al., 1996a) and a blunted ACTH response to the CRH challenge test (Smith et al., 1989) have also been observed among PTSD patients. Overall, the challenge and stimulation tests have provided fairly consistent evidence of an increased negative feedback inhibition of the HPA axis among various PTSD populations. 3. Glucocorticoid Receptors To better understand HPA axis functioning in PTSD, researchers have also studied the number and sensitivity of Type II glucocorticoid receptors, because an increased number of these receptors is likely associated with both lower cortisol output and an increased sensitivity of the negative feedback loop of the HPA axis. In an early study, Vietnam veterans showed an increase in lymphocyte glucocorticoid receptor numbers compared to a normal control group (Yehuda et al., 1991). Recently, hypocortisolism among Bosnian refugees with PTSD was associated with increased glucocorticoid sensitivity of interleukin-6 (IL-6) production (Rohleder et al., 2004). These findings further support the notion of enhanced negative feedback inhibition and hypocortisolism in PTSD. 4. Conclusion: HPAC Section Overall, the majority of studies conducted over the past 20 years seem to show significant alterations in HPA axis functioning among a variety of PTSD populations. HPA axis dysregulation among PTSD patients seems to point primarily to hypocortisolism, enhanced negative feedback inhibition, and an increase in lymphocyte Type II glucocorticoid receptor number and sensitivity. It appears that the HPA axis may become


hypersensitive during and after exposure to trauma, which then perhaps leads to an increase in the negative feedback loop, resulting in lower cortisol levels and an increase in glucocorticoid receptors. However, the exact temporal mechanism involved in this process is unclear at this time. Some studies have also reported altered circadian rhythms of both cortisol secretion and glucocorticoid receptor levels as well. Because PTSD patients often report difficulties with sleep and thus may have altered sleep-wake cycles, studies of circadian rhythms may help further elucidate the HPA axis alterations associated with PTSD. There is also evidence that altered HPA axis functioning developing either before or in response to a traumatic event may be associated with the risk of developing PTSD or PTSD symptoms (Delahanty et al., 2005; Resnick et al., 1995; Yehuda et al., 1998). Some researchers have also hypothesized that the HPA axis alterations described above may also play a role in memory disturbances commonly reported by PTSD patients (Golier and Yehuda, 1998a). In summary, HPA alterations are often evidenced among PTSD patients, although exactly how this process unfolds and the extent of its impact are currently unclear.

B. Sympathetic-Adrenal-Medullary (SAM) System and PTSD PTSD often manifests with multiple symptoms of anxiety, and thus an increase in sympathetic-adrenalmedullary (SAM) system activation is commonly observed among PTSD patients. Among various PTSD populations, an increase in heart rate, blood pressure, startle response, and skin conductance have all been reported, especially in response to loud noises or from exposure to reminders of the traumatic event (Blanchard et al., 1996; Orr et al., 1997a, 1997b; Pitman et al., 1990). Similar to studies of the HPA axis discussed above, researchers have assessed the SAM system functioning of a variety of PTSD populations through a number of methods, such as determining the levels of sympathetic nervous system hormones (e.g., the catecholamines epinephrine and norepinephrine), examining catecholamine levels in response to stimulation tests, and also evaluating the number and sensitivity of noradrenergic receptors. Following is a summary of some of the main findings in this area. 1. Catecholamine Levels Because the release of catecholamines, such as epinephrine and norepinephrine, represents sympathetic activation and triggering of the fight/flight stress response, the vast majority of studies have assessed

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the catecholamine levels of PTSD patients in either urine or plasma samples as a general indicator of sympathetic nervous system functioning. An increase in both 24-hour urinary and plasma catecholamine output has been observed among combat veterans with PTSD compared to combat veterans without PTSD and normal controls (Kosten et al., 1987; Yehuda et al., 1998b), although not in all studies (Pittman and Orr, 1990). Plasma catecholamine levels have also been shown to be elevated in combat veterans with PTSD in response to stress as well (Blanchard et al., 1991; McFall et al., 1990). In a recent examination of urinary catecholamines among a community sample of PTSD patients over a 10-year period, individuals with lifetime PTSD had significantly higher catecholamine levels than the group exposed to trauma without PTSD and also a non-exposed group (Young and Breslau, 2004). In an interesting study, Mellman et al. (1995) collected 24-hour urine in three 8-hour collections from combat related PTSD patients and analyzed these samples for 3-methoxy-4-hydroxyphenylglycol (MHPG), the main metabolite of norepinephrine. They observed a higher level of MHPG in the nocturnal urine sample. In addition, nocturnal MHPG correlated negatively with the total sleep time in the PTSD patients. These findings are important, since awakening from sleep, exhibiting an exaggerated startle response, and having increased vigilance are among the characteristics of PTSD. All these symptoms might be a result of higher levels of nocturnal catecholamines. Elevated urinary norepinephrine had also been observed among maltreated children with PTSD (DeBellis et al., 1997) and among people living in areas of high damage after Hurricane Andrew (Ironson et al., 1996). Overall, the vast majority of studies conducted show significant sympathetic activation and increased catecholamine output among a variety of PTSD populations. 2. Stimulation Tests Researchers have utilized stimulation tests, such as the administration of agents (e.g., yohimbine) that induce SAM system activation, to help fully understand sympathetic nervous system response in PTSD. Yohimbine is an antagonist that blocks alpha-2– adrenergic receptor sites and thus exacerbates the sympathetic nervous system. The use of yohimbine results in panic or anxiety attacks in 70% of combat veterans with PTSD and flashbacks in 40% of this population, which points to a heightened response of the SAM system in PTSD patients (Southwick et al., 1993). This also may indicate that an increased level of catecholamines may be associated with expression of spe-

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cific symptoms and that perhaps at least some PTSD patients are hypersensitive to noradrenergic stimulation. 3. Noradrenergic Receptors Researchers have also examined SAM system functioning of PTSD patients through evaluation of the number of binding sites and sensitivity of noradrenergic receptors. Both combat veterans and children with PTSD have shown fewer alpha-2–adrenergic receptor binding sites per platelet than controls (Perry, 1994; Perry et al., 1987). The reduction in number of receptors in PTSD is likely due to an increased degradation caused by elevated catecholamine levels. Thus, compared to normal control subjects, patients with PTSD may have receptors that are hypersensitive to circulating catecholamines. 4. Conclusion: SAM Section In summary, the vast majority of studies conducted seem to show heightened activation of the SAM system among a variety of PTSD populations. Sympathetic activation, including increased heart rate, blood pressure, startle response, and skin conductance, are consistently reported among PTSD patients. In addition, higher catecholamine output, a supersensitivity to noradrenergic stimulation with yohimbine, and a degradation of alpha-2–adrenergic receptors are also evidence of SAM system activation in PTSD. As expected in a condition commonly characterized by heightened anxiety, fear, and exaggerated startle response, the SAM system appears to be chronically activated and hypersensitive to stress states, including reminders of the initial trauma. Heightened SAM system activation, particularly increased heart rate or catecholamine level, either before or in response to a traumatic event, may be associated with the risk of developing PTSD or PTSD symptoms (Blanchard et al., 1996; Bryant et al., 2000; Delahanty et al., 2003; Hawk et al., 2000; Shalev et al., 1998). Some researchers have also hypothesized that the sympathetic activation described above may reflect an increase in arousal, fear conditioning, and perhaps even problems in memory formation often reported by PTSD patients (O’Donnell et al., 2004). Overall, SAM system activation is commonly observed among PTSD patients, although the extent of its impact is currently unclear.

III. ENDOCRINE-IMMUNE INTERACTIONS IN PTSD Although many studies have examined the connection between the endocrine system and immune func-

tion across a number of psychiatric and medical conditions and also among healthy individuals, relatively few studies have done so among PTSD populations. However, data have started to emerge linking the alterations in the HPA axis and the SAM system noted above with a number of autoimmune and inflammatory diseases regulated by the immune system. A recent study in a national sample of 2,490 Vietnam veterans reported an association with PTSD and the prevalence of common autoimmune diseases, including rheumatoid arthritis, psoriasis, insulindependent diabetes, and thyroid disease (Boscarino et al., 2004). The author of this study also found evidence of endocrine and immune alterations in the sample as well as both cardiovascular and autoimmune diseases. There is also evidence that pro-inflammatory cytokines may play a role in the association between the endocrine-immune interactions (Chrousos, 1998) and perhaps subsequent health status in PTSD. Alterations in cytokines have been implicated in many disorders including rheumatoid arthritis (Feldmann et al., 1996), inflammatory bowel disease (McClane and Rombeau, 1999), and coronary heart disease (Yudkin et al., 2000). HPA axis dysfunction in PTSD, including insufficient glucocorticoid signaling and impaired feedback regulation, resulting in immune activation/inflammation, may in turn contribute to stress-related pathology, including alterations in behavior, insulin sensitivity, bone metabolism, and acquired immune responses (Raison and Miller, 2003). For example, in Bosnian War refugees with PTSD, hypocortisolism was associated with increased glucocorticoid sensitivity of pro-inflammatory cytokine (i.e., interleukin-6, IL-6) production (Rohleder et al., 2004). The relationship between cortisol, norepinephrine, and elevated pro-inflammatory cytokines is an intriguing area to be explored. Despite high NE levels, a measure of central nervous system activation, the HPA axis appears to remain unstimulated, as evidenced by low levels of cortisol. Despite this, there is an elevation of the secretion of TNF-alpha, suggesting possible mechanisms could be either elevated catecholamine or reduced glucocorticoids. There is a good deal of evidence that glucocorticoids suppress production of the IL-1β and TNF-α (Knudson et al., 1987; Lee et al., 1988; Waage and Bakke, 1988; Zuckerman, 1989), so the increase in pro-inflammatory cytokines could potentially be a function of low glucocorticoids. With respect to catecholamines, high catecholamines have been associated with elevated IL-6 in heart failure (MullerWerden and Werden, 2000), and catecholamines stimulate IL-6 production in rat cardiac fibroblasts (Burger et al., 2001). Altered IL-6 production has also been associated with norepinephrine levels among combat


veterans with PTSD as well (Baker et al., 2001), although the sample size for that was very small (n = 11). In addition, Severn et al. (1992) found that preexposure to adrenaline and B-adrenergic agonists increased TNF-alpha production. However, to further complicate matters, a recent study (Rontgen et al., 2004) showed the effects of epinephrine and norepinephrine on monocytes stimulated with LPS were associated acutely with inhibition of IL-6 and TNFalpha. As this was an acute in vitro study, the relevance for PTSD may be limited. Future studies might help to unravel the precise mechanism. Stress-induced changes in hormones (such as cortisol and catecholamines) and also cytokines in Gulf War veterans have been linked to specific immunerelated illnesses reported by these veterans, such as skin and joint problems (Everson et al., 1999). Overall, there appears to be an association between HPA axis and SAM system alterations and changes in immune functioning linked to certain illnesses in PTSD patients, but the work is preliminary, and many of the studies have been done on small sample sizes. Future work is need to help further clarify the mechanisms linking endocrine and immune changes observed in PTSD and physical and medical health status of these individuals.

IV. CONCLUSION It is clear that there are immune and endocrine alterations associated with PTSD. For most of the findings, there are some studies showing a trend in one direction with others showing no difference. Thus, trends are found for an increase in pro-inflammatory cytokines, enhanced DTH, decreased proliferation to PHA, and increased antibody titers to latent viruses. Some of these findings are consistent with enhanced immunity, such as enhanced DTH, an indication of cell-mediated immunity. Others are consistent with poorer immunity, such as decreased proliferation to PHA or increased antibody titers to latent viruses, suggesting poorer control of latent viruses. Alterations in enumerative immune measures have also been found, the most notable of which are increases in leukocytes, lymphocytes, and T-cells. Activation is also a distinct possibility, as indicated by the presence of particular cell surface markers. Finally, alterations of the immune system in PTSD are sometimes consistent with what is found in acute stress, while others are consistent with what is found in chronic stress (Segerstrom and Miller, 2004). The endocrine findings are intriguing as well. Alterations in both HPAC and SAM systems have been found in PTSD. Perhaps the most consistent is elevated

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catecholamines, which is associated with sympathetic activation. A product of another major stress hormonal system (the HPAC system), cortisol, shows less consistent results, although the majority have shown low cortisol. This stands in contrast to the stress literature in which cortisol tends to be elevated. The elucidation of why and when low versus high cortisol is present remains to be discovered. Studies of linkages of immune alterations in PTSD with specific diseases are few. However, there is a potential link between altered cytokines and diseases that are more prevalent in PTSD, such as inflammatory and autoimmune disorders. The relationship between cortisol, norepinephrine, epinephrine, and proinflammatory cytokines is an area that holds potential promise. Much of the above needs to be regarded as preliminary, as many studies with immune measures have been done with very small sample sizes. Some of the larger studies have often been limited to a few immune measures, such as DTH, leukocyte, and lymphocyte counts. It is difficult to make conclusions from much of this literature, as the findings are somewhat inconsistent. At a minimum a few suggestions for future research can be made: First, larger sample sizes are needed. Second, major confounders need to be taken into account in studies of PTSD. These include, at a minimum, psychiatric disorders such as depression, anxiety, and drug and alcohol use/abuse/dependence, all of which are known to be comorbid with PTSD and are known to impact the immune system. While some researchers have attempted to sort this out, more needs to be done. Other confounders impacting on the immune system and particularly relevant to PTSD studies include impaired sleep, poor nutrition (e.g., such as in being a prisoner of war), and age at assessment. In addition, future studies of PTSD need to determine the possible impact of elapsed time between the assessment of the immune system in relation to the original trauma, the duration of the trauma, and the severity of the trauma. Furthermore, it would be useful to have assessments before and after the trauma. While this is not feasible in most traumatic situations, it may be feasible in some (i.e., combat). Choice of immune measures in studies of PTSD should be guided both by the measures of interest for the disease one is studying (i.e., cancer, rheumatoid arthritis, cardiovascular disease), as well as the measures that are likely to be altered in acute or chronic stress (Segerstrom and Miller, 2004). Studies treating trauma and determining the impact on immune measures would also be helpful. This field is really in its early development, and there lies much promise ahead, especially because there are good treatments available for PTSD (Chard and Gilman, 2005; Ironson et al.,

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2002) and the potential impact on illness remains a possibility for exploration.

Acknowledgments The authors wish to thank Grisell Hernandez and Elizabeth Balbin for their help in compiling the literature and constructing the table, and Joe Paroulo for checking references. We also thank NIMH for their support (R01MH066697) to Gail Ironson, P. I.

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nephrine and 3-methoxy-4-hydroxyphenylglycol concentrations and severity of depression in combat posttraumatic stress disorder and major depressive disorder. Biol. Psychiatry, 44, 56–63. Yehuda, R., Southwick, S. M., Krystal, J. H., Bremner, D., Carney, D. S., and Mason, J. W. (1993b). Enhanced suppression of cortisol following dexamethasone administration in posttraumatic stress disorder. Am. J. Psychiatry, 150, 83–86. Yehuda, R., Southwick, S. M., Nussbaum, G., Wahby, V., Giller, E. L., and Mason, J. W. (1990). Low urinary cortisol excretion in patients with posttraumatic stress disorder. J. Nerv. Ment. Dis., 178, 366–369. Yehuda, R., Teicher, M. H., Trestman, R. L., Levengood, R. A., and Siever, L. J. (1996b). Cortisol regulation in posttraumatic stress disorder and major depression: A chronobiological analysis. Biol. Psychiatry, 40, 79–88. Young, E. A., and Breslau, N. (2004). Cortisol and catecholamines in posttraumatic stress disorder: An epidemiologic community study. Arch. Gen. Psychiatry, 61, 394–401. Yudkin, J. S., Kumari, M., Humphries, S. E., and Mohamed-Ali, V. (2000). Inflammation, obesity, stress and coronary heart disease: Is interleukin-6 the link? Atherosclerosis, 148, 209–214. Zuckerman, S. H., Shellhaas, J., and Butler, L. D. (1989). Differential regulation of lipopolysaccharide induced interleukin-1 and TNF synthesis: Effects of endogenous and exogenous glucocorticoids and the role of the pituitary-adrenal axis. Eur. J. Immunol., 19, 301–305.


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26 Psychoneuroimmunologic Aspects of Alcohol and Substance Abuse STEVEN J. SCHLEIFER

I. INTRODUCTION 549 II. ALCOHOL, ALCOHOLISM, AND IMMUNITY 550 III. ALCOHOL, ALCOHOLISM, AND IMMUNITY: MEDIATING AND MODULATING FACTORS 552 IV. ALCOHOL, ALCOHOLISM, AND IMMUNITY: MECHANISMS 553 V. ALCOHOLISM AND IMMUNITY: CONSEQUENCES FOR DISEASE 554 VI. STIMULANTS AND IMMUNITY 555 VII. NICOTINE AND IMMUNITY 556 VIII. OPIATES AND IMMUNITY 556 IX. MARIJUANA AND OTHER SUBSTANCES 557 X. CONCLUDING COMMENTS 557

while acute exposure to alcohol has demonstrable immune effects, chronic alcohol abusers tend to show immune alterations of only modest scope in the absence of liver disease or other comorbid medical conditions (Cook et al., 1997; Kronfol et al., 1993; Schleifer et al., 1999; Stefanini et al., 1989; Watson et al., 1985). Direct effects of alcohol include those resulting from acute exposure of immune tissues to alcohol as well as cumulative effects of chronic exposure. Some substances (e.g., opioids) are most potent in naive subjects, while others (e.g., nicotine) may show immune cell effects only after extended exposure (Sharp, 1998). Effects of acute or subacute substance withdrawal may actually be more pronounced than that of drug exposure itself. Drug exposure and withdrawal may operate centrally (CNS) as well as at the immune-cell level. Chronic exposure may, similarly, lead to immune tolerance through central as well as direct immune-cell processes. Still other categories of indirect immune effects of alcohol and other substances are associated with toxicity to somatic tissues (e.g., liver, hematopoietic system), substance-induced nutritional or hormonal changes, or somatic consequences of druginduced high-risk behavior (resulting in physical trauma or exposure to infection, such as HIV). The immune effects of a substance may be additive or synergistic with those of other drugs used in combination with the index agent (e.g., alcohol plus cocaine) or be additive or interact with immune effects of co-occurring clinical conditions (e.g., depressive disorders).

I. INTRODUCTION Substance use, abuse, and dependence have long been associated with increased risk of disease, some of which (e.g., alcoholic hepatitis, cirrhosis) may be directly attributable to toxic effects of the abused substance. Increased prevalence of medical disorders such as infectious diseases is also associated with alcoholism or other substance abuse, and has been attributed, in part, to altered immunity in substance abusers (Arria et al., 1991; Cook, 1998; Irwin, 2002). Alcohol abuse, the main focus of this chapter, is among the more intensively studied paradigms and is a major public health issue worldwide. Changes in the immune system and in immunerelated medical disorders in alcoholics are associated with complex direct and indirect effects. For example, PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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For alcohol, and possibly other drugs, it may be essential to distinguish effects of alcohol exposure from those associated with alcoholism as a syndrome. Such syndromal states may have genetic and constitutional (e.g., CNS) characteristics with immune correlates independent of the drug exposure. Some syndromes, such as alcoholism, appear to have clinical/biological subtypes, with potentially distinct immune consequences. One clinically important subgrouping distinguishes early- from late-onset alcoholism, early-onset disorders showing distinct cognitive, behavioral, and affective patterns and more prominent CNS and familial effects (Cloninger, 1987). The various clinical dimensions of substance use provide a context for interpreting discordant observations in the psychoimmunologic literature. They also dictate a need for caution in generalizing across clinical settings, especially if the index study assesses acute substance exposure in non-abusing subjects. Selection bias in clinical samples, most notably with crosssectional designs, must also be considered. For example, the only modest immune effects found in studies of otherwise healthy chronic alcoholics would appear to result from adaptation of immune tissues to drug exposure. They may also, however, reflect differential resistance to toxic drug effects, resulting in sustained good health (hence, study eligibility) for chronic abusers with more robust baseline immune function. The current chapter will consider primarily those PNI effects that appear to be a consequence of substance exposure or syndromal substance abuse rather than secondary consequences of medical conditions such as alcoholic liver disease. Immune-related clinical consequences of drug abuse will be reviewed in brief.

II. ALCOHOL, ALCOHOLISM, AND IMMUNITY A. Lymphocytes and Adaptive Immunity Increased circulating immunoglobulins were among the earliest described immune abnormalities in alcoholics. As with many other reported immune changes, these appeared to be largely associated with alcoholic liver disease (Cook, 1998), although increased IgE levels independent of liver dysfunction have been described (Dominguez-Santalla et al., 2001). There are many reports of altered lymphocyte distribution in patients with moderate to advanced alcoholic liver disease (ALD). Roselle and colleagues (1988) found decreased CD4+, CD8+, and CD3+ cells in hospitalized patients with alcoholic hepatitis. In a mixed

sample of alcoholics with and without liver disease, Cook and colleagues (1995) found decreased CD3+ cell subsets (L-selectin CD8+, CD45RA+) compared with non-alcoholic volunteers. Laso and colleagues (1996) found increased HLA DR expression, increased circulating (CD56+) NK cells, and decreased (CD19+) Bcells in persons with alcoholic cirrhosis. In contrast to the changes in persons with alcoholic liver disease or other comorbid disorders, there is only modest evidence for altered peripheral blood lymphocytes and lymphocyte subsets in alcoholic patients without liver disease (AWLDs) (Cook, 1998). Some studies suggest a shift toward an inflammatory state in alcoholics, possibly with “incomplete activation” of T-cells and moderately reduced B-cells. Chronic AWLDs from an ambulatory clinic in Barcelona (Sacanella et al., 1998) showed decreased B-cell markers and increased markers of T-cell activation, including CD69, CD25, sIL-2R, and HLA-DR. Other markers of functional immune activation were not elevated (IL-6, CD71). Cook and co-workers, at a rural U.S. Veterans Hospital, studied AWLDs admitted for inpatient detoxification and similarly found evidence for activated T-cells (Cook et al., 1991) and a reduction in some CD45RA+ markers representing suppressor inducer or naive cells (Cook et al., 1994). Our group examined in vitro immune measures in alcoholdependent persons, without liver or other medical disorders and free of other substance abuse, who were seeking treatment at an inner-city alcohol treatment center. No differences between alcohol-dependent and community control subjects were found in circulating leukocyte and lymphocyte subsets (Schleifer et al., 1999). In contrast, alcohol-dependent persons from the same clinic with evidence of even mild medical disorder (mostly mild liver abnormalities) showed evidence of immune activation, including increased HLA-DR+ cells and decreased total CD45RA+ cells (Schleifer et al., 2002). These subjects, however, also consumed more alcohol than the alcoholics with no evidence of liver disease. (Absolute levels of activation markers intermediate between those of the healthy non-abusers and alcoholics with mild medical abnormalities were found for the AWLDs; however, those differences were not statistically significant.) Adhesion markers in peripheral blood mononuclear cells, which are associated with cellular interactions and passage into the periphery, may also differ in alcoholics. In AWLDs, Cook and co-workers (1995) described changes in the distribution of L-selectin, CD11b, CD31, and CD57 cells, and Sacanella et al. (1998) found increased CD29+ cells and VLA proteins. These observations, together with the increased CD45RO+ (memory) cells found by both groups, are

26. Psychoneuroimmunologic Aspects of Alcohol and Substance Abuse

consistent with increased immune activation even in the absence of liver disease. Mitogen responses have been among the commonly studied non-specific measures of immune activation in PNI research. Lower mitogen response has been reported in alcoholic patients but appears to be a consequence of liver or other organ dysfunction (Lundy et al., 1975; Mutchnick and Lee, 1988; Stefanini et al., 1989; Watson et al., 1985). In our study, AWLDs did not differ from a healthy community comparison sample in PHA, ConA, or PWM responses (Schleifer et al., 1999), nor were effects seen in the alcoholdependent sample with mild liver dysfunction and greater alcohol consumption (Schleifer et al., 2002). Studies of cytokine levels and expression in relation to alcohol exposure and alcoholism, consistent with studies of other immune parameters, suggest that alcoholic patients with liver disease have substantially altered circulating cytokines, with only modest effects in AWLDs. Khoruts and co-workers (1991), in a Midwestern U.S. Veterans Hospital, found that chronic alcoholic patients, 1–2 weeks after admission to a drug dependency unit, did not differ from healthy nonabusers in circulating TNF-α, IL1-α, and IL-6 levels. Patients with alcoholic and other liver diseases, however, showed dramatic increases in these cytokines. A study in Athens of hospitalized AWLDs after about 24 hours of abstinence (Nicolaou et al., 2004) showed no changes in TNF-α, IL-8, IL-10, or IL-12. Moderate increases were found in IL-6, possibly related to the much briefer period of abstinence compared with the earlier study. Accordingly, the subjects in Athens were likely to be experiencing early acute withdrawal, a powerful stressor that may have induced IL-6. Another study of hospitalized AWLDs, in Antwerp (Song et al., 1999), however, found increases in both pro-inflammatory (IL-6, TNF-α) and negative immunomodulators (IL-10, IL-1RA) as long as 1 month after alcohol detoxification (increased circulating neutrophils were also found). In that study, the authors speculated that comorbid depressive disorders contributed to the observations (see below). Finally, studies of actively drinking AWLDs, in Salamanca, showed increased serum IL-12 (Laso et al., 1998) and increased intracellular IFN-γ and IL-2 (Laso et al., 1999). In sum, while both clinical conditions and immune assays are not directly comparable across studies (e.g., Cook, 2000), and the data are still limited in scope, the studies suggest that there is modestly increased Th1 activation in alcoholism even in the absence of liver disease. Experimental studies demonstrating different effects of acute alcohol exposure on Th1–Th2 balance as a function of immunoregulatory status at the time

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of exposure (Girouard et al., 1998) emphasize the need to explore the role of chronic alcohol use from a dynamic perspective.

B. Innate Immunity 1. Natural Killer Cell Activity NK cell activity is among the most sensitive immune components to stress, depression, and other behavioral factors (Herbert and Cohen, 1993a, 1993b). As with other immune measures, decreased NKCA is readily demonstrable in advanced liver disease (Charpentier et al., 1984), with variable findings in alcoholics without liver or other medical disorders. Irwin and co-workers (1990) found decreased NKCA in hospitalized alcoholic men, while Cook and co-workers (1997) found no changes in hospitalized AWLDs in either NKCA or the number of (CD56+) NK cells. In a study of actively drinking ambulatory AWLDs, our group also found no differences from community controls in NKCA and circulating CD56+ cells (Schleifer et al., 1999). Kronfol and co-workers (1993), studying a more medically heterogeneous but mostly healthy alcoholic sample, similarly found no differences in NKCA compared with non-alcoholics. Laso and co-workers (1997), however, found increased NKCA and circulating NK cells in actively drinking AWLDs. In that study, the differences persisted 3 months after alcohol withdrawal, suggesting that effects were unrelated to acute or ongoing alcohol exposure or to acute withdrawal. As with other immune measures, the influence of acute versus chronic alcohol exposure, drug withdrawal, intercurrent medical conditions, and syndromal alcoholism is likely of importance. These and other modulating factors such as age, gender, and genetic factors are reviewed below. 2. Phagocytic Function The studies reviewed have emphasized the modest impact of alcohol use and alcoholism per se on most immune functions. In contrast, alcohol may have a more substantial influence on phagocytic functions (phagocytosis and intracellular killing by phagocytic cells, but not antigen processing by phagocytic cells), granulocytes playing a key role in protecting against the bacterial infections to which alcohol abusers are especially susceptible (Adams and Jordan, 1984; MacGregor, 1986). Alcohol effects on granulocyte function have been reported in vitro, including in human cells (Jareo et al., 1996; MacGregor, 1986; MacGregor et al., 1978; Patel et al., 1996), but there is still limited research data on such functions in alcohol-dependent persons. One study found alterations in chemotaxis but not

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phagocytosis in a small, uncontrolled alcoholic sample (MacGregor et al., 1978). In our study of ambulatory alcohol-dependent persons, decreased phagocytosis was found in alcohol-dependent males, but not females, with no changes in intracellular bacterial killing (Schleifer et al., 1999). Such gender-specific effects are of note considering the differences in alcohol-related behavior in men and women (Schuckit et al., 1995) and gender differences in the immune system itself (Tollerud et al., 1989). There is some evidence that alcohol effects may differ among the phagocytic cells. A recent study of mostly male alcoholics, assessing phagocytosis and the release of reactive oxygen species (necessary for microbial killing), found that alcoholism affected PMNs preferentially (Parlesak et al., 2003). PMN, but not monocyte, phagocytosis was decreased in AWLDs (and ALDs), and chemiluminescence was increased in resting PMNs from AWLDs (and ALDs). Under resting conditions, monocytes from alcoholics showed no differences from healthy controls. Following LPS stimulation, however, monocytes as well as PMNs showed functional changes in the release of reactive oxygen species. Evidence for adaptation to alcohol effects on phagocytes was also found, with acute suppression following drug exposure in non-alcoholic but not alcoholic subjects.

III. ALCOHOL, ALCOHOLISM, AND IMMUNITY: MEDIATING AND MODULATING FACTORS The studies described have identified a number of factors associated with immune effects in patients with alcoholism. These include substantial influences for concurrent medical disorders and organ (e.g., hepatic) dysfunction and important effects associated with the chronicity and volume of alcohol exposure. The role of gender in the immune effects of alcohol and alcoholism in humans has occasioned some discussion, with limited systematic data. Studies of women have been few due to the overwhelming prevalence of males in most clinical patient samples (many U.S. studies from Veterans Administration Hospitals have been largely restricted to males). There is reason to expect different immune effects in women due to baseline gender differences in immunity (e.g., related to estrogen and testosterone levels) and increased susceptibility of women to alcohol-related liver injury (Kovacs and Messingham, 2002). Women also tend to show greater immune activation and inflammation than do men (Kovacs and Messingham, 2002). In rats, acute ethanol administration resulted in increased

PMN phagocytosis in female but not male rats (Spitzer and Zhang, 1996). This may provide a model for exploring the reported decreased phagocytosis in male but not female AWLDs (Schleifer et al., 1999). More research is required concerning the effects of age and aging on immunity in alcoholics. Studies of AWLDs have focused largely on subjects in the 35–50 year age range. Since age may play an important interacting role in PNI processes (Schleifer et al., 1989), studies of alcoholism and immunity across the life span are needed. The design of such research will be challenging since clinical longitudinal studies of aging per se are likely to be confounded by increasing cumulative exposure to alcohol with advancing age. Crosssectional studies are further confounded by sampling procedures that will select for those increasingly atypical subjects who have remained in good health with increasing age. A cohort effect may also complicate such research since, in recent decades, fewer persons abuse alcohol exclusively (e.g., without additional substances such as cocaine). Age-related alcoholism effects may also interact with alcoholism subtypes, some of which are age related (Cloninger, 1987), although we found no association between age of alcoholism onset and several immune measures (Schleifer et al., 1999). Other genetic predisposing factors, whether in relation to the immune system or alcoholism, may influence interactions between alcoholism and immunity. Animal studies have found reciprocal correlations between alcohol sensitivity and immune activity (Morato and Morato, 1993; Petitto et al., 1990). In humans, genetic polymorphisms have been associated with cytokine (TNF and IL-8) changes following alcohol exposure (Gonzalez-Quintela et al., 2004). Allelic differences in the IL-1 receptor have been associated with increased risk of alcoholism (Pastor et al., 2000). Genetic polymorphisms are of additional interest in relation to racial/ethnic background. AfricanAmericans tend to differ from Caucasians in circulating leukocytes (Tollerud et al., 1989) and Irwin and Miller (2000), studying detoxified alcoholics, found decreased NKCA and IL-6 in relation to African American background and alcoholism. The two effects were additive. African American alcoholics also had the highest IL-10 production compared with other groups. These data suggest that health risks may be greater for African Americans compared with Caucasians who become alcohol dependent. Behavioral factors such as stress and depression can also mediate or modulate the association between alcoholism and immunity. Some of these effects may be related to associated somatic symptoms and signs such as sleep disturbance, nutritional change, exercise, and activity level. There is much evidence that stress,


depressive disorders, and altered sleep (especially deprivation) influence the immune system (reviewed elsewhere in this volume), and the increased prevalence of such behavioral conditions in alcoholics may contribute to immune change. Nutritional deprivation in alcoholism may serve as a mediator and modulator of alcohol effects on immunity, as extensively considered in animal studies (e.g., Blank et al., 1991; Watzl and Watson, 1993). Sleep patterns are associated, bidirectionally, with cytokine production and other immune processes, and disordered sleep in alcoholics, a common phenomenon, is associated with altered cytokine levels and NKCA (Redwine et al., 2003). Further interactions result from the frequent experience of physical trauma in intoxicated states, with alcoholics shown to have increased morbidity and mortality following trauma such as burns (McGill et al., 1995). The clinical effects may be associated with additive, possibly synergistic, immunosuppressive effects of the trauma itself and alcohol exposure, and may be gender related (Kovacs et al., 2004; Messingham et al., 2000; Szabo et al., 1994). Approximately one-third of alcoholics suffer from major depression (Kessler et al., 1997; Penick et al., 1994), itself associated with immune system changes including increased circulating leukocytes, monocytes, and neutrophils; increased markers of immune activation and inflammation; and decreased NKCA and mitogen responses (Herbert and Cohen, 1993a; Zorrilla et al., 2001). Some of these effects may be mediated by clinical characteristics such as sleep disturbance that are common in depression (and in alcoholism) (Irwin, 2002). Irwin and co-workers (1990), comparing alcoholics with and without major depression, nonalcoholic depressed patients, and non-depressed non-alcoholics, found independent additive effects for depression and alcoholism in relation to decreased NKCA. Alcoholic and depressed patients also had increased circulating leukocytes and neutrophils, and alcoholics showed increased monocytes and lymphocytes. In our study of ambulatory alcohol-dependent persons, we found similar effects for depression, but more limited differences associated with alcoholism (Schleifer et al., 2006). Depression was associated independently with decreased PHA and possibly NKCA, and increased circulating monocytes. Independent effects of alcohol dependence included decreased Bcells, possibly decreased CD56+ (NK) cells, and increased monocytes, but no effects for NKCA or mitogen response. The immune correlates of alcoholism were largely attributable to recent alcohol exposure. Drug withdrawal is a clinical dimension of considerable importance (Laso et al., 1997). Withdrawal from

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alcohol is a state of extreme physiologic activation and substantial hormonal imbalance. In addition to periodic episodes of acute withdrawal, the day-to-day experience of the alcoholic, especially when access to the agent is variable, may involve continuing shifts in and out of mild to extreme states of withdrawal. These may have substantial immune consequences (Jerrels et al., 1989). Even the mild “hangover” associated with acute exposure in non-alcoholic subjects has been associated with immune alterations (increased IL-10, IL-12, IFN-γ) (Kim et al., 2003).

IV. ALCOHOL, ALCOHOLISM, AND IMMUNITY: MECHANISMS Biological mechanisms associating alcoholism and immunity are likely to vary in relation to the “clinical” dimensions already described (e.g., alcohol exposure, hepatic and hematologic pathology). Mediating and modulating physiologic processes may involve interactions at the systemic/central as well as the cellular level. Studies of direct cellular effects of alcohol exposure suggest that specific receptors are not involved, with drug effects associated with altered membrane fluidity (see, however, below). Ethanol exposure has been associated with decreased adenylyl cyclase activity in lymphocyte membranes (Pauly et al., 1999), altered erythrocyte membrane fatty acid composition, and decreased intracellular free calcium and inositol phosphates following lymphocyte stimulation (Stefanini et al., 1996). Jerrells and co-workers (1989), using an animal model, suggested, however, that the direct cellular effects of alcohol exposure are of limited importance under most conditions and that the most prominent immune effects relate to induction of stress hormones (e.g., corticosteroids) following alcohol withdrawal. Of further note is a report that decreased adenylyl cyclase activity itself is found primarily during acute alcohol withdrawal, with little change in actively drinking alcoholics or persons completely detoxified from the drug (Szegedi et al., 1998). An interesting other model suggests that hormonal effects, such as of opioids, both locally and centrally acting, are key to altered immune activity following chronic alcohol exposure. Rosenberger and colleagues (2003) demonstrated increased lymphocyte POMC mRNA in chronic alcoholics, together with increased LPS-stimulated IL-1RA. Following CRF injection, POMC mRNA increased in non-alcoholics but decreased in alcoholics. Chronic ethanol exposure inhibits hypothalamic CRF terminals, which recover after extended withdrawal from the drug, and both CRF and IL-1 induce the release of POMC-derived

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hormones (Schafer et al., 1996). Another intriguing interaction is suggested by altered levels of the cytokine-like peptide leptin in chronic alcoholics (possibly contributing to malnutrition and varying in relation to alcohol exposure and withdrawal), with a possible association of leptins with altered NKCA and other immune changes (Motivala et al., 2003; Nicolas et al., 2001). Some direct intracellular effects have also been described. Alcohol has been reported to induce suppression of NF-κb mediated production of inflammatory cytokines (Mandrekar et al., 2002). It was also found to augment glucocorticoid receptors (GR) while decreasing transactivation as a result of suppressed nuclear binding (Mandrekar et al., 2002).

V. ALCOHOLISM AND IMMUNITY: CONSEQUENCES FOR DISEASE The psychoneuroimmunology of alcoholism is often linked to its association with immune-related medical disorders. These include infectious diseases (bacterial pneumonia, tuberculosis, hepatitis C, possibly HIV), neoplasia, and autoimmune disorders (Cook, 1998). Autoimmune processes have been implicated in the development of alcoholic hepatitis and cirrhosis (Cook, 1998; Purohit et al., 2005), in turn associated, in a cruel irony, with impaired immunity. The association of alcoholism with morbidity and mortality, however, is not straightforward. There has been some (impassioned) debate concerning the relative health benefits and hazards of light to moderate alcohol use. There is little doubt, however, that alcohol-dependent persons have increased all-cause mortality (Dawson, 2000), including both “unnatural” deaths (traffic accidents, falls, suicide, homicide), of which one-third or more may be alcohol-associated (Sjogren et al., 2000), and death from disease. Mortality among alcoholdependent persons is three to five times higher than that of the general population, and longitudinal studies have found a broad array of (immunologically relevant) factors, from liver disease to divorced/separated status, to be predictive of mortality in alcoholics (Lewis et al., 1995). Although alcoholism is more prevalent in males, female alcoholics seem to be more susceptible to adverse medical outcomes, due in part to higher blood levels per unit ethanol consumed; alcohol-dependent women also face the prospect of inducing fetal alcohol syndromes in offspring (Schenker, 1997). Immune changes in alcoholism most often represent an exacerbating, rather than primary, element in the pathophysiology of infectious diseases (and other

medical conditions), and studies clearly linking alcoholism, immunity, and disease are few. For example, the prominent association between alcoholism and pneumonia relates mostly to the sedative effects of alcohol resulting in diminished clearance of respiratory tract pathogens. Immunologic factors are likely to exacerbate the process, impairing neutrophil migration to the lung. Recognized for many decades, alcohol’s effect on neutrophil migration is now considered not to result from direct cytotoxic effects, but rather from suppressed pulmonary chemokine production and impaired neutrophil adhesion (Boe et al., 2003). Other local immunologic effects, such as impaired alveolar macrophage cytokine production, contribute additionally to reduced host resistance (Omidvari et al., 1998). Alcohol-associated immune changes, including suppressed inflammatory cytokine responses, are especially important in magnifying the risk associated with other co-existing factors. For example, alcohol exacerbates the risk of infection following burns and other physical trauma (Faunce et al., 2003) as well as after elective surgery (Sander et al., 2002; Spies et al., 2004). The role of alcoholism in HIV disease has occasioned much speculation; however, there is only limited specific data concerning this disorder. Alcohol potentiates HIV infection of human monocyte-derived macrophages (Wang et al., 2002), and there is clinical data associating alcohol use with less effective response to anti-retroviral therapy. The latter, however, is likely to be a consequence of less reliable medication adherence rather than alcohol-impaired immune processes (Samet et al., 2003). Alcohol-associated immune alterations contribute to infections of the liver. In hepatitis C virus (HCV)infected patients, alcohol is associated with suppressed antigen-presenting cells, in which viral factors and alcohol interact to impair immune processing (Szabo et al., 2004). Alcohol use can reduce the efficacy of interferon therapy for HCV (Mochida et al., 1996) and of hepatitis B vaccines (Nalpas et al., 1993). Complex immune dysregulation associated with alcohol and alcoholism also appears to contribute to the pathogenesis of alcoholic hepatitis, which has been linked to autoimmune processes since the 1970s (Kakumu and Leevy, 1977). TNF-α and other cytokines are implicated in alcoholic as well as other chronic liver diseases, suggesting directions for therapeutic interventions (Neuman et al., 2001; Tilg and Diehl, 2000). A general exacerbating effect of alcoholism and alcohol use on allergic disorders has been reported, as well as increased IgE levels (Vidal et al., 2002). The association of alcohol and alcoholism with cancer risk is well described, especially for primary neoplasias of the upper gastrointestinal tract. Alcohol-


associated immune changes, however, have received little attention in relation to the pathogenesis of these disorders (Purohit et al., 2005). In contrast, there is intriguing data linking alcohol-induced immune changes with the course of established cancers. Studies in animals have found direct links between alcohol consumption, suppressed NKCA, and increased metastases for NK-sensitive tumors (Ben-Eliyahu et al., 1996; Hebert and Pruett, 2003). Such observations may have substantial clinical implications and call for systematic clinical study. In sum, theoretical and empirical considerations suggest that most immune changes in chronic alcoholics are attributable to clinical factors such as medical disorders, drug withdrawal, depression, sleep disturbances, nutritional state, and level of recent alcohol exposure. To the extent that alcohol-associated immune deficits are not intrinsic to the chronic alcoholic, (guarded) optimism concerning the health benefits of reducing alcohol use, and thereby its comorbidities (e.g., hepatitis), may be warranted.

VI. STIMULANTS AND IMMUNITY Substances with stimulant properties, especially cocaine and amphetamine, are among the more commonly abused recreational drugs. These drugs are associated with dramatic changes in the levels of catecholamines, with both central and peripheral effects and both neurotransmitter and neurohormonal properties. The immunoregulatory effects of catecholamines have been a focus of attention for many decades due to their association with sympathetic nervous system activation, making them important potential mediators of the immune effects of stress. Effects of norepinephrine on the immune system are readily demonstrated; however, they are complex, associated with increases or decreases in Th1 and Th2 responses as a function of exposure condition (Moynihan et al., 2004). Altered leukocyte trafficking is also involved (Dhabhar, 2002). Dopamine has important immunomodulatory effects comparable to those of norepinephrine (Torres et al., 2005). Stimulant exposure is also associated with activation of the HPA axis, which has well-characterized immune effects that interact with the direct immune effects of the catecholamines (Torres et al., 2005). A clinical rationale for studies of stimulants is provided by the increased incidence of HIV infection in cocaine abusers. Experimental studies, however, have failed to demonstrate cocaine effects at non-toxic levels on a number of immune-related medical disorders (Pellegrino and Bayer, 1998).

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Pellegrino and Bayer (1998) reviewed studies of cocaine effects on the immune system, which are not extensive and almost all using animal models. They suggest that cocaine is associated with decreased lymphocyte numbers but increased NK cells. Cocaine can suppress lymphocyte proliferation acutely, but tolerance is induced with repeated administration. The latter is of further interest since chronic cocaine administration induces cross-tolerance to the proliferationsuppressive effects of morphine (Bayer et al., 1996). Effects on cytokine production, even with acute exposure, are still more complex, with increased followed by decreased Th1 cytokines reported (Pellegrino and Bayer, 1998). A recent experimental study in cocaine users suggests that the net effect of acute cocaine exposure is suppressive of Th1 responses, with IL-6 release during acute inflammation substantially blocked by intravenous cocaine injection (Halpern et al., 2003). In contrast to these functions, NKCA does not appear very sensitive to cocaine (Pellegrino and Bayer, 1998). Cocaine effects on immunity are primarily centrally mediated, with shared mechanisms (hence, perhaps, cross-tolerance) with morphine effects, and are related to serotonergic activity (Bayer et al., 1996; Halpern et al., 2003). Nevertheless, in at least one model, cocaine effects on lymphocyte proliferation appeared to be a consequence of peripheral rather than central effects (Pellegrino et al., 2001). While few in number, immune observations in human cocaine abusers have been similar to those with alcoholics, showing little evidence of altered distribution of peripheral blood lymphocytes or lymphocyte responses (Ruiz et al., 1998), but some impairment of phagocytic function (alveolar macrophages) (Baldwin et al., 1997). Studies of amphetamine effects on immunity have also largely been confined to animals, generally as a model of stress. Acute administration is associated with decreased circulating lymphocytes and suppressed mitogen response, effects largely attributable to noradrenergic rather than corticosteroid mechanisms (Pezzone et al., 1992). NKCA was suppressed by amphetamines in some studies, most with acute exposure, and enhanced in others (Wrona et al., 2005). Enhancement is associated with chronic exposure and is more readily found in animals with greater motoric activity and aggressiveness. Amphetamine effects also relate to catecholamine activity, with lesser and indirect corticosteroid effects (Swerdlow et al., 1991; Wrona et al., 2005). Overall, the limited immune studies with stimulants support the pattern seen with alcohol, acute exposure having the clearest effects. The most important mechanisms appear to be associated with cate-

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cholamine release. It is unclear whether the immune effects of stimulant exposure are of substantial clinical relevance.

VII. NICOTINE AND IMMUNITY The recreational use of tobacco is a major risk factor for a host of diseases ranging from lung and other neoplasias to cardiovascular diseases, microbial infections, chronic obstructive pulmonary disease, and diseases of the fetus (Sopori, 2002). In addition to toxic effects on end organs such as the lung, tobacco has also been thought to have suppressive effects on immunity, and to contribute to reduced risk among smokers for some autoimmune disorders (Sopori, 2002). Decreased immune function is often found together with increased numbers of immune cells, possibly reflecting compensatory mechanisms. Since the earliest reports, cigarette smoking has been linked to leukocytosis (Corre et al., 1971). Smoking induces a shift in the balance of T-cell subsets (modulated by factors such as racial background) (Tollerud et al., 1991). It is associated with decreased lymphocyte proliferation (Petersen et al., 1983) and decreased immunoglobulin levels and specific antibody responses (McAllister-Sistilli et al., 1998). Increased numbers and decreased function have been found for alveolar macrophages (Sopori, 2002). Decreased NK cell activity, linked to increased risk for some cancers, has been reported in smokers, although findings have not been uniform (Ferson et al., 1979; Jung and Irwin, 1999). A provocative study by Jung and Irwin (1999), with depressed and non-depressed smokers and non-smokers, found that both leukocytosis and decreased NK activity were a function of the interaction of depression and smoking. Depressed persons have considerably increased rates of tobacco use, adding to the clinical concerns. The nature of the association between cigarette smoke and the immune system requires further study. Nicotine, an important component of cigarette smoke, has been associated with its immune effects; however, the importance of this link is not well established and many other potentially immunoactive substances are contained in cigarettes (McAllister-Sistilli et al., 1998; Sopori, 2002). Effects of nicotine on the immune system may be mediated by both peripheral and central mechanisms (Sopori et al., 1998), analogous to observations with most substances of abuse. Finally, most of the above studies have focused on chronic smokers, and acute exposure to cigarette smoke yields even more variable results (McAllister-Sistilli et al., 1998). As with many of the substances considered in this chapter, the association between tobacco use and

immunity cannot be considered in isolation but must consider the pattern of substance exposure, characteristics of persons who consume cigarettes (as modulating and confounding factors—e.g., depression, genetic/ethnic factors), and the role of direct versus indirect immune effects.

VIII. OPIATES AND IMMUNITY A large literature of more than 25 years (Vallejo et al., 2004; Wybran et al., 1979), with antecedent reports more than a century old (Vallejo et al., 2004), has emphasized the relationship between opioids and immunity. Similarities as well as (bi-directional) interactions between the opioid and immune systems have been noted (Vallejo et al., 2004). As with other substances, the association is complex and mediated by multiple mechanisms. Exogenous opiates, such as morphine, administered in vivo in both acute and chronic schedules, inhibit T- and B-cell and NK activity as well as phagocytic functions (both macrophage and PMN) (Eisenstein and Hilburger, 1998; Vallejo et al., 2004). In contrast, endogenous opioids, such as β-endorphin, appear to enhance immune cell functions, including those of NK cells and phagocytes (Eisenstein and Hilburger, 1998; Vallejo et al., 2004). Important differences can also be demonstrated between in vitro and in vivo conditions, which select for direct versus CNS-mediated processes. Phagocytic functions appear to be directly sensitive to opioids (Eisenstein and Hilburger, 1998), while effects on lymphoid cells, especially NK activity, appear to be indirectly mediated by effects on the CNS (Shavit et al., 1986; Weber and Pert, 1989). The latter involve both sympathetic and HPA axis mechanisms (Eisenstein and Hilburger, 1998; Mellon and Bayer, 1998). The opioid effects, both direct and indirect, also differ in relation to the three primary opioid receptors (Nelson et al., 2000; Vallejo et al., 2004). Reciprocal effects of immune activity on opioid systems result in further interactions that modulate both neural and immune processes. For example, inflammation facilitates pain (through peripheral and central mechanisms) and the induction of morphine tolerance may be influenced by immunologic processes (Shavit et al., 2005; Watkins and Maier, 2002). Immunologic studies in opiate abusers are limited and subject to a host of confounding factors relating to lifestyle (e.g., nutritional effects and infections introduced by contaminated needles). There is, nevertheless, evidence of a general impairment of immune functions in this population. This includes decreased CD4+/CD8+ ratios and other shifts in T-cell subsets,


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decreased mitogen-induced lymphocyte proliferation, and decreased NK activity (Brown et al., 1974; Donahoe et al., 1987; Novick et al., 1989). As with other substances of abuse, the immune changes may also relate to acute and chronic withdrawal and variable schedules of drug exposure/availability and quality with dramatic stress effects (Govitrapong et al., 1998; Rahim et al., 2002; Weber et al., 2004).

kine production, and PMN phagocytic activity (Connor, 2004). Such effects are demonstrable with acute-controlled exposure as well as in habitual users (Connor, 2004; Pacifici et al., 2002). As with many other substances of abuse, the immune effects of MDMA appear largely mediated through the CNS effects of the drug, although PMN phagocytosis (once again) is directly sensitive to this drug (Connor, 2004).

IX. MARIJUANA AND OTHER SUBSTANCES

X. CONCLUDING COMMENTS

While marijuana is among the most commonly used recreational substances and traditionally considered to have only modest medical risk, the medical consequences of marijuana, such as adverse effects on pulmonary function, have become a focus of much attention (Watson et al., 2000). The possibility of cannabinoid effects on the immune system were supported by identification of CB2 receptors on leukocytes (Galiegue et al., 1995). Marijuana has been shown to impair aspects of innate immunity including several alveolar macrophage functions. Such changes may be more extensive than those found following either tobacco or cocaine exposure (Baldwin et al., 1997). Effects on PMNs, however, are not prominent (Deusch et al., 2003). In addition to using the traditional substances already noted, people demonstrate a remarkable ability to adapt naturally occurring and synthetic substances, including those found in the medical pharmacopoeia, to recreational use. Especially when used in combination, some of these are likely to have immune effects (not to mention “dietary” substances such as caffeine). Two substances of abuse will be mentioned as examples of evolving social practice. Anabolic steroids have been popular in recent years, although only indirectly for their CNS effects. Recreational use of these drugs is likely to have immune consequences considering the effects of sex hormones on immunity and the substantial deleterious health effects of these agents (Hughes et al., 1998). Another popular substance of abuse, MDMA (“Ecstasy”), has been studied more extensively, and demonstrates again the multiple mechanisms of immune effects in substance abusers (Connor, 2004). Being related to amphetamine, MDMA is likely to share some immune effects with that agent (see above), and provide insights concerning amphetamine’s immune effects. Numerous immune changes can be demonstrated for MDMA under conditions of controlled exposure, including suppressed CD4+ cells, mitogen-induced lymphocyte proliferation, Th1 cyto-

The nearly ubiquitous use of alcohol and other substances for their CNS-altering properties suggests that all clinical psychoneuroimmunology studies should consider the contribution of such behavior to immunity and health. Immune system effects on substancerelated CNS systems (e.g., opioid) should also be noted. Substance abuse is a major risk factor for many long-standing and emerging threats to public health, including infections and neoplasia, and the present review suggests that psychoneuroimmune processes play some role in these effects. While some immune changes are directly related to immune cell exposure, it appears that the most important immune effects relate to indirect CNS effects shared by most substances of abuse. Many of these drug effects on the immune system are identical to those of stress. Among stressors, drug withdrawal is one of the most powerful experienced by humans under non-traumatic conditions.

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C H A P T E R

27 Schizophrenia and Immunity MATTHIAS ROTHERMUNDT AND VOLKER AROLT

I. II. III. IV. V. VI. VII. VIII. IX.

INTRODUCTION 563 MICROGLIA 564 ASTROGLIA 565 ANTIBODIES 566 BLOOD-BRAIN BARRIER (BBB) 568 HUMORAL IMMUNITY 568 CELLULAR IMMUNITY 569 CYTOKINES 569 HYPOTHESES AND PERSPECTIVES 571

quently involves young adults and can produce severe psychological, social, and vocational disability during the potentially most creative and productive years of a person’s life. Not only is the suicide rate in schizophrenia high, but the deaths tend to occur at a relatively young age. This results in an enormous loss of total years of expected life. Cost-of-illness studies have estimated that approximately 1.5% to 3% of national health expenditures in developed countries and 22% of the costs of mental illness are related to schizophrenia. The National Institute of Mental Health has estimated that schizophrenia costs the United States about $32.5 billion each year for about 2 million patients with the diagnosis. For comparison, the estimated cost of depression is about $30 billion each year for about 19 million patients with that diagnosis (Thieda et al., 2003). About two-thirds of this cost is a consequence of the relative lack of productive employment. In human suffering the social and psychological costs cannot be expressed monetarily. Despite major research efforts, the etiology and pathogenesis have yet to be defined. Two major hypotheses on the pathogenesis of schizophrenia have been discussed. (1) The neurodevelopmental hypothesis suggests that schizophrenia is a disorder based on disturbances in the early development of neurons and glial cells starting in the second trimenon of intrauterine life. It is supported by several pieces of evidence: (a) the presence of minor physical anomalies; (b) the presence of neurological, cognitive, and behavioral dysfunction long before illness onset; (c) a course and outcome of the illness itself that is predominantly incompatible with a classical degenerative disorder; (d) the presence of ventricular

ABSTRACT The involvement of immunological and immunopathological mechanisms in the etiopathogenesis of schizophrenia has been a matter of research with recently increasing effort. This chapter reviews the findings focusing on microglia, astroglia, antibodies against brain structures and neurotropic viruses, blood-brain barrier, humoral immunity, cellular immunity, and cytokines. Evidence for the three primarily postulated hypotheses (infectious hypothesis, autoimmune hypothesis, Th1/Th2 imbalance) is critically discussed. On the basis of the findings, perspectives for future research are outlined, aiming at a precise and consequent strategy to elucidate a potential involvement of immune mechanisms in the etiopathogenesis of schizophrenia.

I. INTRODUCTION Schizophrenia is a severe psychiatric disorder with a worldwide prevalence of about 1%. The illness frePSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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enlargement and decreased cortical volume at onset of symptoms, if not earlier; (e) the presence and nature of cytoarchitectural abnormalities (such as neuron density, number and morphology, dendritic arbors and spines, synapse-related proteins); and (f) the absence of postmortem evidence of neurodegeneration comprising gliosis as a sequelae of a classic degenerative mechanism (Arnold, 1999; Harrison, 1999; Marenco and Weinberger, 2000). The second (2) and, for a long time, opposing approach was called the neurodegenerative hypothesis. More than 100 years ago Kraepelin and Alzheimer were convinced that schizophrenia was an organic disease involving destruction of neural tissue. This hypothesis fell behind when researchers failed to consistently demonstrate a destruction of neurons and glial cells or gliosis as a consequence of a passed neurodegenerative process. The implementation of imaging techniques such as CT and MRI as well as improved methodologies and new techniques in postmortem studies, however, has confirmed that neurodegeneration is present in at least a subgroup of schizophrenic patients. There are convincing data showing (a) progression of cerebral volume reduction during the course of disease (DeLisi et al., 1999; Gur et al., 1998; Lieberman et al., 2001; Pearlson and Marsh, 1999; Shenton et al., 2001). A classic neurodegenerative mechanism involving loss of neurons and development of gliosis, however, appears unlikely to be relevant for schizophrenia. Brains from patients who had suffered from schizophrenia mostly show (b) unchanged numbers of neurons but a reduction of neuronal cell size and neuropil that accounts for the loss of brain volume (Harrison, 1999; McGlashan and Hoffman, 2000; Powers, 1999; Selemon and GoldmanRakic, 1999). A reduction of neuropil involves compromised cell structure and decrease of neuronal connectivity, resulting in a presumed loss of functional communication between neurons. Beyond that, several studies reported (c) changes in synaptic proteins and their gene expression (Glantz and Lewis, 2000; Harrison and Eastwood, 2001; Karson et al., 1999; Kung et al., 1998; Mirnics et al., 2001). These findings support a diminution or dysfunction of dendrites, neurites, and synapses in schizophrenia (Jones et al., 2002). Recently, several authors endeavored to integrate the neurodevelopmental and the neurodegenerative aspects into a comprehensive pathogenetic hypothesis of schizophrenia (Bartzokis et al., 2002; Lieberman et al., 1997; Mirnics et al., 2001; Woods et al., 1998). Immune factors play an important role in neurodevelopmental as well as neurodegenerative processes. Inflammatory and immune reactions directly influence

neuronal proliferation, differentiation, migration, and apoptosis. Therefore, it is obvious that immune mechanisms have to be considered as potential pathogenic factors of schizophrenia. This has been a matter of scientific discussion since Esquirol described an “epidemic” appearance of psychotic disorders in 1845. The review summarizes the current knowledge of altered immunity in schizophrenia and evaluates the contribution of immune factors to the pathogenesis of schizophrenia (Figure 1).

II. MICROGLIA Microglial cells represent a major part of the cerebral immune system and are located within the brain parenchyme behind the blood-brain barrier. They originate from mesodermal hemapoietic precursors and are slowly turned over and replenished by proliferation in the adult central nervous system. In the healthy brain resting, ramified microglia function as supportive glia cells, and their activation status is regulated by neurons through soluble mediators and cell-cell contact. However, in response to brain pathology, microglia become activated: acquisition of innate immune cell functions render microglia competent to react toward brain injury through tissue repair or induction of immune responses. They are capable of phagocytosis, secretion of cytokines and neural growth Lymphocytes NK Cells Cytokines

Astroglia

Microglia Monocytes

B R A I N

Neurons

Antibodies

Viruses

Blood-Brain Barrier Immunoglobulins

BLOOD

FIGURE 1 Interaction between various immune subsystems in schizophrenia.

27. Schizophrenia and Immunity

factors, and antigen presentation. In addition, microglia are involved in central nervous system development and remodeling. In certain pathological conditions, however, microglia activation may sustain a chronic inflammation of the brain, leading to neuronal dysfunction and cell death. This might be mediated by the microglial release of extracellular toxic reactive oxygen and nitrogen species (Vilhardt, 2005). Despite the important role of microglia in inflammation of the brain, only a few studies focused on these cells in schizophrenia. Recently, it was demonstrated in two postmortem studies (Bayer et al., 1999; Radewicz et al., 2000) that activated microglia cells can be observed in a subset of patients with schizophrenia. Hirsch (2004) undertook a PET study using (R)PK11195 to label activated microglia cells. Patients with schizophrenia showed increased isotope binding throughout the cortex with some regional accentuation in the frontal lobes. A significant correlation was found between the reduction in the amplitude of mismatch negativity and increased (R)-PK11195 binding. Solov’eva and Orlovskaia (1979) reported that embryos of schizophrenic mothers show signs of increased microglia activity. In elderly schizophrenic patients, Falke et al. (2000) found no signs of microgliosis, while Wierzba-Bobrowicz and colleagues (2004) described signs of microglia degeneration (cytoplasm shrinkage, thinning, shortening, and fragmentation of their processes). The evidence for an activation of microglia cells in schizophrenia is not sufficient so far. However, the application of advanced techniques such as (R)PK11195 PET is promising, especially since it can be performed in various states of disease and correlated with functional aspects.

III. ASTROGLIA Astrocytes (macroglia) are the major glial cells within the central nervous system (CNS) and have a number of important physiological properties related to CNS homeostasis. Besides regulating the extracellular ionic and chemical environment, they serve as immunocompetent cells within the brain. They are able to express class II major histocompatibility complex (MHC) antigens and co-stimulatory molecules (B7 and CD40) that are critical for antigen presentation and T-cell activation. As immune effector cells, they influence aspects of inflammation and immune reactivity within the brain, e.g., by promoting Th2 responses. In addition, astrocytes produce a wide array of chemokines and cytokines (Dong and Benveniste, 2001).

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Early studies focusing on astrocytes in schizophrenia were initiated to search for astrogliosis as a sign of neurodegeneration supporting the neurodegenerative hypothesis (Arnold et al., 1996, 1998; Falkai et al., 1999). None of these studies could demonstrate an increase of astrocyte numbers. Quite contrary, Webster et al. (2001) reported a reduction of glial fibrillary acidic protein (GFAP)-immunoreactive astroglia adjacent to blood vessels of the prefrontal cortex. Rajkowska et al. (2002) also found decreased GFAP-positive astrocytes, but in layer V of the dorsolateral prefrontal cortex. In the serum of schizophrenic patients, antibodies against astrocyte muscarinic cholinoceptors were reported (Borda et al., 2004). A DNA microarray study revealed that the highest frequency of mRNA expression alterations occurred in astrocyte- and oligodendrocyte-related genes in the prefrontal cortex of schizophrenic patients (Sugai et al., 2004). Matute and colleagues (2005) discovered a 2.5-fold increase in astrocytic glutamate transporter (GLT-1) mRNA in the prefrontal cortex of schizophrenics. Protein concentration and function of the transporter were also elevated. A different approach to assess the functionality of astrocytes in patients suffering from schizophrenia is to measure astrocytic markers that can be detected in CSF and serum. This offers the opportunity to investigate patients in various stages of disease, including drug-naïve first-episode patients. S100B can serve as such a marker. S100B–-a small, Ca2+-binding, astrocytic protein–plays an important role modulating the proliferation and differentiation of neurons and glia cells. It is involved in the regulation of the cellular energy metabolism and interacts with many immunological functions of the brain (Adami et al., 2001; Vives et al., 2003). It is highly brain specific and easily passes the bloodbrain barrier. Just recently, Liu and colleagues (2005) reported an association between a certain haplotype of the S100B-gene (four SNPs examined) and schizophrenia, making the S100B-gene a susceptibility gene. For S100B protein concentrations in schizophrenia, the data base is quite consistent. Rothermundt et al. (2004a) showed that S100B is increased in the CSF and serum of schizophrenic patients. CSF and serum levels are in high correlation, and the S100B concentration is correlated with the severity of symptoms. Several other studies focused solely on serum concentrations. Wiesmann and co-workers (1999) presented a significantly increased serum concentration of S100B in schizophrenic subjects under neuroleptic medication. S100B levels tended to be higher in patients with residual symptomatology and long-term continuous psychotic symptoms. The study by Gattaz et al. (2000)

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showing decreased S100B levels in chronic schizophrenic patients has to be cautiously interpreted since the samples used (citrate plasma) were not adequate for the assays used. In 2001 the same group published data on medication-free schizophrenic patients (Lara et al., 2001). The serum of the patients contained significantly higher concentrations of S100B than that of the matched healthy controls and was negatively correlated with illness duration. Schroeter and colleagues (2003) reported increased S100B serum concentrations in schizophrenic patients treated with anti-psychotic drugs, while untreated patients showed normal values. Patients with deficit schizophrenia had higher S100B concentrations than non-deficit subtypes. In a longitudinal study paranoid schizophrenics were examined in acute stage of disease without medication and after 6 weeks of anti-psychotic treatment (Rothermundt et al., 2001b). Upon admission, the S100B serum level was significantly higher compared to the matched healthy controls. After 6 weeks of treatment, the level of significance was no longer reached. However, S100B levels after treatment were positively correlated with severity of negative symptoms after 6 weeks, indicating that little change or even deterioration of the negative symptomatology was associated with high S100B levels (Rothermundt et al., 2001b). Another longitudinal study focusing on chronic schizophrenic patients with primarily negative symptoms supported these findings. In a large sample, serum S100B concentrations were increased at the beginning of the study as well as after 12 and 24 weeks of standardized treatment. Patients with high S100B concentration at intake showed a slower improvement of symptoms than patients with normal S100B levels (Rothermundt et al., 2004b). In summarizing the evidence, it is likely that a dysfunction of astrocytes might be a pathogenic factor for schizophrenia. However, replication studies in independent samples are needed, and the character of the astrocytic dysfunction remains to be concretized.

IV. ANTIBODIES In 1937 the German neuropsychiatrist LehmannFacius (1937) pointed to the possible role of an autoimmune process in the etiology of schizophrenia. This assumption is supported by certain clinical features that can be observed in both schizophrenia and autoimmune diseases. Both categories of diseases have onset in late adolescence or early adulthood, often triggered by psychosocial distress, drug abuse, and physical injury. In both cases, variability, of course, often with acute disease episodes and subsequent deficit

remittance, is regularly observed. Such clinical features can also be observed in acute and chronic (viral) infections. Menninger (1919, 1926, 1928) reported the occurrence of schizophrenia-like psychoses in victims of the influenza pandemic which occurred after the end of World War I. Furthermore, there is an ongoing discussion on the association between viral infections (influenza) during pregnancy (second trimester) and the birth of a child who later developed schizophrenia (Torrey et al., 1997). If this were the case, a line of evidence would point to the possibility that a viral contact with an extremely immature immune system would have taken place, leading to enduring immunological abnormalities. These observations have resulted in different forms of infectious/autoimmune hypotheses of schizophrenia. One hypothesis states that a virus, possibly a retrovirus, causes direct structural or functional damage to the brain, eventually leading to schizophrenic psychosis (for review, see Kirch, 1993). Another hypothesis suggests that an early viral contact of the fetal brain induces a dysfunctional reaction of the immature immune system, leading to autoimmune pathology (for review, see Kirch, 1993).

A. Antibodies against Neurotropic Viruses Antibodies against several neurotropic viruses have been the subject of research throughout the years. Most studies have focused on the family of herpes viruses. Bartova et al. (1987) reported antibodies against herpes simplex virus (HSV) in the CSF of 16% of studied patients with schizophrenia. Pelonero et al. (1990) found increased HSV antibody levels in the serum of 32% of investigated schizophrenics. An increased CSF/serum antibody ratio suggesting local antibody production in the brain was found in 4% of studied schizophrenic subjects (Albrecht et al., 1980). Pandurangi et al. (1994) reported an association between high HSV antibody titers and left frontal cortical atrophy. Dickerson and colleagues (2003) demonstrated an association between serologic evidence of HSV infection and cognitive impairment in schizophrenia. The offspring of mothers with antibodies to HSV appear to be at increased risk for the development of schizophrenia in adulthood (Buka et al., 2001). On the other hand, many researchers have failed to demonstrate increased serum HSV antibody titers in schizophrenia (review: Rothermundt et al., 2001a). Cytomegalovirus (CMV) antibodies were discovered in the CSF of 11% of schizophrenics (Torrey et al., 1982), and Albrecht et al. (1980) reported signs of local CMV antibody production in the brains of 68% of


patients. In the serum of schizophrenic subjects, no changes in antibody titers were observed (review: Rothermundt et al., 2001a). DeLisi et al. (1986) found increased quantities of Epstein Barr Virus (EBV) antibodies in the serum of schizophrenics. Decreased titers against mumps virus and unchanged titers against rubella and measles were reported. Measles antibodies in the CSF were increased in 35% of patients (review: Rothermundt et al., 2001a). Fukuda et al. (1999) discovered no changes in antibody titers against HSV, CMV, VZV, EBV, measles, rubella, mumps, influenza, and Japanese encephalitis virus between an acutely psychotic state and after 8 weeks of treatment in schizophrenic patients. More recent studies have focused on Borna Disease Virus (BDV), a neurotropic, single-stranded RNA virus. Between 3% and 45% of schizophrenic patients were reported to have BDV serum antibodies compared to 0–5% in various samples of controls without psychiatric disorder (review: Rothermundt et al., 2001a; Terayama et al., 2003; Yang et al., 2003). Waltrip et al. (1997) observed higher BDV antibody levels in schizophrenic patients with deficit syndrome, whereas Richt et al. (1997) discovered no BDV antibodies in patients suffering from psychosis. Antibodies against retroviral antigens were reported in 52% of studied psychotic patients (Hart et al., 1999). Karlsson et al. (2001) demonstrated sequences homologous to retroviral pol genes in the CSF of 28.6% of patients with recent onset of schizophrenia. Antibodies against non-human primate retroviruses (MasonPfizer monkey virus [MPMV], baboon endogenous virus [BaEV], simian retrovirus type 5 [SRV-5]) were observed to be more frequent in schizophrenic patients than healthy controls (Lillehoj et al., 2000), e.g., MPMV antibodies in 28.9% first-episode schizophrenia patients as compared to 3.7% of the unaffected controls. Serum antibodies against the viruses of the herpes family are very common in the general population worldwide. Most of the antibody carriers are asymptomatic, and the assumption of active disease in individuals should be based on increasing specific antibody titers in the same individual over a defined period of time. Changes in antibody production at local sites are helpful in substantiating an ongoing infectious pathology in the brains of tested individuals. Only a few of the above-mentioned studies adhered to these criteria. This fact might be primarily responsible for the inconsistency of the findings. In addition, inconsistent laboratory methods used and differing definitions of antibody reactivity contribute to the diversity of findings. However, it is a promising strategy to associate antibody titers or virus load with various phenotypes such

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as cognitive impairment (Dickerson et al., 2003) or electrophysiological abnormalities. This strategy will help to identify a subgroup of patients which is more likely to have an infectious pathogenesis for their disorder.

B. Autoantibodies against Brain Structures Several studies have focused on specific antibodies against brain structures. Serum anti-brain antibodies were positive in 28–95% of studied schizophrenics, while in several other investigations this finding could not be reproduced (review: Rothermundt et al., 2001a). Pandey et al. (1981) reported anti-brain antibodies in serum and CSF of 48% of studied schizophrenic patients. More recent studies have investigated specific brain regions, with serum antibodies against the following regions being reported: hippocampus, septum, cingulate gyrus, amygdala, and frontal cortex (review: Rothermundt et al., 2001a). Another approach to specifying the early finding was to study specific structures of cells in the brain. Most of these findings have not been reproduced so far: antibodies against membrane antigen, ganglioside, nicotinic acetylcholine receptor, GFAP, S100, neuronspecific enolase, p80–85 protein of a human neuroblastoma cell line, myelin basic protein (review: Rothermundt et al., 2001a), cholinergic muscarinic receptor (Borda et al., 2002), and nerve growth factor (NGF) (Shcherbakova et al., 2003, 2004). Anti-neuronal cell antibodies (review: Rothermundt et al., 2001a) and antibodies against astrocytes (Borda et al., 2004) were also reported. The search for antibodies against specific neuronal, glial, or microglial structures represents a fundamentally interesting scientific approach in the search for etiopathogenetic factors of schizophrenia. An antigenantibody interaction might be responsible for functional changes in the brains of a subgroup of patients suffering from schizophrenia. This hypothesis is compatible with the fact that the brains of schizophrenics usually show no glial scars or major defects but rather cellular pruning, a diminution of dendrites, and synaptic contacts. Unfortunately, the search for antibodies has not yet gone beyond the screening stage. Most findings have not been replicated in independent samples, and possible pathophysiological consequences of antigen-antibody-interactions in the brains of schizophrenic patients have not been explored. Cross-reactivity of each tested antibody has to be considered to prevent false positive results and to document the specificity of the antibody. Until now

568


researchers have not been successful in identifying clinical or biological subtypes of schizophrenic patients by means of specific antibodies. It appears reasonable to correlate specific phenotypes identified by imaging, electrophysiology, or neuropsychology with antibodies against specific brain regions, cell types, or molecular structures. Subtyping merely according to psychopathology obviously is not promising.

V. BLOOD-BRAIN BARRIER (BBB) In several studies an impairment of the blood-brain barrier in schizophrenia has been discussed. Between 18% and 31% of studied subjects showed signs of increased permeability of the BBB (review: Rothermundt et al., 2001a). In these patients reduced concentrations of the soluble intercellular adhesion molecule-1 (sICAM-1) (Krönig et al., 2005; Schwarz et al., 2000) and the very late antigen-4 (VLA-4) positive CD4+ and CD8+ cells were reported (Müller et al., 1999). Krönig and co-workers tried to find a correlation between sICAM-1 concentrations and a polymorphism of the respective gene. This could be seen in healthy controls but not in schizophrenic patients (Krönig et al., 2005). Furthermore, endogenous CNS IgG-production was detected (review: Rothermundt et al., 2001a). Heat shock protein 60 (hsp60) antibodies were discovered in 10–62% of subjects suffering from schizophrenia (review: Rothermundt et al., 2001a); antibodies against hsp70 and hsp90 were detected in 31% or 20%, respectively (Kim et al., 2001). Autoantibodies against a 60 kDa protein were demonstrated by Wang et al. (2003). Potential target proteins include hsp60 and protein associated with MYC (PAM). These findings might indicate that an increased permeability of the BBB could play an important role in the understanding of possible immunopathogenic mechanisms in schizophrenia. It facilitates the penetration of T-cells to fight an inflammatory CNS process. Interleukin-2 (IL-2) is also known to modify the permeability of the BBB. An increased permeability of the TABLE 1 IgA

IgE

IgG

Increased Normal Decreased

Number of studies reporting evidence.

IgM

BBB facilitates the spread of a primarily local CNS process into the periphery or the invasion of infectious pathogens, causing damage to the CNS.

VI. HUMORAL IMMUNITY Several studies have focused on serum immunoglobulins and acute phase proteins in schizophrenic patients. Immunoglobulin M (IgM) may be an index of acute infection, while immunoglobulin G (IgG) titers are more persistently elevated after infection. The results of immunoglobulins are inconsistent (Table 1). IgG, IgM, and IgA concentrations in the serum of patients were reported to be increased, normal, or decreased (review: Rothermundt et al., 2001a). IgE levels were reported to be increased (Ramchand et al., 1994; Sugerman et al., 1982) in patients with poor therapeutic response or normal (Cazzullo et al., 1998). The producers of immunoglobulins, the B-lymphocytes, were shown to be increased at least in subgroups of studied patients, while others found no differences between patients and healthy controls (review: Rothermundt et al., 2001a). Maes et al. (1993, 1995) reported increased transferrin receptor serum levels. Neopterin was normal in urine, normal, or increased in serum (review: Rothermundt et al., 2001a). Overall complement activity (review: Rothermundt et al., 2001a) was normal although certain parts of the complement were up(C1, C3, C4) or downregulated (C2) (Hakobyan et al., 2005). C-reactive protein was increased (Mazzarello et al., 2004) or normal (Wilke et al., 1996). Acute phase proteins and immunoglobulins are non-specific markers of changes in the immune system of the body. Their concentrations can be changed under many different conditions such as infection, inflammation, and stress. As separate parameters, they cannot be specifically related to the development of schizophrenic psychosis but might be helpful as additional parameters in characterizing the role of specific immune subsystems.

Humoral Immunity

B-lymphocytes

C-reactive protein

Neopterin

Transferrin receptor

569


VII. CELLULAR IMMUNITY Leukocytes and especially lymphocytes have been a matter of research interest in schizophrenic psychosis for quite some time. In one of the first immunopathological studies on schizophrenia ever published, Bruce and Peebles (1903) observed increased leukocyte counts especially during the acute phase of the illness. This finding was reproduced by Zorilla et al. (1996), while Rothermundt et al. (1998) found no leukocytosis in schizophrenia. Lymphocytes were extensively studied but with conflicting results (Table 2). The contradictions could not be solved as yet, not even by lymphocyte subtyping. Lymphocytes were reported to be unchanged or increased (review: Rothermundt et al., 2001a). Tlymphocytes were shown to be increased, decreased, or unchanged. The determination of T-helper cells (CD4) also resulted in conflicting results showing increased as well as decreased or unchanged numbers. The same holds true for T-suppressor cells (CD8) (review: Rothermundt et al., 2001a). Nikkilä et al. (1995) saw increased CD4 and CD8 cells in the CSF of some patients, while others showed decreased numbers. Monocytes were reported to be increased in blood (Zorilla et al., 1996) and CSF (Nikkilä et al., 1999). Natural killer (NK) cells were found to be increased (Sasaki et al., 1994) or decreased (Sperner-Unterweger et al., 1999). Counting absolute or relative numbers of immunocompetent cells in the blood of patients obviously does not help to unravel whether there is an immunopathology that might be linked to the etiopathogenesis of schizophrenia. This method is too non-specific and may be too sensitive concerning confounding variables. It is unlikely that a defect in the immune system reflected by the counts of defined groups of immunocompetent cells is relevant in the pathogenesis of schizophrenia. Furthermore, minor infections not involving the central nervous system as well as stress or medication largely influence the numbers and composition of immunocompetent cells without any releTABLE 2 Lymphocytes

T-lymphocytes

Increased Normal Decreased


vance regarding the pathophysiology of schizophrenia. Focusing on the functional capacity and the degree of activation of certain specific subsystems appears to be more promising (see the next section, titled “Cytokines”). Changes in the counts of immunocompetent cells in CSF indicate ongoing pathology in the central nervous system (CNS). The potential confounding effect of infections outside the CNS is minimized by focusing on CSF measurements. However, these findings do not indicate specificity regarding the pathophysiology of schizophrenia but rather show unspecific signs of CNS infection.

VIII. CYTOKINES The function of T-lymphocytes as a major part of the adaptive immune system has become a focus of interest for immunological research in schizophrenia during the last 15 years. Technical advances have permitted different cytokines and activation markers to be studied. Cytokines represent a class of signaling proteins produced by immunocompetent cells with numerous important functions in immune regulation. In various animal experiments, intrauterine or perinatal inflammatory exposure was shown to cause changes in cytokine concentrations and produce similar behavioral changes in the offspring as seen in schizophrenia. Maternal infection increases TNF-α and decreases BDNF and NGF (Gilmore et al., 2005). A disruption of prepulse inhibition in the offspring (Borell et al., 2002; Tohmi et al., 2004) could be shown, which was reversed by application of anti-psychotic medication. In humans, Brown et al. (2004) reported increased IL-8 serum concentrations in the second trimester of pregnancy in mothers whose offspring later developed schizophrenia. The cytokines interleukin-2 (IL-2) and interferongamma (IFN-γ) are produced by T-helper cells and are functionally closely related. They play a major role in the regulation of T-cell–mediated immunity. In CSF,

Cellular Immunity T-helper cells

T-suppressor cells

Monocytes

NK cells

570


three studies demonstrated unchanged IL-2 levels (Barak et al., 1995; El-Mallakh et al., 1993; Rapaport et al., 1997), while two studies reported increased concentrations of IL-2 (Licinio et al., 1993; McAllister et al., 1995); in one study an increase in IL-2 predicted the recurrence of psychotic symptoms in relapse-prone schizophrenics (McAllister et al., 1995). The measurement of serum concentrations of these cytokines was not a promising method, showing mostly normal values (Barak et al., 1995; Becker et al., 1990; Gattaz et al., 1992; Xu et al., 1994) with two exceptions (Theodoropoulou et al., 2001; Zhang et al., 2003: IL-2 increased). Several groups then started to investigate the capacity of T-cells to produce cytokines upon mitogen stimulation. In many studies it was shown that schizophrenic patients have a reduced capacity to produce IL-2 (review: Kaminska et al., 2001; Koliaskina et al., 2004; Mahendran et al., 2004; Rothermundt et al., 2001a; Zhang et al., 2002) and IFN-g (review: Kaminska et al., 2001; Koliaskina et al., 2004; Rothermundt et al., 2001a). However, O’Donnell (1996) and Cazzullo et al. (1998, 2001, 2002) were unable to reproduce these findings (Table 3). The serum concentration of the soluble IL-2 receptor (sIL-2R) indicating the degree of activation of T-helper cells was reported by most groups to be increased (review: Rothermundt et al., 2001a). However, some studies reported normal sIL-2R concentrations (Erbagci et al., 2001; Haack et al., 1999; Naudin et al., 1997; O’Donnell et al., 1996; Villemain et al., 1989); one

TABLE 3 IL-2 CSF

IL-2 serum

IL-2 production

Increased

Normal

Decreased


IFN-g production

study reported decreased levels (Kowalski et al., 2000). It was discussed whether the increase of sIL-2R might be a medication artifact since unmedicated patients had normal levels but medicated patients had increased concentrations in some studies (Maes et al., 1996; Müller et al., 1997b). Gaughran and colleagues (2002) found elevated sIL-2R levels in first-degree relatives of patients with schizophrenia. T-helper cells are subdivided into functionally different subgroups. Type 1 helper cells (Th1) evolve mainly from pro-inflammatory action, producing IL-2 and IFN-γ among others. The products of the type 2 helper cells (Th2) such as interleukin-10 (IL-10) are antagonists of the type 1 cytokines. The production of IL-10 was reported to be increased (Cazzullo et al., 1998), unchanged (Kaminska et al., 2001), or decreased (Koliaskina et al., 2004; Rothermundt et al., 1996); serum concentration was increased (Maes et al., 2002). The serum concentration of interleukin-6 (IL-6), a cytokine produced by several different immunocompetent cells, was reported to be increased (review: Rothermundt et al., 2001a; Kaminska et al., 2001; Zhang et al., 2003) or normal (review: Erbagci et al., 2001; Rothermundt et al., 2001a). Frommberger et al. (1997) showed increased levels of IL-6 in acutely psychotic patients with normalization in remission. The soluble IL-6 receptor (sIL-6R) was shown to be increased (Lin et al., 1998) or decreased (Maes et al., 1994), while Müller et al. (1997a, 1997b) reported normal concentra-

Cytokines sIL-2R

IL-10 production

IL-6

sIL-6R

IL-1

TNF-a

571


tions in unmedicated and decreased levels in medicated schizophrenics. Conflicting results were reported concerning serum concentrations of the fast-acting monokines (produced by monocytes), interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-a) (review: Rothermundt et al., 2001a). Various other cytokines were investigated, showing increased (Kaminska et al., 2001; Maes et al., 2002; Zhang et al., 2002) or normal (Erbagci et al., 2001) concentrations of IL-8, normal IL-12 (Kim et al., 2002), and elevated IL-18 (Tanaka et al., 2000). Measuring the plasma cytokine response to surgical stress, Kudoh et al. (2001) found decreased levels of IL-6 and IL-8 in schizophrenic patients, while TNF-α did not change. Genetic studies investigating polymorphisms of genes encoding cytokines or cytokine receptors have recently been started. For IL-1 (Chowdari et al., 2001) and IL-4 (Jun et al., 2003a), no associations were found, while a polymorphism of the IL-1 receptor antagonist (IL-1RA) was identified in a Korean population with an odds ratio of 2.24 (Kim et al., 2004). For IL-10, significant findings were reported by Chiavetto et al. (2002) and Yu et al. (2004), while Jun and co-workers (2003b) failed to reproduce this finding. In the light of these findings, up to now two subsystems show convincing results to strengthen the hypothesis that an immunopathology might be involved in the etiopathogenesis of schizophrenia: the IL-6 system and even more so the IL-2/IFN-γ system (Table 3). The inconsistencies in the IL-6 system might be clarified by studying schizophrenic phenotypes characterized by imaging, electrophysiology, and neuropsychology. The IL-2/IFN-γ system offers fairly consistent findings in schizophrenic patients and compatible data from CSF, serum, and cell culture stimulation experiments. There is, to date, considerable evidence that an altered regulation of the IL-2/IFN-γ system plays a role in the development of autoimmune disorders. An in vivo activation of T-cells in autoimmune disorders is typically reflected in increased serum levels of their products, namely interleukin-2 (IL-2) and the soluble interleukin-2 receptor (sIL-2R). Paradoxically, a decreased in vitro production of IL-2 upon IFN mitogen stimulation is found in these patients. These common features of autoimmune disorders (Ganguli et al., 1993; Kroemer and Martinez, 1991) have been supplemented by the demonstration of a decreased in vitro production of interferon-gamma (IFN-γ) upon mitogen stimulation (Ruschen et al., 1992). Genetic studies need to be amended, and a phenotypic subclassification of schizophrenia appears helpful, since most likely only in a subgroup of patients, cytokine dysfunction represents a pathogenic factor.

IX. HYPOTHESES AND PERSPECTIVES Compiling the findings of immunological and immunopathological research in schizophrenia reveals several lines of evidence that an infectious or autoimmune process might play a role in the etiopathogenesis of a subgroup of schizophrenic disorders. Specific dysfunctions of immunological subsystems have been demonstrated repeatedly and coherently, as well as some non-specific markers of immune activation. Signs of inflammation, microglia and astroglia activation in postmortem brains and in the CSF with a dysfunctional BBB, increased retroviral activity, proof of antibodies against brain structures and neurotropic viruses, enhanced cytokine levels in the CSF preceding psychotic episodes, signs of T-cell system activation, and decreased cytokine production capacity in the peripheral blood point to impressive parallels between schizophrenia and autoimmune/infectious disorders. However, although some striking findings cannot be overlooked, there is as yet no direct proof of an immune pathogenesis of schizophrenia. On the basis of the findings, various hypotheses have been formulated and are currently discussed.

A. Infection Hypothesis In the event of an infection being responsible for the development of a schizophrenic psychosis, it seems important to identify the infectious agent. The search for antibodies as well as for antigens should be included to find this agent. So far no virus or bacterium could be identified as a causative agent in the development of schizophrenia. If an infectious agent with widespread occurrence is suspected to be involved in the pathology, increases in antibody levels over time are essential to prove an ongoing infection. Findings in CSF appear more reliable since peripheral signs of infection do not necessarily imply CNS involvement. The majority of published studies did not consider these criteria and are therefore not able to prove an infectious pathology. A CNS infection is usually associated with microglia and/or astroglia activation as presented for schizophrenia directly or indirectly in various studies. An impairment of the BBB is to be expected in case of a relevant CNS infection, as has been shown in several studies which indeed need reproduction. However, more subtle infections might not necessarily cause a detectable change in the permeability of the BBB. If the infection is limited to the CNS, changes in acute phase proteins are not to be expected. Therefore, serum measurements of these proteins are helpful only as additional markers to complete the knowledge of

572


the actual immune situation. Immunocompetent cells and markers of pro-inflammatory immune cell activation such as cytokines can be expected to be increased in the CSF in case of a CNS infection, as has been shown by several studies in schizophrenia. The functional capacity of the peripheral immunocompetent cells reflected by cytokine production experiments gives important indications concerning the individual’s potential to fight an infection.

B. Autoimmune Hypothesis It has been hypothesized that an autoimmune process might be involved in the etiopathogenesis of schizophrenia. The microglia activation reported in two studies might be due to an autoimmune pathology, although microglia activation is an unspecific inflammation marker. Signs of BBB impairment with increased permeability are also seen in autoimmune diseases of the brain. So far it is unknown how an individual develops an autoimmune disorder. One hypothesis suggests that a foreign protein induces an immune reaction. Subsequently, the produced antibodies cross-react with specific proteins of the individual. In schizophrenia, cross-reacting antibodies are assumed to target specific brain structures. Therefore, the search for autoantibodies against specific brain structures represents an important approach. However, the reported findings on antibodies against specific structures of brain cells have not yet been reliably reproduced. It appears to be more relevant to focus on cellular structures rather than on topographic regions. As soon as antibodies against certain cell structures have been repeatedly found in schizophrenic individuals, it will be essential to elucidate their functional role. If the autoimmune reaction is limited to the CNS, changes in acute phase proteins are not inevitably to be expected. Counting immunocompetent cells does not help to identify autoimmune pathology, but it is rather the characterization of the functional capacity of those cells that is relevant. A decreased production capacity of Th1 cytokines in the periphery associated with increased cytokine levels on the inflammatory site, as has been repeatedly reported in schizophrenia, can be understood as a typical feature of an autoimmune pathology (Ganguli et al., 1993; Kroemer and Martinez, 1991; Ruschen et al., 1992).

C. Th1/Th2 Hypothesis Schwarz and colleagues (2001) formulated the Th1/ Th2 hypothesis in schizophrenia. This hypothesis is

based on the fact that T-lymphocytes can be divided in subclasses with at least partially antagonistic functions. Th1-like action is mainly pro-inflammatory; Th2like activity, predominantly anti-inflammatory. There is evidence for a downregulation of Th1-like (decreased IFN-γ and IL-2 production ex vivo, elevated sIL-2R serum levels) and an upregulation of Th2-like immune parameters (increased serum concentrations of IgE, IL6, antibodies against several antigens, elevated IgG and IL-4 CSF levels) in psychosis patients. They suggested that there might be a Th2 shift in schizophrenia, especially in patients with predominant negative symptoms and treatment resistance. However, inconsistent data or even controversial results question the hypothesis. It is necessary and trendsetting to formulate wellfounded hypotheses to enable a hypothesis-driven approach. This method appears much more promising than remaining in a screening stage. Utilizing advanced techniques in cell cultures and animal models will be helpful in studying immune subsystems under welldefined conditions. What mechanisms generate the pattern of microglial and astroglial activation found in the brains of schizophrenics? How does a specific antibrain antibody interfere with the function of neurons or glia cells? Is there a specific retroviral activity in the brains of schizophrenic patients? How do increased CSF cytokine concentrations influence neurons and glia cells? What are the molecular mechanisms underlying the decreased peripheral cytokine production in schizophrenic patients? Increasing our knowledge about functional interactions on a molecular basis and the application of appropriate animal models will enable us to interpret the indirect parameters available from CSF, blood, or functional MRI studies in schizophrenic patients. Clinical studies should focus on the identification of a subgroup of patients with schizophrenia showing immunological abnormalities. It is obvious that many reported studies did not include sophisticated psychopathological examinations. Acute and chronic, psychotic and residual, paranoid and disorganized, schizophrenic and schizoaffective patients were often compiled into one sample. Associations of psychopathological and immunological findings have not been sufficiently reproduced. A psychopathological characterization may well not be enough to permit subtyping. Advanced neuropsychological and electrophysiological methodology, structural and functional MRI techniques, evaluation of the course of disease and treatment response should be included to identify specific phenotypes of schizophrenic patients showing immunopathology. Investigation of gene polymorphisms of immune modulators in connection with


their transcription and translation products might provide further insight in specific subtypes and the functional relevance for the pathogenesis of schizophrenia. First-episode or even prodromal patients appear to be promising for study purposes, as an active immunopathology would be expected in the early stage of the disease, probably even before the manifestation of overt clinical symptoms. Focusing on patients with a recent onset of disease also helps in reducing confounding variables such as medication, diet, or exposure to a hospital environment. However, since schizophrenia is often a chronic disorder, mechanisms causing the progressive deterioration of several brain functions and maintaining inflammatory pathology throughout the course of disease need to be investigated. Applying novel methods and technologies in basic and clinical sciences will eventually enable researchers to characterize immunopathological mechanisms involved in the etiopathogenesis of schizophrenic psychoses and to delineate a subtype of schizophrenia in which infectious or autoimmune pathology is relevant.

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C H A P T E R

28 Sleep and the Immune System MARK R. OPP, JAN BORN, AND MICHAEL R. IRWIN

I. INTRODUCTION 579 II. OVERVIEW OF SLEEP AND ITS CHARACTERISTICS 580 III. EFFECTS OF INFECTIONS ON SLEEP 584 IV. CYTOKINE REGULATION OF SLEEP 586 V. SLEEP MODULATION OF IMMUNITY 591 VI. DISORDERED SLEEP AND IMMUNITY: CLINICAL POPULATIONS 606 VII. SUMMARY 609

possibly disease risk (Ancoli-Israel and Cooke, 2005; Lager, 1994). Epidemiological data increasingly implicate insomnia as a predictor of cardiovascular and non-cardiovascular disease mortality, particularly in community elderly populations (Bryant et al., 2004; Foley et al., 1995; Kripke et al., 2002; Mallon et al., 2000; Mallon et al., 2002; Pollak et al., 1990). Furthermore, objective electroencephalographic (EEG) measures of sleep amounts and quality show that prolonged sleep latency (i.e., a difficulty falling asleep) predicts a nearly twofold increase in all-cause mortality over and above the contribution of demographic confounds and medical factors in healthy older adults (Dew et al., 2003). In 2004, the National Sleep Foundation in the United Stated specifically promoted the importance of sleep for good health. Hence, it is increasingly important to consider the possible physiological effects of sleep and the consequences of sleep loss for homeostatic regulation of systemic processes including the immune system. It is commonly thought that persons are more susceptible to infections when deprived of sleep, and that infections seem to increase somnolence (Bryant et al., 2004). However, the evidence to support these anecdotal observations has only recently begun to emerge. In this chapter, we consider the reciprocal influences of sleep and the immune system. Specifically, this review addresses whether the immune system causes changes in sleep, and whether sleep has a role in the modulation of the immune system with possible consequences for disease risk. Finally, we consider clinical conditions in which disordered sleep and disordered

I. INTRODUCTION About one-quarter of the population of the United States experiences sleep problems, with the prevalence of at least one insomnia complaint occurring in up to one-third of the population (Ohayon, 1996; Ohayon, 2002). Nearly 10% of all persons fulfill diagnostic criteria for chronic syndromal insomnia characterized by symptoms of insomnia of greater than 6 months duration with difficulties in sleep initiation or maintenance (e.g., greater than 30 minutes awake per night), frequent sleep problems (e.g., greater than three times per week), and associated distress and clinical significant impairments in daytime functioning (e.g., fatigue) (Ancoli-Israel and Cooke, 2005; Ohayon, 2002; Savard and Morin, 2001). Moreover, during the past 90 years the amount of time that we spend sleeping has steadily decreased (Jean-Louis et al., 2000), and average nightly sleep has declined from 9 hours in 1910 to 7 hours in 2002. Together, the high prevalence of insomnia and the curtailment of sleep amounts pose a large economic burden to society with reduced productivity, accidents, behavioral and cognitive consequences, and PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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immune function co-occur, as such disorders provide naturalistic evidence for a role of sleep in the regulation of the immune system and possibly in mediating increases in the risk infectious disease and inflammatory disorders in these populations.

II. OVERVIEW OF SLEEP AND ITS CHARACTERISTICS

human SWS the slow waves predominate for less than half of a 30-second period, that period is defined as stage 3. If slow waves predominate for more than half of a 30-second period, the period is considered stage 4. The arbitrary criteria that are used to define phases and stages of sleep give the impression that sleep consists of discrete units that are quantal in nature. This is not the case. In healthy humans and laboratory

A. Sleep Characteristics Arousal states compose a continuum from fully awake and alert to deep sleep. Until 1953, sleep was thought to result passively from withdrawal of wakefulness-promoting stimuli. The discovery of rapid eye movement (REM) sleep by Aserinsky and Kleitman (1953) demonstrated that sleep is not a passive process; during REM sleep several brain regions are as active, or more so, than during wakefulness. The fact that REM sleep was only recently discovered (1953) is remarkable considering that brain electrical activity [the electroencephalogram (EEG)] was recorded as early as 1875. By the 1920s, the Austrian psychiatrist Hans Berger was recording human EEG. During the 1930s several seminal papers were published, notably by Loomis and colleagues (Davis et al., 1938; Loomis et al., 1938) and Blake, Gerard, and Kleitman (Blake and Gerard, 1937; Blake et al., 1939) demonstrating changes in the EEG through the course of a night’s sleep. In none of these early works was the possibility of distinct phases of sleep acknowledged. We now consider sleep to consist of two major phases: REM sleep and non-rapid eye movement (NREM) sleep. Most investigators conducting preclinical studies using laboratory animals find it sufficient to define only two phases of sleep. In sleep research using human subjects and in sleep disorders medicine, the phase of NREM sleep is further subdivided into four stages: stages 1, 2, 3, and 4. The four stages of NREM sleep may generally be considered to parallel a continuum of sleep depth with stage 2 being lighter sleep than stage 4. Stage 1 is the transition between wakefulness and sleep, whereas sleep onset is generally considered to occur when two EEG features characteristic of stage 2 appear—spindles and K-complexes. Spindles are bursts of sinusoidal waves of about 12–14 cycles/second (Hz), whereas K-complexes are transient high-voltage biphasic waves. Sleep spindles and K-complexes are superimposed on a background of low voltage EEG (Figure 1). Human stages 3 and 4 NREM sleep are referred to as slowwave sleep (SWS) due to the preponderance of highamplitude low-frequency components characteristic of the EEG. Differentiating between stages of human sleep is done on the basis of arbitrary criteria. If during

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FIGURE 1 Stages of sleep in humans. The relationship between human sleep, level of consciousness, and electroencephalogram (EEG) patterns. Stages of sleep are characterized by differences in the frequency and amplitude of EEG waves. Stage 1 comprises light sleep with lowamplitude waveforms. Stage 2 is characterized by sleep spindles (the higher frequency waves) and K-complexes (not shown). Stages 3 and 4 comprise slow-wave sleep (SWS) with high-amplitude waves and a deeper level of unconsciousness. These four stages comprise non–rapid eye movement (NREM) sleep. The final stage is rapid eye movement (REM) sleep, which is associated with θ-activity (all waves shown) and, in some individuals, with lowamplitude sawtooth waves (not shown) and a higher level of consciousness. [From Horne, J. A. Why We Sleep: The Functions of Sleep in Humans and Other Mammals. Oxford University Press, Oxford. 1988. Reproduced by permission of Oxford University Press.]

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animals, there is an orderly progression from wakefulness to NREM sleep and REM sleep. Healthy animals and humans enter NREM sleep from wakefulness. From NREM sleep there is a transition to REM sleep. After a period of REM sleep, there may or may not be a brief arousal or awakening (Figure 2). These progressions from wakefulness through stages of sleep repeat to form cycles. The number and duration of cycles that occur during a period of sleep are species-specific; healthy human adults will have four to six NREM– REM cycles of about 80–110 minutes duration, whereas rats will have many NREM–REM cycles, each lasting about 8–10 minutes. In addition to species-specific features with respect to the number and duration of cycles that occur during a sleep period, there are species-specific aspects to the timing and distribution of sleep across the 24-hour day (Campbell and Tobler, 1984; Tobler, 1989; Tobler, 1995). Sleep distributions of vertebrates may generally be considered to be either monophasic or polyphasic. If all sleep across a 24-hour day occurs during a single period, an animal is said to be monophasic with respect to sleep. Sleep distributed during multiple periods across the 24-hour day constitutes a polyphasic sleep pattern (Tobler, 1989). Humans are generally thought to be monophasic, with sleep normally occurring only at night, but the monophasic patterning of human sleep may be a societal artifact and not necessarily a manifestation of fundamental physiological processes. Laboratory rodents (rats, mice), the subjects of most pre-clinical sleep studies, are polyphasic, sleeping during both the light and dark periods of a 24-hour

light:dark cycle. However, though rats and mice sleep during both the light and dark periods, their sleepwake behavior is strongly influenced by circadian factors, and they are generally considered to be nocturnal; rats and mice sleep more during the light period of a light:dark cycle than they do during the dark period. As will be detailed later, the differences in the distribution of sleep across the 24-hour period is an important consideration when conducting studies on the relationship between sleep-wake behavior and immunomodulators.

B. Assessment of Sleep Sleep is first and foremost a behavior, and can be defined by behavioral criteria. Body posture and eye state are primary indicators that may be used to differentiate between sleep and wakefulness. It is also possible to differentiate between NREM sleep and REM sleep of mammals and birds on the basis of changes in muscle tone; during REM sleep postural muscle tone is lost. The most observable consequence of loss of postural muscle tone is a drooping of the head. The use of behavioral criteria as the only determinants of arousal state, however, has little utility for exploration of underlying physiological processes. To systematically determine those neuroanatomical and neurochemical substrates involved in the regulation of arousal state, it is necessary to instrument the subject, whether human or animal, so that multiple physiological parameters may be recorded for relatively long periods.

FIGURE 2 The progression of sleep stages across a single night in a normal young adult. The hypnogram is based on a continuous overnight recording from a 19-year-old man as assessed in 30-second epochs. [From Carskadon, M. A., and W. C. Dement. Normal human sleep: an overview. Chapter 2, in: Principles and Practices of Sleep Medicine, Fourth Edition. (eds. Kryger, Roth, Dement). W. B. Saunders, Philadephia. 2005. Reproduced by permission of W. B. Saunders].

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Every physiological parameter studied to date has been demonstrated to differ depending on whether the animal is awake or asleep. As such, there are numerous parameters that could be recorded that would allow instantaneous assessment of arousal state, and there are now well-defined associations between multiple physiological parameters and arousal state. Some parameters, however, are easier to record and require less instrumentation. The most frequently recorded parameters include the EEG and one or more of the following: muscle tone [electromyogram (EMG)], eye movements [electrooculogram (EOG)], respiration, body movements, and body or brain temperature. In human studies, whether for research purposes or for diagnostic assessment of sleep disorders, multiple surface electrodes are used to record the EEG over several regions of the cerebral cortex. Pre-clinical studies using laboratory animals generally employ surgically implanted electrodes, and only one or two channels of EEG are recorded. For most pre-clinical studies using laboratory animals, recording the EEG and either the EMG or body movements provides information sufficient to allow determination of phases of sleep or wakefulness. The determination of arousal state in laboratory rodents is readily done on the basis of the relationship between specific amplitude and frequency characteristics of the EEG and the EMG or body movements. When awake and active, the EEG of a laboratory rodent is characterized by a low amplitude (voltage) waveform composed of mixed frequencies, while the EMG recorded from the neck muscles of these animals will be activated due to use of postural muscles and movement. During wakefulness, generalized activity (feeding, locomotion, grooming) of the animal within the recording cage will also be detected. Upon a transition from wakefulness to NREM sleep, generalized activity and the amplitude of the EMG in the neck muscles will begin to diminish. Spectral analyses of several types may be used to characterize amplitude and/or frequency components of the EEG waveform. Spectral analyses of the EEG of a rodent that is awake reveal mixed frequency components. During the transition from wakefulness to NREM sleep, the amplitude of the EEG will increase, and spectral analyses reveal shifts from mixed EEG frequencies to predominately lower EEG frequencies (72 h↑

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↔ ↑

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↑

↔

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↔ ↔ ↔ ↔

↔ 96 h↓ ↔ ↔ ↔ ↔

↓ ↓ ↔ ↔ 96 h↑ ↓ 72 h↓ 63 h↑ ↓ ↔ ↑ >39 h↑ ↑ ↔ ↑ 63 h↑ ↑ >24 h↑

24 h/every 3 h 120–168 h/6:30 h 64 h/22:00 h 40 h/7:00 h 72–126 h/variable 40 h/every 30–120 min 48 h/8:00 h 77 h/12:30 h 88 h/every 6 h 24 h/every 30–60 min 40 h/every 60 min 8 d partial SD/every 30 min Born et al., 1997 [1,2] Boyum et al., 1996 [3] Dinges et al., 1994, 1995 Heiser et al., 2000 Kuhn et al., 1969 Moldofsky et al., 1989 Öztürk et al., 1999 Palmblad et al., 1976 [2] Shearer et al., 2001 Späth-Schwalbe et al., 1992 [2] Vgontzas et al., 1999 Vgontzas et al., 2004a [4]

IFN-g IL-2 TNF-a IL-6 IL-1b Lymphocytes NK cells Monocytes Granulocytes (neutr.) Duration of SD/ time of blood sampling

Effects of Sleep Deprivation (SD) of More Than 10 hours on White Blood Cell Counts and Cytokines TABLE 1

cytokine IL-10, as compared to wakefulness. These results are similar to those of Redwine et al. (Redwine, Dang et al., 2003), who found a nocturnal shift in the ratio of stimulated production of IFN-γ/IL-10 using mixed mononuclear cell populations. Taken together, these observations suggest a preferential facilitation of type 1 cytokine activity and point to a general supportive influence of sleep on launching a cellular adaptive immune response. A considerable number of studies in healthy humans have examined effects of prolonged periods of sleep deprivation, lasting between 24 hours and 168 hours, on immune functions (Table 1). Numbers of circulating granulocytes and monocytes increase after extended periods of total or partial sleep deprivation (e.g., Boyum et al., 1996; Dinges et al., 1994; Heiser et al., 2000; Kuhn et al., 1969). The effects of sleep loss on granulocytes and monocytes appear to take some time to develop. In the study by Dinges et al., monocytes did differ until 39 hours of sleep deprivation, and the increase in granulocyte counts required 63 hours of deprivation to be altered. These long-term sleep deprivation studies also rather consistently indicate a more or less pronounced decrease in the number of NK cells, in NK activity, and in the number of the major lymphocyte subpopulations that gradually develops during the sleep-deprivation period (Boyum et al., 1996; Dinges et al., 1994; Öztürk et al., 1999). Examination of cytokine activity likewise suggests an activation of innate immune functions after extended sleep deprivation. Thus, an early study by Moldofsky et al. (1989) reported increases in IL-1–like and IL-2– like activity in plasma after 40 hours of wakefulness. Another study examining effects of 4 days of partial sleep deprivation (allowing 2-hour naps each day) and of total sleep deprivation revealed an increase in soluble TNF-α RI (p55) and in IL-6 plasma concentration following total sleep deprivation (Shearer et al., 2001). Levels of TNF-α, TNF-α RII (p75), and of IL-10 and sIL-2R remained unaffected by prolonged sleep deprivation in this study. Plasma concentrations of IL-6 increase after 1 week of sleep restriction to 6 hours a night (Vgontzas et al., 2004a). In contrast, Boyum et al. (1996) reported decreased plasma IL-6 concentrations after 96 hours of sleep deprivation in young men participating in a military training course. However, in addition to being sleep deprived, these men were also subjected to physical exercise and reduced food intake. No consistent changes in T-cell–derived cytokines such as IL-2, IL-4, and IFN-γ were found after prolonged sleep deprivation (Boyum et al., 1996; Dinges et al., 1995). In summary, in comparison with sleep restriction or short periods of sleep deprivation (one night),

Significant effects of SD in comparison with regular sleep-wake conditions are indicated. [1] Note, only effects after the first night (i.e., >10 hours of SD) are shown. Effects occurring during the first night are partly opposite and not shown (see text). [2] Production of cytokines was measured after in vitro mitogen stimulation of whole blood or PBMC. [3] Most of the effects in this study were observed after 24 hours of sleep deprivation. [4] Measures were taken before and after sleep restriction to 6 hours for 1 week.

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nocturnal sleep promotes a decrease in circulating lymphocytes presumably reflecting an enhanced accumulation of these cells into extravascular lymphoid tissues. Moreover, apart from some studies indicating increased IL-6 concentrations during sleep, sleep has been found to increase sIL-6R concentrations, enabling an integration of IL-6 actions on brain and other nonimmune functions, aside from its effects on immune cells through the membrane-bound IL-6R. Finally, distinct sleep-dependent increases in some type 1–related cytokines such as IL-2 and IL-12 have been demonstrated. Overall, these changes point toward a supportive influence of sleep on the initial steps of an adaptive cellular immune response, i.e., on the priming of naive lymphocytes by antigen presenting cells in secondary lymphatic tissues. Extended periods of sleep deprivation, on the other hand, primarily manifest themselves in a distinct granulocytosis, decreased NK cell numbers and activity, and other signs of disturbed innate immunity.

C. Sleep Effects on Infectious Processes The profound alterations in sleep that occur during an infection have led to the hypothesis that sleep promotes recovery. Though such an hypothesis has high face validity and is the basis of folklore, there is little empirical evidence to support such an hypothesis. Most studies of interactions between sleep and the immune system have used one of two approaches: Animals or human volunteers are subjected to immune challenge and the impact on sleep determined, or subjects are sleep deprived and impact on immune function determined. As reviewed in the previous section, most studies of immune function and sleep have documented changes in cytokine levels or profiles, changes in numbers or cytotoxic activity of NK cells, or changes in numbers of cell types associated with specific aspects of immunity. While determination of these parameters provides information about many aspects of the immune system, they do not necessarily indicate there are functional consequences of infection-induced alterations in sleep. That is, do the changes in these immune parameters during the course of infection facilitate recovery? To determine if there are functional consequences to infection-induced alterations in sleep, it is necessary to alter sleep during the course of infection and determine if there are effects on outcome. An early pre-clinical study by Brown et al. (Brown, Pang et al., 1989) evaluated the effects of sleep deprivation of mice on responses to influenza virus challenge. Mice that were sleep deprived before inoculation with influenza virus did not clear the virus as efficiently as non–sleep-deprived animals (Brown, Pang et al., 1989). However, subsequent studies failed to replicate these

provocative results (Renegar et al., 1998a; Renegar et al., 1998b; Renegar et al., 2000), and it is not clear whether sleep loss alters influenza risk in animals. In another pre-clinical study, Toth and Opp (Toth et al., 1995a) sleep deprived rabbits for 4 hours either before or immediately after inoculation with Escherichia coli. Rabbits sleep deprived before infection demonstrated increases in NREM sleep, delta power during NREM sleep, and NREM sleep bout lengths that were greater than observed after sleep deprivation alone or E. coli alone. Animals that were sleep deprived after infection with E. coli exhibited only transient increases in NREM sleep and delta power during NREM sleep, suggesting these rabbits maintained a sleep deficit. Rabbits inoculated with E. coli and then sleep deprived developed fevers that were greater in magnitude than either the hyperthermia elicited by sleep deprivation or the fever elicited by infection alone. Other clinical symptoms of E. coli infection (plasma IL-1β, corticosterone, cortisol, and triglycerides; total white blood cell counts; nucleated red blood cells; lymphocytes; neutrophils) were not dramatically altered by the combination of sleep deprivation and infection. As such, under the conditions of this pre-clinical study, sleep deprivation of rabbits alters changes in sleep that occur during bacterial infection but does not alter clinical illness. Two recent studies of human subjects also examined the question of whether sleep loss is of consequence with respect to immune function. Spiegel et al. invited healthy subjects into the sleep laboratory, where they were either allowed 8 hours sleep each night for 14 days or were subjected to 14 days of sleep restriction to 4 hours per night. After 4 days of this protocol, individuals of both groups were immunized against infection to influenza A virus; that is, they received their flu shots (Spiegel, Sheridan et al., 2002). In those persons who underwent sleep restriction, virus-specific antibody titers were less than 50% of those in non–sleep-deprived persons. In a similar study, Lange et al. found that total sleep deprivation for one night after vaccination lead to low virusspecific antibody titers after immunization against hepatitis A virus (Lange, Perras et al., 2003). These two studies are intriguing because they demonstrate impairment of immune function by short-term sleep loss that is of functional consequence to public health. It may be that prior sleep history is a factor to be considered in vaccination programs, in that sleep loss prior to immunization may reduce the effectiveness of antibody production against the vaccine and result in individuals being at risk for these viral diseases. As sleep loss appears to impact at least some aspects of responses to true infections of viral vaccinations, the converse, that changes in sleep during the course of


infection aid in the recovery process, may also be true. Though few studies have focused on this aspect of the relationship between sleep and immune function, there is tantalizing evidence this may indeed be the case. Toth et al. (1993) retrospectively analyzed data obtained from rabbits that had been infected with E. coli, Staphylococcus aureus, or Candida albicans and calculated a sleep quality score for each animal. The sleep quality score incorporated both the amount of time spent in NREM sleep and delta power during NREM sleep. Using the sleep quality score, the animals were classified as exhibiting minimal sleep changes during infection, enhanced sleep during infection, or suppressed sleep during infection. Enhanced sleep during infection was associated with more favorable prognosis (survival) and less severe clinical illness, whereas suppressed sleep was associated with increased mortality and more severe clinical illness. The results of these analyses support the hypothesis that changes in sleep through the course of an infectious challenge facilitate recuperation.

D. Mechanisms Linking Sleep and Immunity Humoral and neural influences are the principal mediators of bi-directional sleep-immune interactions (Besedovsky and delRey, 1996). Both the endocrine and autonomic nervous systems convey efferent information from the brain to the periphery. The endocrine and autonomic nervous systems also convey afferent information from peripheral systems to the brain. Accordingly, these systems mediate the brain’s control over immune processes during sleep and also mediate influences of systemic immune responses on the central nervous sleep process. There may be additional mechanisms linking sleep and immunity. For example, it has been suggested that sleep-promoting actions of TNF-α signaling are conveyed via an influence on hypothalamic thermoregulation (Haack et al., 2004). The disturbance of sleep observed after administration of endotoxin and proinflammatory cytokines in humans could well be a consequence of increased body temperature in response to these agents. Likewise, increases in NREM sleep of laboratory animals after administration of various cytokines (IL-1, IL-2, IL-6, IL-8, IL-15, IL-18, TNF-α) are associated with distinct febrile responses (reviewed Krueger et al., 2001; Opp, 2005; Opp and Toth, 2003). However, the NREM sleep-promoting effects of IL-1β and TNF-α are not a byproduct of changes in body or brain temperature (Krueger and Takahashi, 1997). For example, CNS inhibition of IL-1β and TNF-α reduces cytokine-induced increases in NREM sleep but does not alter febrile responses (Takahashi et al., 1999). Low

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doses of IL-1 administered to laboratory animals and of endotoxin injected into human volunteers increase NREM sleep and SWS, respectively, without affecting brain or body temperature (Mullington et al., 2000; Opp et al., 1991). Though sleep and thermoregulation are closely linked, the role of pro-inflammatory cytokines in the regulation of sleep can indeed be separated from their actions on body temperature (Krueger and Takahashi, 1997). In the following section we will focus on neuroendocrine and vegetative mechanisms linking sleep and immune function. 1. Neuroendocrine Nocturnal sleep in humans is characterized by a unique orchestration in the neuroendocrine activity of a number of hormones well known for their immunomodulatory properties (Born and Fehm, 1998; Steiger, 2003). The most relevant of these hormones and their temporal secretory patterns during human sleep are briefly described (Figure 8). There is a strong surge in secretory activity of the somatotropic system during the early period of sleep, which in humans is typically dominated by SWS (stages 3 and 4). Activity of this system, i.e., hypothalamic release of growth hormonereleasing hormone (GHRH) and, in turn, pituitary release of growth hormone (GH), decreases to a minimum during the late part of nocturnal sleep, during which REM sleep is predominant. This pattern is primarily determined by sleep processes, and independent of circadian oscillators; a surge in GH in the early period of sleep occurs also during daytime sleep. Like somatotropic activity, release of prolactin is also enhanced during sleep and particularly so during periods of SWS (Spiegel et al., 1995). Activity of the hypothalamic-pituitary-adrenal (HPA) system shows a reversed temporal pattern. The release of hypothalamic corticotropin-releasing hormone (CRH), of pituitary corticotropin (ACTH), and of adrenal corticosteroids reaches an absolute minimum during the early SWS-rich part of nocturnal sleep, and increases during the late, REM sleep-rich part of sleep, reaching a maximum at about the time of morning awakening. HPA secretory activity during nocturnal sleep is determined both by circadian and sleep-dependent mechanisms. SWS inhibits HPA secretory activity, as can be demonstrated by experimental stimulation of the system during SWS (Bierwolf et al., 1997; SpäthSchwalbe et al., 1993). However, normally sleep and circadian oscillators act synergistically so that sleep deprivation during a single night does not induce a substantial increase in cortisol release during the early night. Release of epinephrine and norepineprhine is generally greatly reduced during sleep as compared to levels during waking (Dodt et al., 1997). Minimum

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FIGURE 8 (Top) Representative profiles of nocturnal sleep and (Bottom) associated plasma concentrations of immunoactive hormones (cortisol, growth hormone [GH], epinephrine, and norepinephrine) in a healthy young man. Abscissa indicates time of day, ordinate indicates sleep stage (W, wake; REM, rapid eye movement sleep; S1, S2, S3, S4, sleep stages 1–4; M, movements). S3 + S4 represents the time in slow-wave sleep (SWS). Note that the first half of sleep is dominated by SWS; the second half, by extended epochs of REM sleep. Minimum plasma concentrations of cortisol and maximum concentrations of GH occur during early sleep. Catecholamine concentrations are reduced during sleep and, in particular, during REM sleep. [From Marshall and Born. Brain-immune interactions in sleep. Int’l. Rev. Neurobiol. 52: 93–131, 2002. Reproduced by permission of Elsevier].


plasma catecholamine concentrations were found during periods of SWS in some studies (Irwin et al., 1999) and during REM sleep in others (Dodt et al., 1997), although single sympathetic nerve fiber activity decreases during SWS, but not REM sleep (Somers et al., 1993). Plasma concentrations of thyroid stimulating hormone (TSH) normally are increased in the evening hours before sleep. Subsequent sleep decreases TSH levels, in comparison with persistent wakefulness (Allan and Czeisler, 1994; Goichot et al., 1992). Melatonin, a pineal hormone that has attracted much interest in sleep research, is controlled by the dark:light cycle, with light inhibiting its release. Accordingly, melatonin concentrations show strong circadian rhythm with peak concentrations around midnight (Arendt, 2000). Melatonin facilitates sleep. However, the effect of sleep per se on melatonin seems negligible. Immune cells express specific receptors for all of these steroid and peptide hormones (Blalock, 1994). Effects of the hormones on immune function have been mostly studied in vitro and often with pharmacological doses far beyond the normal physiological range of variation. Hence, conclusions with regard to the role of these hormones in mediating effects of sleep on immune function under normal conditions must be tempered with caution. The effects of GH, prolactin, and melatonin appear to be generally immunosupportive, whereas ACTH, cortisol, and catecholamines are considered immunosuppressive hormones (Besedovsky and delRey, 1996; Brzezinski, 1997; Chikanza, 1999; Gala, 1991). In general, during the early, SWSrich part of sleep, enhanced release of GH and prolactin in combination with the suppression of HPA secretory activity may convey primarily immunosupportive influences. During nocturnal sleep late in the night, characterized by strongly enhanced release of glucocorticoids and distinctly lowered release of GH and, to a lesser degree, reduced prolactin, these influences on immune function become reversed. Among the hormones modulated by sleep, the corticosteroids are the best-studied regulators of immune functions. Cortisol affects the distribution of white blood cells by inducing a pronounced decrease in the numbers of circulating lymphocytes due to an enhanced trafficking of the cells to the bone marrow (Fauci et al., 1976; Ottaway and Husband, 1992; Sackstein and Borenstein, 1995). The effect of cortisol on lymphocyte distribution occurs with some delay; the nadir in the number of lymphocytes circulating in blood follows by 2–4 hours the maximum cortisol concentration (Gatti et al., 1992; Kronfol et al., 1997). While these temporal relationships indicate a strong contribution of cortisol to circadian rhythms in lymphocyte counts, they

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cannot explain sleep-dependent reductions in lymphocyte and monocyte counts, since, as mentioned earlier, differences in nocturnal plasma cortisol concentrations between awake and sleeping subjects are normally marginal, and not altered by such experimental strategies as partial-night sleep deprivation (Redwine et al., 2000). GH, prolactin, and the reduced concentrations of catecholamines possibly contribute to these changes during sleep. However, with the exception of catecholamines (see the subsection titled “Autonomic” in Part V), the influence of these hormones on cell trafficking has so far not been thoroughly investigated. Glucocorticoids, even at relatively low concentrations, suppress the production of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6 (Besedovsky and delRey, 1996; Petrovsky and Harrison, 1998). In contrast, melatonin is reported to stimulate pro-inflammatory cytokine activity (e.g., Sutherland et al., 2002) and to decrease the production of the Th2 cytokine IL-10 (Kuhlwein and Irwin, 2001). In combination, reduced glucocorticoid levels and increased melatonin levels during the night may well account for the circadian increase in IL-6 activity, and the shift toward Th1 cytokine dominance that is consistently observed during the night. There are also reports of a stimulating influence of GH on monocyte/ macrophage activity (Edwards, III et al., 1992; Kelley et al., 1992) and of increased IL-1β and TNF-α activity after repeated GH administration in children (Bozzola et al., 2003). However, since sleep-dependent increases in the activity of these cytokines have so far not consistently been demonstrated in humans, the relationship of the sleep-associated surge in GH concentrations to the systemic regulation of these cytokines remains to be fully elucidated. However, in combination with reduced catecholamine levels during sleep (see the subsection titled “Autonomic” in Part V), GH might contribute to an enhancement in NK cell activity observed upon awakening (Bidlingmaier et al., 1997). Sleep-dependent alterations in T-cell function have been primarily linked to the enhanced release of GH and prolactin during sleep. GH and prolactin, which act via receptors of the same cytokine/hemopoietin receptor superfamily and have similar binding affinities and intracellular signal transduction (Matera et al., 2000; Yu-Lee, 2002), have enhanced T-cell proliferation and type 1 cytokine activity (Chikanza, 1999; Dimitrov et al., 2004b; Mellado et al., 1998; Postel-Vinay et al., 1997; Rook et al., 1994; Takagi et al., 1998). Concurrently, type 2 cytokine activity may be reduced by GH and prolactin. Both hormones stimulate the differentiation of T-cells and enhance the primary antibody response when given within a short period after

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vaccination (Chikanza, 1999; Spangelo et al., 1987; Stephenson et al., 1991). Accordingly, the distinctly enhanced release of these hormones may not only account for the immediate sleep-dependent shift toward a predominance of type 1 cytokine activity, but also for a supportive effect of sleep on longer-lasting adaptive immune defense and memory. The sleep-dependent effects of GH and prolactin on T-cell function arise on a background of circadian modulation that appears to reflect, in part, effects of glucocorticoids and melatonin on these functions. At higher concentrations, glucocorticoids also suppress the production of T-cell–derived cytokines such as IL-2 and IFN-γ. In contrast, at low nanomolar concentrations, glucocorticoids may enhance the in vitro production of IL-2 (Snijdewint et al., 1995). Petrovsky and Harrison (1997) showed in healthy humans that the circadian shift in the type1/type 2 cytokine balance towards increased type 1 cytokine activity during early nocturnal sleep can be effectively inhibited by prior administration of a high dose of cortisone. Apart from the inhibition of adrenal corticosteroid release during the early night, the nocturnal rise in melatonin has been considered another factor that contributes to the nocturnal predominance of type 1 cytokines like IL-2 (Lissoni et al., 1998). In vitro, melatonin stimulates lymphocyte proliferation and reduces production of IL-10, a cytokine with strong inhibitory actions on the synthesis of type 1–related cytokines, whereas IFN-γ activity remains unaffected by melatonin application (Kuhlwein and Irwin, 2001). Two recent studies directly addressed the issue of the extent to which the hormonal milieu established during nocturnal sleep, particularly the early SWS-rich sleep of humans, controls T-cell–derived cytokine activity (Dimitrov et al., 2004b; Lange et al., 2005). The first study analyzed intracellular cytokine production in morning blood samples from awake healthy humans by multi-parametric flow cytometry. Sleep-like hormonal conditions were established by adding GH and prolactin at concentrations mimicking levels present during normal SWS. High glucocorticoid activity present in these morning samples was suppressed by adding corticosteroid receptor blockers. Under these conditions, prolactin and GH increase the percentage of IFN-γ producing CD4+ T-cells. Moreover, prolactin increases the percentage of CD4+ T-cells producing IL-2 and TNF-α, as well as the percentage of CD8+ Tcells producing IFN-γ and IL-2. In the second of these studies, blood samples were collected during the early night while subjects were in a period of SWS. Intracellular cytokine production was determined after a 6hour incubation of the samples with GH-neutralizing

antibody and with cortisol at concentrations typical for blood levels in awake subjects in the morning. Incubation with GH antibody led to a clear reduction in CD4+ T-cells producing IFN-γ. However, other cytokines, like IL-2 and IL-4, were not influenced. Collectively, results of these two studies provide supporting evidence for the view that during normal SWS, prolactin and GH release by increasing type 1 cytokine activity contributes to a sleep-dependent shift in the type 1/ type 2 cytokine balance. Noteworthy are the results of these studies with regard to the function of cortisol, which suggest actions of this hormone are mediated not only via glucocorticoid receptors (GR) but also via mineralocorticoid receptors (MR). Binding affinity of the major endogenous corticosteroids, i.e., cortisol in humans and corticosterone in the rat, is about 10-fold higher for MR than GR (De Kloet et al., 1998). Accordingly, about 70–80% of MR are continuously occupied throughout the 24-hour cycle, whereas GR become predominantly occupied during periods of distinctly increased corticosteroid concentrations, i.e., during stress and around the maximum of the circadian oscillation. The blockade of MR in blood from awake subjects (characterized by high cortisol concentrations) markedly enhances CD4+ and CD8+ T-cells producing IL-2, IFN-γ, and TNF-α, and thereby also induces a shift toward increased type 1 cytokine activity as measured by the ratio of IFN-γ/IL-4 producing CD4+ T-cells. The effects of MR blockade are stronger than those of the selective GR blockade (with RU-486), suggesting that the suppressing effects of cortisol on T-cell–derived cytokines are to a substantial extent mediated via MR activation. Adding cortisol to blood sampled during SWS (characterized by minimum cortisol concentrations) confirmed a decrease in the percentage of CD4+ and CD8+ T-cells producing IFN-γ, IL-2, and TNF-α, as well as a decrease in CD4+ T-cells producing the type 2 cytokine IL-4. Collectively, data from these two ex vivo studies in humans indicate a specific role of sleepassociated GH and prolactin release for inducing a shift toward type 1 cytokine activity, whereas the simultaneous suppression of endogenous cortisol release during the early night appears to serve a general facilitation of both production of type 1 and type 2 cytokines. Hormones are also involved in signaling infection to the brain. The acute response to infection includes an activation of different hormonal systems, primarily the hypothalamic-pituitary-adrenal (HPA) stress system and the adrenal release of catecholamines. Experimental administration of endotoxin in humans and animals increases plasma concentrations of ACTH,


cortisol, and GH (e.g., Daniel et al., 2005; Lang et al., 1997; Mullington et al., 2000). Administration of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 also stimulates activity of the HPA system at its different levels (Turnbull and Rivier, 1999). Simultaneously, these cytokines appear to suppress somatotropic activity, although this action is complex (e.g., Jones and Kennedy, 1993). In rats, ICV injection of IL-1β increases expression of GHRH receptors but does not change GHRH expression (Taishi et al., 2004). Whereas low doses of IL-1β administered ICV into rats increase GH release, high ICV doses suppress GH release. In humans, administration of IL-6 does not alter plasma GH concentrations but does increase plasma ACTH and cortisol concentrations (Späth-Schwalbe et al., 1996). 2. Autonomic Lymphoid tissues are densely enervated by nerve endings of the autonomic nervous system, and leukocytes express adrenergic and cholinergic receptors. These conditions enable an efferent control of lymphocyte function via paracrine release of neurotransmitters and peptidergic neuromodulators. These efferent neuronal pathways appear to convey predominantly influences of the sympathetic nervous system (Elenkov et al., 2000; Ottaway and Husband, 1992; Straub et al., 1998). A parasympathetic enervation of lymphoid tissues has been demonstrated in some studies (Madden and Felten, 1995) but questioned by others (e.g., Qiu et al., 2005). However, there is also some evidence suggesting that lymphocytes express cholinergic receptors to a greater extent than adrenergic receptors, which are prevalent in granulocytes (Abo and Kawamura, 2002). Analyses of various physiological functions (e.g., heart rate variability), which allow the separation of parasympathetic and sympathetic activation, have indicated that sympathetic tone is synergistically controlled both by circadian oscillators and sleep, whereas parasympathetic tone appears to be controlled mainly by circadian factors (e.g., Burgess et al., 1997). Moreover, these studies show that nocturnal sleep in humans is characterized by a general reduction in sympathetic activity and a relatively enhanced parasympathetic tone. The sympathetic nervous system exerts a great variety of influences on the immune system, which globally manifest themselves in an enhancing effect on innate immune functions. Responses to experimental administration of agonists and antagonists of adrenergic receptors have consistently supported the view that sympathetic activation, among others, induces

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leukocytosis and transient lymphocytosis, enforcing primarily a mobilization of NK cells and granulocytes to the circulation (Benschop et al., 1996; Ottaway and Husband, 1992; Ottaway and Husband, 1994). Administration of β-adrenergic blockers increases the emigration of lymphocytes to extravascular tissues. Moreover, β-adrenergic blockers enhance, and catecholamines reduce, the in vitro production of T-cell–derived IL-2 and IFN-γ (Maisel et al., 1991; Malec et al., 1990; Sanders, 1995). There have been also a number of reports on effects of cholinergic agonists and antagonists on immune cell function, reflecting possible contributions of parasympathetic activity. Thus, an early study showed that over several days in healthy humans administration of the muscarinic cholinergic blocker atropine left the number of circulating lymphocytes unaffected but impaired in vitro PHA-stimulated lymphocyte proliferation (Pluzanska and Mazurowa, 1977). On the other hand, stimulation of (muscarinic) acetylcholine receptors increases mitogen-stimulated proliferation of T-cells and the production of IL-2 (Fujino et al., 1997; Nomura et al., 2003; Qiu et al., 2005). Acetylcholinergic agonists attenuate the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-18) in human macrophages (Borovikova et al., 2000). In vivo electrical stimulation of the vagus in rats during endotoxinemia likewise inhibits production and reduces circulating TNF-α. Moreover, animal studies suggest that parasympathomimetic treatment improves the antibody immune response to vaccination (Besedovsky and delRey, 1992). Collectively, these observations support the view that parasympathetic activity globally acts to attenuate systemic pro-inflammatory responses of innate immunity and supports aspects of T-cell–mediated adaptive immune functions. The regulatory influence of sympathetic and parasympathetic activity on immune functions has so far been rarely studied in the context of sleep. However, from the available evidence, it can be hypothesized that the profound sleep-associated reduction in sympathetic tone, together with a circadian increase in parasympathetic tone during the night, affects adaptive T-cell function by increasing type 1 cytokines and decreases aspects of innate immunity (Besedovsky and delRey, 1996; Petrovsky, 2001; Sanders, 1995). In addition, this constellation of changes may permit an enhanced homing of lymphocytes in secondary lymphatic tissues, which is probably controlled cooperatively via endocrine factors (Abo and Kawamura, 2002; Ottaway and Husband, 1994). Considering the size of the hypothesized effects, more thorough investigations to elucidate the influences of the autonomous

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nervous system on immune functions directly in the context of sleep appear promising.

VI. DISORDERED SLEEP AND IMMUNITY: CLINICAL POPULATIONS To determine whether the experimental associations between sleep disturbance, sleep loss, and immunity translate to a naturalistic or “real life” setting, evidence has been sought from clinical conditions in which disturbances of sleep are found along with alterations in immunity. Such work is important, as it carries forward the acute effects of sleep disturbances and especially partial sleep loss, and evaluates whether chronic loss of sleep as found in many clinical disorders impacts the immune system, which might play a role in the increased incidence of infectious diseases and inflammatory disorders in at-risk populations such as depressed persons or older adults. In addition, such studies have the potential to provide insights into the reciprocal influence of cytokines on sleep, and whether increases in inflammatory markers in many of these disorders (e.g., major depression, aging, rheumatoid arthritis) might contribute to abnormalities in sleep continuity or sleep depth in these populations.

A. Primary Insomnia Chronic primary insomnia is characterized by the presence of difficulties in sleep initiation or maintenance and frequent sleep problems with associated distress and clinical significant impairments in daytime functioning that persist for greater than 6 months. Importantly, primary insomnia, by definition, cannot be accounted for by the confounding presence of another psychiatric disorder, such as depression, or underlying medical conditions. Hence, evaluation of immune alterations in primary insomnia patients lends compelling evidence that chronic sleep disturbances lead to changes in immune responses. Consistent with the acute effects of partial-night sleep deprivation, patients with chronic insomnia show significant decreases in the numbers of CD3+, CD4+, and CD8+ T-cells (Savard et al., 2003) along with decreases in NK cell responses (Irwin et al., 2003). In the latter study, primary insomnia patients also showed nocturnal elevations of circulating levels of norepinephrine, which correlated with difficulties with sleep maintenance. Together with evidence that β-adrenergic activation mediates a suppression of NK activity in humans (Friedman and Irwin, 1997), these data suggest that abnormalities in sleep continuity might mediate declines in NK responses via sympathetic nervous system activation. In animals, substantial evidence

suggests that NK cell responses are an important marker of immunological defenses against tumors (Ben-Eliyahu et al., 2000; Shakhar and Ben-Eliyahu, 1998). The present data show that insomnia is associated with declines of NK activity, suggesting a possible pathway in the link between insomnia and cancer morbidity (Savard et al., 1999) and mortality (Kripke et al., 1979). Vitaliano and colleagues have also found declines of NK activity in individuals with cancer histories which are exacerbated by the presence of sleep problems (Vitaliano et al., 1999). Cytokine abnormalities have also been reported in association with chronic insomnia. Consistent with the effects of sleep deprivation to shift the Th1/Th2 cytokine balance, insomnia correlates with low levels of IFN-γ and a low IFN-γ to IL-4 ratio. There is also evidence that primary insomnia may lead to increases in circulating levels of inflammatory cytokines. Vgontzas et al. (2002) reported increases in daytime circulating levels of IL-6, and Burgos et al. (2005) extended these findings to show that IL-6 levels are prominently elevated during the nocturnal period. Furthermore, nocturnal IL-6 amounts negatively correlated with perceived subjective sleep quality. In another study of healthy persons, sleep quality also negatively correlated with morning IL-6 levels (Hong et al., 2005). Further temporal characterization of profile of IL-6 secretion in relationship to sleep and/or the use of experimental administration of cytokine antagonists is needed to evaluate whether sleep impairments activate IL-6 analogous to sleep deprivation, or whether increases of IL-6 have sleep-disturbing effects in this population, as has been described following the infusion of IL-6 (Späth-Schwalbe et al., 1998). However, consistent with the effects of acute IL-6 infusion, the amount of SWS in primary insomnia patients was negatively correlated with nocturnal IL-6 levels (Burgos et al., 2005). Given that immune activation is implicated in atherosclerosis and the progression of cardiovascular disease (Ridker, 2001), these findings have implications for understanding the mechanisms that might underlie the association between insomnia and cardiovascular disease mortality. For example in a 12year follow-up of 1,870 middle-aged men and women, sleep complaints (e.g., difficulty maintaining sleep) predicted coronary artery disease mortality (Mallon et al., 2002).

B. Aging Poor sleep is one of the most common complaints in older adults. Even in healthy seniors, populations with prevalence rates of diagnostic insomnia exceed 20–30%, greater in frequency and severity than any

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other age group (Ancoli-Israel, 2000; Foley et al., 1995; Petit et al., 2003). Independent of medical and psychiatric illness, medication use, circadian rhythm changes, and psychosocial factors, which can all contribute to complaints of insomnia, primary insomnia is highly prevalent in older adults (Ancoli-Israel, 2000). Indeed, normal aging is associated with declines in subjective sleep quality, sleep fragmentation, increases of light (stages 1 and 2) sleep, and decreases of deep slowwave sleep (Benca et al., 1992; Ehlers and Kupfer, 1989; Ehlers and Kupfer, 1997; Van Cauter et al., 2000), and these sleep abnormalities are thought to contribute to daytime fatigue, depression, and impairments in health functioning. Elderly persons exhibit many of the prominent immune changes observed after acute or prolonged sleep deprivation in younger persons, i.e., an impaired adaptive immune response conjoined with signs of disturbed innate immune function. Like acute sleep deprivation, aging is associated with reduced T-cell function that includes a shift in the type 1/type 2 balance toward enhanced type 2 cytokine activity, and in some studies has been found also to be associated with decreased production of IL-2 and IFN-γ (e.g., Ginaldi et al., 1999b; Ginaldi et al., 1999c; Perras and Born, 2005; Shearer, 1997). Regarding innate immune functions, aging, like sleep deprivation, leads to diminished NK cell activity when considered on a per cell basis (Irwin et al., 1994; Solana and Mariani, 2000). Blood counts of activated T-cells (HLA-DR) have likewise been found to be increased after short periods of sleep deprivation as well as in the aged, and there are also hints at increased activity of pro-inflammatory cytokines like IL-6, IL-1β, and TNF-α in both conditions. Poor sleep in the aged may in fact contribute to these alterations. For example, in one study, healthy elderly in comparison with young subjects showed an increased production of IL-1β and TNF-α, which appeared to be particularly pronounced during nocturnal sleep (Born et al., 1995). In addition, nocturnal levels of IL-6 are lower during SWS as compared to Stages 1–2 sleep, and the deficits of SWS in older adults may be associated with overall increases in circulating levels of this pro-inflammatory cytokine. However, other changes in the aged, like the reduced number of circulating lymphocytes thought to originate from a thymic involution, are clearly opposite to what is observed following sleep deprivation. Accordingly, it should be cautioned against considering alterations in immune function in the aged as a consequence of a “chronic sleep deprivation,” since the effects of poor sleep in elderly persons interact with a number of other physiological conditions not related to sleep.

C. Major Depressive Disorder Insomnia is also one of the most common complaints of depressed subjects, and emerging evidence indicates that disturbances of sleep in depression moderate and/or mediate immune alterations in these patients. For example, as with primary insomnia, subjective sleep quality and insomnia complaints negatively correlate with NK activity in depression, whereas other depressive symptoms including somatization, weight loss, cognitive disturbance, or diurnal variation do not show a similar relationship (Irwin et al., 2003). Likewise, EEG studies reveal that disturbances of sleep continuity (e.g., prolonged sleep latency, declines of total sleep time) correlated with alterations of natural and cellular immune function among depressed patients (Cover and Irwin, 1994; Irwin et al., 1992). Moreover, in bereaved subjects, causal statistical analyses have shown that disordered sleep mediates the relationship between severe life stress and a decline of NK responses (Hall et al., 1998). Recent attention has focused on the links between sleep, IL-6, and sICAM in depression, as these markers of immune activation have important implications for cardiovascular disease in depression. For example, in patients recovering from an acute coronary artery event, comorbidity for depression is associated with elevated levels of sICAM independent of smoking status, obesity, and other traditional risk markers (Lesperance et al., 2004). Motivala and colleagues (Motivala et al., 2005) found that prolonged sleep latency and increases of REM density are associated with elevated levels of IL-6 and sICAM in depression. Moreover, these EEG sleep measures fully account for the association between depression and IL-6, indicating that sleep disturbances have a key role in alterations of inflammatory markers in depression.

D. Alcohol Dependence A more extensive line of research has focused on alcohol-dependent patient populations, who have profound disturbances of sleep continuity and sleep architecture, including decreases of total sleep time, declines of delta sleep, and increases of REM (Irwin et al., 2000; Irwin et al., 2002). To determine the consequences of disordered sleep on immunity and to explore whether abnormal cytokine expression is associated with alterations in sleep continuity or sleep depth in alcohol dependence, the relationships between EEG-defined sleep and NK cell responses and nocturnal cytokine expression have been investigated in alcoholdependent persons as compared to controls. In addition, by blood sampling across the nocturnal period

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before sleep onset and during sleep, these studies have evaluated the hypothesized bi-directional relationships between sleep and cytokines. In alcohol-dependent men who have profound disturbances of sleep depth, there is a differential change in the nocturnal expression of IFN-γ/IL-10 ratio in which alcohol-dependent persons showed a shift toward a Th2 cytokine response after the onset of sleep and a predominance of this Th2 response throughout the night (Figure 9). In contrast, controls showed an increase in the relative expression of Th1 cytokines during the later half of the nocturnal period. The nocturnal production of IL-6 also changed differently in the alcohol-dependent persons versus controls; during the early part of the night, alcohol-dependent subjects showed lower amounts of IL-6 expression, but then higher amounts during the second half of the night as compared to controls. Finally, alcohol-dependent persons showed low levels of NK activity across the night as compared to controls, with increases of NK activity occurring across the night in the controls but not in the alcohol-dependent persons. Taken together, this pattern of abnormal immune responses in alcoholdependent persons is similar to the immune changes during sleep deprivation. Abnormalities of sleep architecture were also associated with measures of nocturnal immunity, and REM sleep amounts predicted morning increases in stimulated IL-6 production and decreases of NK activity. These data, along with evidence that IL-6 is elevated during REM sleep (Redwine et al., 2000), raise the possibility that greater amounts of REM sleep during the

FIGURE 9 The TH1/TH2 ratio increased from 03:00 to 06:30 hours (p < 0.01) in the controls, whereas the IFN-γ/IL10 ratio decreased from 23:00 to 03:00 hours (p < 0.05) and remained low from 03:00 to 06:30 hours in the alcoholics. [From Redwine et al., 2003. Reproduced by permission of Lippincott, Williams and Wilkins].

late part of the night contribute to the nocturnal rise of IL-6 in alcohol-dependent persons. Deficits in SWS in alcohol dependence may also lead to abnormal elevations of IL-6 and TNF. For example, experimental sleep deprivation typically induces a rebound increase in SWS, as described above. However, alcoholics evidence a defect in SWS recovery (Irwin et al., 2002), which is coupled with an exaggerated recovery night increase in circulating levels of IL-6 and TNF in alcoholics as compared to controls (Irwin et al., 2004). Finally, given that daytime elevations of IL-6 correlate with fatigue (Vgontzas et al., 1997), such increases in the production or circulating levels of IL-6 may have implications for daytime fatigue in alcohol-dependent persons. Studies in alcohol-dependent patients have also provided insights into the possible influence of cytokine abnormalities on occurrence of disordered sleep in this population. In studies in which measures of circulating levels of pro-inflammatory cytokines and stimulated cytokine production were taken prior to the onset of sleep onset as well as during sleep, tests were conducted to determine whether levels at evening awake, as opposed to nocturnal sleep or morning awake, predicted measures of sleep continuity or sleep architecture. Using a regression model, evening awake levels of circulating IL-6 were associated with prolonged sleep latency in alcohol-dependent persons independent of confounding influences and other times of cytokine assessment (Irwin et al., 2004) (Figure 10). In addition, IL-6 is associated with increases in REM sleep during the later half of the night. In contrast, expression of the anti-inflammatory cytokine, IL10, before sleep predicted increases in delta sleep, accounting for over 23% of the variance in delta sleep independent of age and alcohol consumption (Redwine et al., 2003). Further experimental studies are needed to determine whether anti-inflammatory cytokines augment delta sleep and whether pro-inflammatory cytokines inhibit delta sleep in human beings. Nevertheless, these observations suggest that cytokine abnormalities temporally predict changes in sleep in clinical populations such as alcohol-dependent persons, and that these immune abnormalities are not merely a consequence of disordered sleep. As reviewed elsewhere in this text, peripheral pro-inflammatory cytokines are capable of exerting direct effects on central nervous system function. Bower et al. (2002) found that immune activation is associated with severity of fatigue symptoms in women recovering from breast cancer, and Capuron et al. (Capuron et al., 2002) suggest that immune activation is associated with cognitive impairments. Likewise, experimental immune activation

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FIGURE 10 Associations between IL-6 at 23:00 h prior to sleep onset and sleep latency in the baseline night (r = 0.37, p = 0.08), PSD-E night (r = 0.56, p < 0.01), and recovery night (r = 0.53, p < 0.01) in alcoholics and controls . [From Irwin et al., 2004. Reproduced by permission of Elsevier].

results in increases of anxiety and depressed mood, which are coupled with declines of memory functions (Reichenberg et al., 2001). Whether disordered sleep and daytime elevations of pro-inflammatory cytokines contribute to depressive symptom severity and cognitive disturbances in recovering alcoholics is not known. Importantly, the contribution of cytokines in the regulation of sleep suggests that cytokine mechanisms are a possible target for interventions to optimize sleep initiation and sleep quality in alcoholic and other atrisk populations.

E. Rheumatoid Arthritis The prevalence of self-reported sleep disturbances in the inflammatory disorder rheumatoid arthritis (RA) is very high, with 50–75% of patients complaining of difficulties initiating or maintaining sleep (Drewes, 1999). In one study, RA patients had significantly longer sleep latency and poorer sleep efficiency with more awakenings during the night as compared to controls (Hirsch et al., 1994). Two other studies similarly found fragmented sleep in RA patients with evidence of alpha-EEG arousals during slow-wave sleep (Drewes et al., 1998; Mahowald et al., 1989). In this population, sleep disturbance is also hypothesized to contribute to complaints of pain, fatigue, and depressed mood in patients with RA, and a number of studies show that subjective sleep complaints correlate with fatigue, functional disability, greater joint pain, and more depressive symptoms in rheumatic patients (Moldofsky, 2001). Indeed, sleep difficulties, pain, depressed mood, and fatigue appear to cluster together in RA; depression is associated with greater pain (Frank et al., 1988; Murphy et al., 1988), whereas sleep difficulties are associated with fatigue, depression, and

pain (Drewes et al., 1998; Nicassio and Wallston, 1992). There is a striking absence of studies examining the role of inflammatory processes in mediating these links between sleep and disease symptoms in RA patients. Prospective and/or experimental studies are needed which simultaneously assess multiple symptoms and inflammatory mechanisms in order to advance our understanding of sleep and its association with other RA symptoms. One study measured sleep disturbance and found that sleep loss was associated with an overnight increase in tenderness in the peripheral joints in RA patients who are experiencing an acute flare (Moldofsky et al., 1983); no assessment of cytokines was completed. In a non-clinical sample of healthy men, experimental disruption of NREM sleep over 3 nights induced increases in objective and subjective measures of pain sensitivity and muscle tenderness (Moldofsky et al., 1975), a finding replicated in healthy middle-aged women (Lentz et al., 1999). Again, studies have not examined the biological mechanisms that underlie the link between sleep loss and/or sleep recovery deficits and increases of pain and fatigue, although elevations in pro-inflammatory cytokine activity are implicated (Moldofsky, 2001).

VII. SUMMARY In this chapter, we have reviewed evidence of a reciprocal relationship between sleep and immunity, and that sleep loss influences immune responses with potential impacts on infectious and inflammatory disease processes. Some evidence in humans suggests sleep preferentially supports aspects of adaptive immune function. Given the association between

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insomnia and mortality, sleep disturbance that is suffered by those with chronic disease, depression, or other disorders may have substantial heath implications due to the effects of sleep on the regulation of the immune system, although the underlying reasons linking insomnia and morbidity remain unclear. The possible effect of sleep disruption has tremendous import because of the pervasiveness of insomnia complaints, with up to 69% of adults reporting having sleep problems on a few nights a week or more. Moreover, in recent decades, decreases in the mean duration of sleep and an increase in shift work have occurred, which might pose substantial public health burden that has not yet been evaluated. Together, these data indicate that sleep is a new “vital sign” that should be more carefully characterized in the management of patients, particularly in vulnerable populations such as the elderly or the immune compromised. To this end, medical environments are needed which will minimize the likelihood of sleep disruption as patients recover from infections or surgery and other medical procedures. Ongoing basic and clinical research aims to provide insight into the molecular and cellular pathways by which sleep and the immune system are interrelated, which will ultimately lead to novel interventions and therapies for insomnia which have the potential to promote health.

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C H A P T E R

29 Emotions and the Immune System MARGARET E. KEMENY

I. INTRODUCTION 619 II. IMPORTANCE OF STUDYING AFFECT IN PNI 619 III. CAN THE IMMUNE SYSTEM PLAY A ROLE IN SUPPORTING THE ADAPTIVE BEHAVIORAL CHANGES ORCHESTRATED BY EMOTIONS? 620 IV. NEGATIVE AFFECTIVE STATES AND THE IMMUNE SYSTEM 621 V. DEPRESSION AND THE IMMUNE SYSTEM 622 VI. ANXIETY AND THE IMMUNE SYSTEM 622 VII. ANGER, HOSTILITY, AND THE IMMUNE SYSTEM 623 VIII. SELF-CONSCIOUS EMOTIONS AND THE IMMUNE SYSTEM 624 IX. POSITIVE AFFECT AND THE IMMUNE SYSTEM 624 X. DIFFERENTIAL IMMUNE CORRELATES OF AFFECTIVE STATES 625 XI. SUMMARY 625 XII. FUTURE DIRECTIONS 626

extensive over the past 30 years. The majority of this research focuses on the immunological effects of exposure to naturalistic or laboratory-induced stressors (Segerstrom and Miller, 2004). A limitation in this literature is the lack of a consistent theoretical framework for understanding the psychological mediators of the effects of stressors on the immune system (Kemeny and Laudenslager, 1999; Segerstrom et al., 2001). While the stress and coping model is often invoked (Lazarus and Folkman, 1984), adoption of this model has tended to result in studies which measure one or more of a long list of potential psychological variables consistent with the model (e.g., anxiety, depression, active coping, passive coping, denial, social support, support seeking, threat appraisal) and one or more of a long list of indices of enumerative or functional immunity, resulting in a very large matrix of findings. From this matrix, it is difficult to discern the critical psychological variables that have reliable links to specific immune pathways.

I. INTRODUCTION II. IMPORTANCE OF STUDYING AFFECT IN PNI

This chapter will discuss the importance of studying affect in the field of psychoneuroimmunology, consider the possible adaptive nature of immunologic changes that have been associated with specific affective or motivational responses, and review the literature linking specific affective states to immune changes, including potential mediators of these relations. Finally, future directions in this area of research will be considered. Research on the relationship between psychological factors and the immune system has become quite PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

One strategy for grappling with the heterogeneity in psychological variables that could be examined in this area is to focus on the most proximal end of the psychological continuum. Research could focus on those psychological states that are posited to be the final links in the pathway from psychological response to the CNS orchestration of the neuroendocrine and immune effects of that response. Many stress and coping theorists consider affective states to be the final

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link in the chain from the environmental perturbation to the biological response (Lazarus and Folkman, 1984). In other words, coping processes and social support, for example, are posited to act on physiology by modifying the affective response to a given context. A greater understanding of the links between affect and peripheral systems might help to organize and focus this complex literature. This chapter will discuss frameworks for considering the relationship between affect and the immune system and review the literature linking affective experience to indicators of immune status. Affective experience includes a number of categories of experience. Emotions are short, intensely felt, affective states that are associated with distinctive facial expressions and behavioral predispositions; emotions have evolved to coordinate response to specific eliciting conditions (e.g., fear in response to threat of attack) (Ekman, 1994; Levenson, 1992; Segerstrom et al., 2001). Moods are longer-term affective states that do not necessarily have a specific trigger, may involve a blend of different affective experiences, do not have unique facial expressions (Ekman, 1994), and often are accompanied by persistent and distinctive cognitions (e.g., negative thoughts about the self and the future with depressed mood; cynicism with hostility). Affective traits involve the dispositional tendency to experience specific emotions and moods across situations and over time. Affective disorders, such as major depressive disorder and generalized anxiety disorder, are pathological forms of affective experience that can interfere with daily life and may require intervention. Affect has been categorized in a variety of ways, for example, along the dimension of valence (positive and negative) and arousal (high or low). While there is a great deal of research on the link between depressive disorders and immune parameters (see Chapter 24, this volume), research on immune correlates of affective responses outside the context of a psychiatric illness is limited.

III. CAN THE IMMUNE SYSTEM PLAY A ROLE IN SUPPORTING THE ADAPTIVE BEHAVIORAL CHANGES ORCHESTRATED BY EMOTIONS? Currently, it is presumed by most investigators that all negative affective experiences would have the same peripheral physiological effects. However, there is increasing evidence that distinct emotions have different physiological correlates. For example, specific emotions are associated with distinct patterns of activation in the central nervous system (Canli et al., 2001;

Damasio et al., 2000; Lane et al., 1997) and in the autonomic nervous system (although the findings in this area are sometimes inconsistent) (Ekman et al., 1983; Herrald and Tomaka, 2002). Given these distinctions in neural systems, it is possible that peripheral physiological systems such as the immune system that are impacted by these neural systems could also respond with different patterns of activation depending on the affect experienced. In considering what physiological changes would be predicted to correlate with an affective response to a given context, it may be useful to consider what the adaptive psychological/behavioral and physiological responses to that context may be. Fleeing from a predator, grieving a loss, or demonstrating one’s social status require different kinds of psychological, physiological, and behavioral changes to respond adaptively to the situation (Weiner, 1992). Emotions may play a central role in orchestrating these coordinated psychobiological responses. For example, fear in response to a threat to survival is associated with specific behavioral and physiological changes that facilitate flight. It would be expected that these behavioral and physiological changes would differ from those associated with sadness, for example (Ekman, 1999; Levenson, 1994). There are at least three adaptive affective/motivational response patterns that become activated in response to specific eliciting conditions that may involve the immune system: the fight/flight response, the recuperative response, and the behavioral disengagement response (Kemeny, in press). The fight/flight response involves recognition of a threat that requires physical action and the mobilization of physiological resources to promote such activity, while at the same time downregulating other systems, for example, resource building or restorative systems, that are not central in the context of a threat (Sapolsky, 1993). This shift from resource building to resource utilization during a threat has been shown to activate the sympathetic nervous system (SNS), the hypothalamicpituitary-adrenal (HPA) axis, and other physiological systems. Immune changes that have been observed during fight/flight responses have mostly been conceived of as negative side effects of the adaptive activation of mobilization systems such as the SNS. However, Dhabhar and McEwen (2001) argue that some of these immune changes are actually an integrated part of the physiological response to such threats. Specifically, they argue that leukocytes are shunted from the bloodstream to immune organs in the context of this type of threat and that this redistribution is an integral part of the fight-flight response, preparing the animal for pos-

29. Emotions and the Immune System

sible wounding or infection as a result of activities related to fighting or fleeing (Dhabhar, 2003; Dhabhar and McEwen, 2001). In addition, “first line of defense” cells such as natural killer cells are increased in number in the bloodstream with these forms of acute threat, and a rapid increase in these first line of defense cells has been argued to allow for quick, non-specific killing of organisms that puncture the skin via wounds or other injuries (Kemeny and Gruenewald, 2000). This acute increase in NK cells is mediated by activation of the SNS; products of the SNS increase heart rate, cause this redistribution of white blood cells including NK cells, and modulate the expression of immune molecules that control trafficking (Ottaway and Husband, 1992). Fear, which is a central component of the fight/ flight response, plays an important role in coordinating the SNS activation that occurs with such a threat. These SNS changes then modulate the activity and distribution of immunologic cells, possibly in the service of promoting adaptation in the context of this particular type of threat. The immune system also supports another adaptive behavioral response called behavioral recuperation following infection or injury. Pro-inflammatory cytokines are proteins that are released during an infection or injury and play an important role in facilitating the destruction of the pathogen and tissue repair. These cytokines also act on the brain and cause “sickness behavior,” which includes an increase in sleep and a decrease in social, sexual, aggressive, exploratory, and other behaviors, and leads to a withdrawal from normal activities. Overall, these behavioral changes are an adaptive mechanism for supporting recuperation from injury or illness by lowering energy utilization so that energy-related systems can support the physiological response to infection (e.g., fever) and also facilitate other restorative processes (Maier and Watkins, 1998). Researchers have argued that disengagement-related affective responses, in particular depression, share a number of features with sickness behavior, as will be described below, and may be a central part of this response. Pro-inflammatory cytokines may play a role in other forms of behavioral disengagement as well. There are a variety of physiological changes that occur with goal disengagement in animals, such as increases in ACTH and cortisol, opioid-mediated analgesia, release of dopamine from the prefrontal cortex, and lowered cellular immune function (Fleshner et al., 1989; Miczek et al., 1982; Stefanski and Ben-Eliyahu, 1996). More recently, evidence suggests a link between goal disengagement and pro-inflammatory cytokine levels. It appears that elevated levels of these cytokines are more likely to be occur when the animal is confronted

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with a stressor that is uncontrollable—a context in which behavioral disengagement would be adaptive (Kemeny et al., 2006; Dickerson et al., 2004). For example, in an encounter with a dominant and aggressive male, cytokine-induced behavioral withdrawal and reduction in aggressive and social behavior would be adaptive because it would reduce the chances of attack. This link between behavioral disengagement and pro-inflammatory production has been demonstrated using a social reorganization model, which involves the introduction of a dominant animal into the home cage of other animals. The subordinate animals show glucocorticoid resistance (GCR), a condition in which the glucocorticoid receptor on immune cells is downregulated and the cells become unresponsive to glucocorticoids, resulting in unrestrained production of pro-inflammatory cytokines. The level of GCR is correlated with the frequency of submissive behavior (Avitsur et al., 2001). Also, animals injected with a pro-inflammatory cytokine display no offensive behavior in an aggressive encounter but only defensive elements, such as upright defensive posture and submissive posture, a pattern of behavior that is usually exhibited in uncontrollable contests with other animals in which they are likely to lose (Cirulli et al., 1998). Thus, increases in pro-inflammatory cytokines may be an adaptive response to an uncontrollable social status threat because an increase in these cytokines can induce or maintain behavioral disengagement, which would reduce the likelihood that the animal would provoke an attack by the dominant animal (Dickerson et al., 2004a). Also, the release of these molecules during such an encounter would promote wound healing, since subordinates are more likely to be wounded in such encounters. Acute social threats in humans have also been shown to increase levels of these cytokines (Ackerman et al., 1998; Dickerson et al., in press). Overall, uncontrollable social and other threats may be capable of increasing levels of pro-inflammatory cytokines and eliciting behavioral disengagement in animals and humans. These immunological changes may be orchestrated by the disengagement-related affective/motivational states that are elicited in these conditions (as described below).

IV. NEGATIVE AFFECTIVE STATES AND THE IMMUNE SYSTEM There is a growing literature linking negative affective states to various immune parameters. Stable individual differences in negative affect, called negative affectivity, have been studied in psychoneuroimmunology. Negative affectivity is strongly associated with

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the personality dimension of neuroticism (Watson and Clark, 1992). Negative affectivity and/or neuroticism has been associated with decrements in functional immune measures (Segerstrom, Kemency, and Laudenslager, 2001). More recently, task-based neurophysiological measures of negative affectivity have been used to understand the link between this construct and peripheral physiological systems. For example, using an EEG measure of cortical asymmetries, Davidson (1998) found that higher levels of right-prefrontal activation is associated with less positive and more negative affect and that individual differences in this pattern of asymmetry predict responses to negative emotion tasks. This pattern of cerebral activation has been associated with lower antibody titers after vaccination for influenza (Davidson et al., 2003). Greater relative right-sided prefrontal activation has also been found to be associated with lower natural killer cell activity (Davidson et al., 1999; Kang et al., 1991). These are important studies because they utilized non-self report measures of negative affect and suggest potential CNS mediators of the link between affect and peripheral physiological response patterns.

V. DEPRESSION AND THE IMMUNE SYSTEM The most commonly and carefully studied affective state in the field of PNI is depression. Studies have examined correlations between major depressive episodes and immune status as well the associations between immune processes and other forms of psychiatric depression and depressed mood. In addition, because depression has been associated with risk of mortality from cardiovascular disease (Glassman and Shapiro, 1998) as well as the onset and course of other illnesses and all cause mortality in some studies (Wulsin et al., 1999), the potential immunologic mediators of these effects are a new area of investigation. Major depression has been associated with a variety of functional and phenotypic immune alterations, in some cases mirroring the changes observed with exposure to major life events, such as bereavement (Irwin et al., 1986; Schleifer et al., 1984). Depression has been associated with reliable increases in immune subsets including neutrophils and decreases in B- and T-cells, as well as deficiencies in the proliferative response to mitogenic stimulation and natural killer cell activity, based on a meta-analytic review in this area (Herbert and Cohen, 1993). In some cases, these changes can resolve with psychiatric treatment (Irwin et al., 1992).

Depressed individuals most likely to show immune impairment are those who are older, with a more severe form of depression (for example, melancholia), and those who also have a sleep disorder or alcoholism (Irwin, 2001). See Chapter 24, this volume for a full discussion of immune correlates of depression. In addition to the function decrements described above, there is increasing evidence that depression is associated with increased levels and/or production of pro-inflammatory cytokines. Maes and his colleagues (1994) found that patients with depressive symptoms or those with a depression syndrome showed elevated levels of these cytokines, although the findings across studies have not always been consistent. Other researchers have demonstrated this pattern of inflammatory activity in patients with clinical depression compared to demographically matched controls, controlling for other medical conditions, medications, and health behaviors (Miller et al., 2002). Researchers have proposed the intriguing hypothesis that pro-inflammatory cytokines may play an etiologic role in depression (Dantzer et al., 2001; Yirmiya, 1996; Yirmiya et al., 1999). In addition to the findings linking depression with higher levels of these cytokines as described above, there are three other sources of evidence for this hypothesis. First, patients with inflammatory diseases often experience depressed affect, which could be due, in part, to the inflammatory cytokines that play a role in the pathophysiology of their condition. Second, individuals injected with these cytokines, as part of cancer treatment, for example, show psychological and behavioral changes that are similar to depression, such as dysphoria, anhedonia, fatigue, apathy, and helplessness, which disappear when treatment has ended. Third, animals injected with these cytokines show the behavioral changes called sickness behavior, including locomotor retardation; immobility; anorexia; sleep disorders; decreased sexual, social, and aggressive behavior; and anhedonia. It is interesting that the behavioral changes observed with sickness behavior bear a striking resemblance to the vegetative signs and symptoms of some forms of depression and, in many cases, are part of the diagnostic criteria for a clinical depression. Work is ongoing that attempts to link pro-inflammatory cytokines causally to depression in humans.

VI. ANXIETY AND THE IMMUNE SYSTEM Anxiety is a reaction to a real or perceived threat that can include fear and apprehension about current and future circumstances, and includes actions that relate to escape or avoidance of the threat. There is


evidence that anxiety is associated with risk for cardiovascular endpoints, including morbidity and mortality, although the findings are not as strong as they are for depression (Kubzansky et al., 1998). There is as yet no clear pattern of association between immune system alterations and anxiety disorders. In one study of panic disorder (PD) patients, changes in anxiety levels from pre to post-intervention were associated with changes in proliferative responses to mitogens and IL-2 cytokine production (Koh and Lee, 2004), suggesting that anxiety may have mediated effects of the intervention on immune functions. However, overall, for each study that has shown an immune difference in PD versus controls, another study has failed to replicate the finding. In a carefully conducted study of a range of phenotypic and functional immune parameters in medication-free patients with PD compared to a carefully matched control group without psychiatric illness, there were no significant differences in proliferative response to different mitogens or NKCA and no differences in the numbers of most lymphocyte subsets (Schleifer et al., 2002). However, these studies are conducted in patients who are not undergoing a panic attack and do not provide evidence for or against the possibility of immune effects of the experience of panic during an actual episode. A variety of studies have correlated anxiety levels with immune parameters in individuals without a psychiatric disorder. Again, the studies do not present a cohesive picture of the relationship between anxiety and the immune system. In some studies, increased levels of anxiety are associated with a similar pattern of immune changes seen with exposure to an acute laboratory stressor. In other studies, the opposite pattern is observed. When individuals in an actively anxiety-producing naturalistic situation are studied, there is an increased likelihood of finding immune effects. For example, patients at their first visit to an outpatient clinic were asked about their anxiety about having cancer. Those reporting higher levels of anxiety about cancer had significantly lower levels of NKCA, which suggests that new and acute anxiety may have immunological correlates (Koga et al., 2001). In addition, some intervention studies that have demonstrated reductions in anxiety have also shown increases in functional measures of the immune system (Anderson et al., 2004). Worry can be differentiated from anxiety. Trait worriers are those who are more likely to anticipate and imagine potential future negative events. Individuals with trait worry (controlling for anxiety) have demonstrated distinct immune changes (e.g., fewer NK cells) in response to naturalistic stressors such as the after-

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math of an earthquake (Segerstrom et al., 1998) as well as immune alterations in response to acute laboratoryinduced stressors (Segerstrom et al., 1999).

VII. ANGER, HOSTILITY, AND THE IMMUNE SYSTEM The anger family of affective experience includes anger, hostility, and aggression (Spielberger et al., 1995). Anger is an emotion that often results from exposure to a situation that interferes with one’s goals, particularly if the interference is experienced as deliberate (Ekman, 2003). It can also be elicited from the perception that one has been treated unfairly; it is associated with the tendency toward aggressive behavior. Hostility has been considered both a mood state and an affective trait and often includes specific cognitions about others, in particular cynical mistrust. Both anger and hostility can lead to aggressive behavior, which involves the intention to harm. There is now an extensive literature suggesting that hostility is as an independent risk factor for coronary heart disease (Miller et al., 1996). Outcomes in these studies have included measures of blood pressure, atherosclerosis, coronary artery calcification, mortality, and other endpoints (Suinn, 2001). Anger, particularly trait anger, has also been linked to cardiovascular endpoints, such as the incidence of cardiac events, but the relationship has been studied less often (Kubzansky and Kawachi, 2000). Evidence is beginning to suggest that immune factors may play a role in these relationships (Murasko et al., 1988). For example, it is now recognized that inflammation can play a key role in plaque formation, and studies are beginning to provide the links between hostility, inflammatory processes, and cardiovascular disease. In a related literature, anger has been associated with greater cardiovascular reactivity, and cardiovascular reactivity has been related to declines in cellular immunity (Matthews et al., 1995). In an excellent model for understanding the relationship between acute real-world anger and the immune system, Kiecolt-Glaser and colleagues (1993) asked married couples to engage in a standardized marital interaction task which involved attempting to resolve a conflict in their marriage for 20 minutes. They found that hostility coded from verbal and nonverbal indicators during the conflict task predicted decreases in a number of functional immune parameters (NKCA, proliferative response to mitogen stimulation), and increases in antibody titers to latent viruses (an indicator of failure of immune control over this latent virus) over a 24-hour period.

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One intervention study suggests the possibility that anger experience may have some beneficial physiological effects. In a cognitive behavioral intervention in cancer patients, results indicated that those assigned to the intervention, compared to the control condition, showed a decrease in negative mood over a 6-month follow-up period as well as an increase in the percent of NK cells and an increase in the ability of the NK cell activity to be augmented with the addition of IFNα. In other words, the cells were more responsive to cytokine signals following the intervention (Fawzy et al., 1990). In addition, patients assigned to the intervention showed a greater disease-free interval and greater survival over a 6-year follow-up (Fawzy et al., 1993). With regards to psychological mediators of NK effects, the patients most likely to show increases in NK cell percent or function following the intervention were those who showed a decrease in depressed and anxious mood over the 6-month period and an increase in anger. Thus, it may be that interventions of this kind help those with difficulty acknowledging, experiencing, or expressing anger, and that changes in their response to their own anger experience are associated with physiological benefits.

et al., 2004; Dickerson and Kemeny, 2004; Dickerson et al., 2004b). Chronic or repeated exposure to social evaluative or rejecting contexts, a sensitivity to evaluation or rejection, or a vulnerability to experience shame might result in persistent activations of these systems with potential health consequences. In partial support of this hypothesis, rejection sensitivity around homosexuality predicted CD4 T-cell decline, time to the onset of AIDS, and mortality in a 9-year follow-up of HIV positive gay and bisexual men (Cole et al., 1997). Moreover, rejection sensitivity also predicted elevated levels of HIV viral load following antiretroviral treatment (Cole et al., 2003), controlling for demographic, behavioral, and medical confounds. Persistent HIV-related shame over a 1-year period predicted CD4 decline over 7 years in an HIV positive sample (Weitzman et al., 2005). Thus, shame, as a disengagement-related emotion, is related to a physiological system that supports disengagement and may also be associated with health effects when experienced persistently.

VIII. SELF-CONSCIOUS EMOTIONS AND THE IMMUNE SYSTEM

A growing but still limited literature suggests that positive affective states may also be associated with immune system changes (Pressman and Cohen, 2005). Older literature suggests that positive affect is associated with increases in secretory Immunoglobulin A (SIgA), an antibody found in the mucosal immune system (e.g., in saliva) (Hucklebridge et al., 2000; Lambert and Lambert, 1995; McClelland and Cheriff, 1997). These findings are difficult to interpret. In most cases, total salivary protein levels were not controlled for and increases in salivary rate could explain some of these findings (decreases in salivation are associated with anxiety). A much stronger approach involves the administration of an antigen and the examination of SIgA specific to that antigen. In two studies, daily positive affect was associated with elevated levels of SIgA to a specific antigen measured on a daily basis (Stone et al., 1987; Stone et al., 1994); one study did not find this association (Evans et al., 1993). Inducing positive affect has been associated with increases in the number of certain subsets of white blood cells, although the results are not entirely consistent. It is interesting that the direction of relationships is similar to that seen with exposure to an acute stressor (Segerstrom and Miller, 2004). Studies of immune function are rare in this area, but evidence thus far suggests that laboratory induction of

More recently, a set of studies has examined the physiological correlates of shame and other selfconscious emotions and the possible health consequences of persistent shame-related affective experience. Shame is a self-conscious emotion, like guilt and embarrassment, that is elicited in social evaluative or rejecting contexts, in response to appraisals of a loss of social esteem (evaluations of how others view the self), social status, or social acceptance (Dickerson et al., 2004a; Gruenewald et al., 2004). “Social Self Preservation Theory” argues that threats to one’s social self are associated with the experience of shame-related emotions and a coordinated set of behavioral and physiological responses that relate to the motivation to withdraw, appease, or submit (Dickerson et al., 2004a). A key component of the physiological response is an increase in pro-inflammatory cytokines. An increase in pro-inflammatory cytokines would support the behavioral disengagement that occurs in this context (as described above). In a number of studies of acute laboratory-induced social evaluative threat, increases in social evaluation and shame have been associated with increases in cortisol and/or pro-inflammatory cytokines (Gruenewald

IX. POSITIVE AFFECT AND THE IMMUNE SYSTEM

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positive affect may result in increases in lymphocyte proliferative response to mitogenic stimulation (Futterman et al., 1992). These effects were not mediated by changes in cortisol in response to the manipulation. Positive mood induction may also result in increased levels of certain cytokines (interleukin [IL]-2, IL-3) while resulting in decreases in others (i.e., interferon-γ and tumor necrosis factor-α (Mittwoch-Jaffe et al., 1995). However, some studies have found no correlates (Pressman and Cohen, 2005; Ryff et al., 2004). In studies of a whole ex vivo immune responses—type I hypersensitivity to an allergen—positive states of mind have been associated with reductions in response in some studies (Kimata, 2001; Laidlaw et al., 1996) but not in others (Zachariae et al., 2001). There is a consistent increase in the number of NK cells and often in NKCA in laboratory studies of stress induction (Segerstrom and Miller, 2004). It is interesting that induction of positive affect can also result in an increase in NK function. One study found such an increase in NKCA with both positive and negative affect induction; both appeared to be mediated by sympathetic nervous system arousal (Futterman et al., 1994). It is possible that changes in patterns of activation of the HPA, sympathetic nervous system, and/ or parasympathetic nervous system could mediate some of the associations found above. A number of studies have shown decreases in cortisol levels following positive mood induction or correlated with trait positive affect in naturalistic studies (Berk et al., 1989; Buchanan et al., 1999; Zachariae et al., 1991), and some of the immune parameters described above can be affected by the HPA (e.g., cytokine production). However, others have found increases in cortisol with induced positive affect, which may result from the arousal that is experienced (Pressman and Cohen, 2005). Positive affect induction has been shown to increase heart rate and blood pressure in a number of studies (Ekman et al., 1983; Futterman et al., 1994; Schwartz et al., 1981) particularly in tasks that involve active rather than passive involvement of participants (Pressman and Cohen, 2005). This pattern of changes is consistent with ANS effects of stress or negative affect manipulations. However, in studies that attempted to test for endocrine or autonomic mediation of the relationship between positive affect and the immune system, the results are mixed. For example, in the study comparing positive and negative affect induction, cellular proliferation indices were reduced with positive affect, but cortisol levels and SNS arousal were increased (Futterman et al., 1994).

X. DIFFERENTIAL IMMUNE CORRELATES OF AFFECTIVE STATES Very few studies have directly tested the premise that different affective states may have different immunological effects. There is, however, some evidence that induced positive and negative affect result in different acute immunological changes. Futterman and colleagues (1994) found an increase in NKCA with both positive and negative affect induction over a 25minute period. However, positive affect was associated with an increase in the proliferative response to mitogens, while negative affect was associated with a decrease in this immune function. These data are consistent with a larger literature indicating that the number of NK cells and, in some cases, their activity increase in response to a wide variety of emotionally evocative stimuli. However, these data suggest that proliferative capacity may depend on the valence of the emotions experienced. Also, studies of daily mood ratings and measures of antigen-specific IgA antibody response have shown increases in this antibody response with positive affect and decreases with negative (Stone et al., 1987). There is initial evidence of different immune correlates of bereavement exposure depending on whether an individual responds with depression and/or grief (Irwin et al., 1987; Kemeny et al., 1995; Kemeny et al., 1994). And, in one study designed to induce shame and guilt by asking individuals to write about a situation in which they blamed themselves on three different days in one week or to write about a neutral topic, the self-blame condition elicited increases in TNF-α receptor levels in oral mucosal transudate—a marker of pro-inflammatory cytokine activity—and the increases were correlated with increases in shame but not guilt (or other negative emotions) (Dickerson et al., 2004b).

XI. SUMMARY Evidence suggests that immune system changes are associated with affective states, including depression, anxiety, anger/hostility, shame, and positive affect. As yet, studies have not been able to determine whether the nature of the changes depend on the nature of the affective experience or are a function, for example, of the arousal that occurs across many forms of emotional experience. There is also not sufficient evidence to support the hypothesis that adaptive immunologic changes occur in the context of certain emotional/ motivational states, namely the fight/flight response, the recuperative responses, and the behavioral disen-

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gagement response. While initial evidence suggests adaptive trafficking effects of white blood cells with fight/flight responses and increases in the production of pro-inflammatory cytokines with disengagement responses, this area requires further study to fully test these hypotheses.

XII. FUTURE DIRECTIONS Research on emotion and the immune system to date has suffered from significant limitations that hinder development in this important area. For example, many studies have relied on self-report of emotion and mood. Self-reports suffer from a number of methodological problems. Most such scales require individuals to describe their emotional response over an extended time period, e.g., level of sadness over the past week on a scale from 1 to 7. It is unclear how individuals do this and what cognitive processes are utilized to make this determination. Do people rely on their current state of mind or try to count up the number of sad episodes over the time frame? Kahneman and colleagues (Kahneman and Riis, 2005) argue that there are three “selves” that can be tapped by a given measurement tool: the experiencing self, the evaluative self, and the anticipating self. Self-reports of mood capture the evaluative self—i.e., how we evaluate our experience and summarize it—but do not capture the experiencing self—what we actually experience at a given moment in time. And there are a number of biases that have been demonstrated when individuals attempt to evaluate their subjective states over a period of time. For example, after a medical procedure, pain ratings of the procedure reflect the most recent level of pain experienced and the most intense pain experienced during the procedure but do not capture duration and other dynamics. This pattern has been called the “peak end theory” of bias in reports of emotional experience (Kahneman et al., 1993). Thus, it may be very difficult to link changes in self-reported affect and changes in physiological parameters when the affect reports do not capture the actual affect experienced over the time frame of interest. To capture something closer to the actual emotional experience over a given period, all moments in a given episode would be assessed. The closest approximation to this approach is experience sampling, where online assessments of emotion are captured at specific moments throughout the day, with various collection methods like diaries or PDAs. There are a few studies in the emotion and immunity literature that utilize this technique; rather, the vast majority use scales that require evaluation and summarization and do not

adequately represent the emotional experience. This limitation may explain some of the difficulty investigators have found in correlating changes in affective state with changes in peripheral physiological indicators. Second, there are individual difference factors that complicate these self-report affect assessments. For example, many people are not psychologically minded and do not differentiate their feelings in the way questionnaires require. Some individuals have rarely reflected on what they are feeling and may not be able to make the emotional distinctions required in a mood measure. Emotion reporting is confounded by emotional styles; e.g., those with a repressive style will under-report their emotional experience in contrast to those with a sensitizer style. Other biases, such as the motive to respond in a socially desirable way or to please the investigator, complicate self-reports in this area. There are now a number of alternatives to selfreports that may increase the chances of detecting relationships between emotion and immune parameters. Tools used in other fields of psychology, for example, cognitive psychology and affective neuroscience, are beginning to be employed by investigators interested in predicting patterns of peripheral physiological responding. Examples of more behavioral measures of emotional responding include emotion-induction techniques such as emotion films or other tasks that generate emotion and involve ratings of facial expressions of emotion, tasks that prime emotion, implicit measures that capture “non-conscious” emotional experience, and startle eye blink tasks to measure anxiety-related experience. Despite the fact that human research in PNI began to flourish in the 1980s, it is difficult to paint a coherent picture of the primary psychological processes that can affect the immune system, outside the work on depression and “stress.” Studies of affective experience, including affective pathologies, individual differences in affectivity, and emotional responses, could play a critical role in bringing clarity to this important area. Such research could help to link other important psychological factors such as personality, coping processes, social context, and appraisals to peripheral physiological responses, including the immune system.

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C H A P T E R

30 Behaviorally Conditioned Enhancement of Immune Responses GUSTAVO PACHECO-LÓPEZ, MAJ-BRITT NIEMI, HARALD ENGLER, AND MANFRED SCHEDLOWSKI

I. INTRODUCTION WITH A HISTORICAL OVERVIEW 631 II. THE PHENOMENON OF BEHAVIORALLY CONDITIONED ENHANCEMENT OF IMMUNE RESPONSES 636 III. CLINICAL AND ADAPTIVE RELEVANCE OF CONDITIONED IMMUNE ENHANCEMENT 652 IV. SUMMARY 654

is beginning to emerge, with potential clinical applications.

I. INTRODUCTION WITH A HISTORICAL OVERVIEW Ambulatory organisms evolved to face a rapidly changing environment, thus acquiring the ability to learn. Most learning situations comprise one or more initially neutral stimuli (later becoming the conditioned stimulus, CS), the animal’s responses (behavioral and others), and a biologically significant stimulus (unconditioned stimulus, US). Classical or Pavlovian conditioning is often described as the transfer of the response-eliciting property of a biologically significant stimulus (US) to another stimulus (CS) without that property (Carew and Sahley, 1986; Domjan, 2005; Fanselow and Poulos, 2005; Hawkins et al., 1983; Pavlov, 1927). This transfer is thought to occur only if the CS serves as a predictor of the US (Pearce, 1987; Rescorla, 1988; Rescorla and Wagner, 1972). Thus, classical conditioning can be understood as learning about the temporal or causal relationships between external and internal stimuli to allow for the appropriate preparatory set of responses before biologically significant events occur. Much progress has been made in eluci-

ABSTRACT Classical conditioning can be understood as learning about the temporal or causal relationships between external and internal stimuli to allow for the appropriate preparatory set of responses before biologically significant events occur. In this regard, the capacity to associate a certain immune response or status (e.g., allergens, toxins, antigens) with a specific stimulus (e.g., environments or flavors) is of high adaptive value; we consider that it was acquired during evolution as an adaptive strategy in order to protect the organism and/or prepare it for danger. In this chapter current data are reviewed and summarized to indicate that both innate and adaptive immune responses can be enhanced by behavioral conditioning protocols. An understanding of the effects of behavioral conditioning on immune functions PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

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dating the neuronal and molecular events that take place during association and consolidation of the memory trace in classical conditioning paradigms (Berman and Dudai, 2001; Bermúdez-Rattoni, 2004; Glanzman, 1995; Kandel et al., 1983; Menzel and Muller, 1996; Tully et al., 1990).

A. Definition of Behavioral Immunoconditioning An immunomodulatory stimulus (e.g., antigen, immunostimulating, or immunosuppressive drug) is used as a US and paired with a neutral stimulus. After this phase, the neutral stimulus becomes a conditioned stimulus (CS), which modifies the immune response (conditioned response, CR), as the US did before conditioning. The influences of behavioral conditioning on immune responses have been reviewed several times before (Ader and Cohen, 1991; Ader and Cohen, 2001; Brittain and Wiener, 1985; Hucklebridge, 2002; Markovic et al., 1993). In this chapter we will focus on conditioning protocols eliciting an enhancement of immune responses.

B. Associative Learning Rules Two basic steps compose any conditioning protocol: an association phase in which one or more CS-US pairings occur inducing an associative learning process, and a recall phase in which the memory of such an association is retrieved after exposure to the CS. In order to successfully acquire a typical Pavlovian CR (e.g., salivation, eye blink, nictitating membrane, freezing behavior), many CS-US pairings or trials are necessary, occurring in a contingent manner, with a fixed order (forward association: CS precedes US), and with a short inter-stimuli interval (seconds or even shorter intervals). Typically, a backward association scheme where the US precedes the CS will not induce an associative learning process. The features of CS-US timing and predictability affecting classical conditioning have been reviewed elsewhere (Rescorla, 1988). Regarding the CS, it is important to mention that features such as novelty, intensity, duration, and naturalistic relation to the US may explain the feasibility of associating the CS with the US, and the stability and strength of such an association. The rest interval between association and recall is another key factor explaining the CR magnitude: Typically, the longer the rest interval, the weaker the CR (passive forgetting). Moreover, extinction of the CR is a classical feature of behavioral conditioning. This phenomenon occurs at recall time if the CS is repeatedly presented without the US (active forgetting), reducing the CR magnitude, and finally extin-

guishing it. Pre-exposure to the CS before the association phase usually induces a reduction in the CR, which is called latent inhibition. Additionally, it is important to mention that the CR and the response elicited by the US (unconditioned response, UR) are not necessarily of a similar magnitude nor of the same direction, and the kinetics of each response may also differ (Rescorla, 1988).

C. Experimental Design in Immunoconditioning When I. P. Pavlov (1849–1936) studied the conditioned salivary response in dogs, it was sufficient to note that before conditioning a dog would not salivate after the sound of a bell, but that after conditioning the dog would do so. All that was needed as a control was this before-after comparison with another dog not exposed to the conditioning protocol. The emphasis was on observing conditioning in the individual animal, and it was noted that some animals conditioned better than others (Pavlov, 1927). Dealing with immune functions involves further challenges for the researcher attempting to demonstrate a behaviorally conditioned immune response. The immune system itself shows “memory” to be independent of neural activity. This means that the experimenter not only has to control for psychological sensitization and habituation, but also for immune memory and tolerance. In addition, immunoconditioning protocols involving taste/odor-visceral associative learning may require more consideration than standard conditioning protocols. The fact that flavor trace memory lasts for a number of hours should be taken into account in the experimental design of immunoconditioning protocols as well as in the analysis of the results (Garcia et al., 1966; Schafe et al., 1995). Considering all these particularities with regard to the behavioral conditioning of immune functions, R. Ader and N. Cohen provided the following guidelines for the experimental design of such protocols (Ader, 2003; Ader and Cohen, 2001): • Conditioned group: The primary experimental group

that is conditioned by pairing CS and US, and at recall time is exposed to the CS. • Conditioned not evoked group: At association time this group is treated identically to the conditioned group, but not exposed to the CS at recall phase. This group serves as a control for any direct or indirect immunomodulating effects of the association procedure per se, as well as for possible residual effects of the US at recall phase upon re-exposure to the CS.

30. Conditioned Immune Enhancement • Unconditioned response group: At association time

this group is exposed to the US and CS at the same times as the conditioned group. At recall time it should be exposed to the US in the absence of the CS. This control group defines the magnitude and direction of the UR. • Latent inhibition group: In order to assure an associative learning process, this group is pre-exposed to the CS at least once before being submitted to the same association and recall regimen as the conditioned group, since pre-exposure to the CS before the association phase usually induces a reduction in the CR. • Non-conditioned but evoked group: At association time this group is exposed to the CS and US as many times as the conditioned group, but in a non-contingent manner. At recall time, the subjects in this group are exposed to the CS. This group is mainly intended to control for non-associative factors, and also certifies the immunological neutrality of the CS during association and recall phases as well. • Placebo group: In the association and recall phases this group is exposed to the CS, which is paired with an immunological neutral stimulus (e.g., sterile saline or phosphate-buffered saline) as a placebo. This group controls for residual effects of the CS and possible artifacts due to the procedure (e.g., handling, injection, etc.). It should be noted that there may be experimental situations in which it is not possible to include certain control conditions, in particular in human studies. However, we recommend that future studies on immunoconditioning include all necessary control groups, since omissions will be detrimental to the interpretation and validation of the resulting data.

D. Theoretical Framework for Behavioral Immunoconditioning Before the existing data about conditioned enhancement of immune responses can be analyzed in detail, it is important to draw up some guidelines for the general mechanisms underlying behavioral immunoconditioning (summarized in Figure 1). Part of this conceptualization has already been elaborated (Bovbjerg, 2003; Eikelboom and Stewart, 1982). According to this theory, in the terminology of behavioral conditioning, both the CS (i.e., changes in the external environment) and the US (i.e., changes in the internal environment) must be inputs to the central nervous system (CNS), which in turn processes and associates

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this information. Thus, at association time only a change in the immune system that is sensed by the CNS can serve as a US. Furthermore, both the CR and the UR must be outputs of the CNS. Thus, only an immune parameter that can be modulated by the CNS can serve as a UR for conditioning, and at recall time the CR will resemble such a UR. Association: There are two possible unconditioned stimuli employed to acquire a behaviorally conditioned immune response. The US that is directly detected by the CNS is defined as a genuine US, whereas the one that needs one or more intermediary molecules, released by another system, in order to be detected by the CNS, is called a sham US. For any US, genuine or sham, there are two possible afferent pathways to the CNS: (a) a neural afferent pathway and (b) a humoral afferent pathway. The neural afferent pathway may detect the US and translate this information into neural activity. This sensory process implies the CNS’s interoceptive capacities including immunoception (Blalock, 2005; Goehler et al., 2000). Theoretically, this afferent pathway may also be able to codify the location of stimulation (local vs. systemic). The humoral afferent pathway is required for any US that is not detected locally or for the molecules induced by sham US that reach the CNS via blood stream. If a sham US has effects on several cell types, it is reasonable to assume that several molecules are candidates for the genuine US perceived by the CNS. Figure 1 indicates that for the sham US the pathway to the CNS is complex and longer than for the genuine US. In this regard, we hypothesize that it takes longer for the CNS to detect the sham US after administration. This feature can be employed in the experimental design to elucidate the nature of the US. For instance, backward conditioning should not be possible in the case of the genuine US, whereas the sham US could induce a CR under such conditions. For those sham USs that affect immune functions, it is necessary to consider that the immune history (tolerance and memory) of the subject may interfere with the response to such USs, resulting in a different immune reaction and different signal intensity to the CNS. For example, the immune response to a given antigen differs completely between the first and the second exposure, since an immune memory process takes place after the first exposure to an antigen. Another example is the phenomenon of immune tolerance developed after repeated exposition to the same drug. Therefore, applying the same conditioning protocol to two subjects with varying immune histories may result in quite different associative learning processes, which in turn may affect the conditioned immune responses in diverse ways.

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FIGURE 1 Theoretical framework for immunoconditioning. At association time there are two possible unconditioned stimuli (US) to be associated with a conditioned stimulus (CS). The US that is directly detected by the central nervous system (CNS) is defined as a genuine US, whereas the one that needs one or more intermediary molecules, released by another system, in order to be detected by the CNS is called a sham US. For any US, genuine or sham, there are two possible afferent pathways to the CNS: a neural afferent pathway and a humoral afferent pathway. At recall time there are two possible pathways by which the CNS can modulate immune functions: the humoral efferent pathway and the neural efferent pathway. The humoral efferent pathway may imply changes in neurohormones that directly or indirectly modify the immune response. The neural efferent pathway is supported by the direct innervations of primary and secondary lymphoid organs. Regarding the CS, it is important to mention that features such as novelty, intensity, duration, and naturalistic relation to the US may explain the feasibility of associating the CS with the US, as well as the stability and strength of such an association. In addition, it is necessary to consider that the immune history (tolerance and memory) of the subject may vary the response to the US, and from the CNS, resulting in a different associative learning at association time and/or in a different conditioned response at recall time.

In Pavlovian conditioning, the strength of the association between the CS and the US is affected by the temporal relation between these stimuli, the interstimulus interval. The orthodox theory predicts that when the US precedes the presentation of the CS (backward conditioning) learning is poor. In addition, when the CS precedes the US by increasing intervals, the probability of a CR declines (Rescorla, 1988). The specific inter-stimulus interval that yields the most pronounced CR varies with the organism and responses studied. For behavioral immunoconditioning, this associative feature could be employed to delineate the nature of the US (genuine vs. sham) by systematically

varying the inter-stimulus interval between the CS and US and describing the UR kinetics. Regarding the CS, it is important to mention that features such as novelty, intensity, duration, and naturalistic relation to the US may explain the feasibility of associating the CS with the US, as well as the stability and strength of such an association. For instance, it is well documented that gustatory or olfactory stimuli are strongly and easily associated with visceral US, in contrast to tactile, visual, or auditory stimuli (Domjan, 1973; Domjan, 2005). The strength and lastingness of flavor-visceral associations are reflected in several features of the CR, such as the high magnitude and the

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low forgetting rate. Additionally, the neutrality of the CS on the immune system needs to be assured for each experimental setting. Recall: The CR is the tacit and unique proof that an associative learning process has occurred at association time. In several immunoconditioning protocols the experimental subjects display a complex CR, with behavioral and neuroendocrine components in addition to the conditioned effects observed in the immune system. Furthermore, it is necessary to consider that the immune system itself displays sensitization (memory) and habituation (tolerance) responses to specific stimuli and that, in addition, many immune parameters underlie a circadian rhythm. Thus, the specific time-points for association and recall, as well as the immunological history of each experimental subject, may be important variables for the final outcome of the conditioned immune response. The portrait of some of the conditioned immune responses reported, therefore, might be the bizarre reflection of neural activity, which cannot be explained by the established learning and memory rules. For example, in a given conditioning protocol, the neural activity responsible for the conditioned immune response may follow a normal extinction process during consecutive recall trials, whereas the conditioned immune response itself may not display the same extinction slope, or may even be enhanced. Such peculiar results might be explained in two ways: It is possible that the interval between the recall trials is not long enough for the specific immune measure to return to its baseline, resulting in an additive effect of the conditioned immune response during consecutive, near-recall trials. Another possibility is related to immune processes that may be modulated by neural activity; however, once such a process has started, it is basically independent of neural modulation (e.g., hematopoiesis, immune memory, anaphylactic response). Thus, some changes in the immune response cannot strictly be called a CR (Eikelboom and Stewart, 1982). In summary, immune responses can be affected by behavioral conditioning protocols, but this does not necessarily imply that such immune responses were behaviorally conditioned. Regarding the conditioned immune response, there are specific features that may give important clues to its nature, and should be considered in the experimental design in order to differentiate the underlying mechanisms. There are two possible pathways by which the CNS can modulate immune functions: (a) the humoral efferent pathway and (b) the neural efferent pathway. The humoral efferent pathway may imply changes in neurohormones that directly or indirectly modify the immune response at recall time. The peripheral effects evoked after activation of this pathway would be diffuse

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and long-lasting like any neuroendocrine response. The neural efferent pathway is supported by the direct innervations of primary and secondary lymphoid organs (Elenkov et al., 2000; Mignini et al., 2003). There are several immune parameters that are subject to neural modulation, e.g., T-cell differentiation (Sanders and Kohm, 2002; Sanders and Straub, 2002), hematopoiesis (Artico et al., 2002; Maestroni, 1998; Miyan et al., 1998), T-cell activity, B-cell activity (Downing and Miyan, 2000), NK cell activity (Hori et al., 1995; Katafuchi et al., 1993a; Katafuchi et al., 1993b), and inflammatory response (Czura and Tracey, 2005; Pavlov and Tracey, 2005; Tracey, 2002). We hypothesize that many of these neuroimmune interactions may be affected by behavioral conditioning protocols, but few have been investigated experimentally so far. The extinction of the conditioned immune response is another feature that may give some hints about the underlying mechanisms. It has been demonstrated that for behavioral conditioning, in which the nervous system directly modulates the CR (e.g., nictitating membrane, gastric secretion, aversive behavior), an extinction process occurs when the CS is repeatedly presented without the US (active forgetting). However, if the CR observed is the reflection of neural activity on different types of cells, like immune cells that exhibit tolerance and memory processes, then CR extinction will not necessarily be elicited in an orthodox Pavlovian manner. Moreover, if the CR observed reflects the neural activity on two or more cellular types, then the picture may be more abnormal (e.g., neuroendocrine efferent pathway affecting immune functions). Further consideration is required since the life span of immune cells is short. Thus, each recall trial might affect a different cellular set.

E. Historical Development of Immunoconditioning S. Metalnikov and V. Chorine are generally credited with having conducted the first studies on behaviorally conditioned immune effects (Metalnikov and Chorine, 1926). However, V. I. Luk’ianenko cites observations in the dissertation of I. I. Makukhin (1911) at the University of St. Petersburg, and the report by A. Voronov and I. Riskin (1925) as perhaps the first ones demonstrating “conditioned leukocytic reaction” (Luk’ianenko, 1961). From the beginning, behavioral immunoconditioning has had an interdisciplinary history. The enzymatic-immunologist Sergey Metalnikov was born in 1870 in St. Petersburg, Russia. After finishing his studies at the University of St. Petersburg, he worked for many years with the embryologist A. Kowalevsky. In 1897 he moved to the University of

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Heidelberg, with the zoologist O. Bütschli, and then in 1901 he went to work with the famous immunologist E. Metchnikov at the Pasteur Institute in Paris. In 1907 he was appointed professor at the University of St. Petersburg, and in 1910 he became director of the Institute of Biology. At that time I. P. Pavlov was already a famous physiologist, who had been awarded the Nobel Prize in 1904, and certainly S. Metalnikov would have been aware of the conditioning paradigms. During the Bolshevistic revolution in 1919, he decided to leave Russia and returned to continue his work at the Pasteur Institute in Paris. Due to the importance of these visionary findings, it is worthwhile taking a closer look at the first reports on conditioned enhancement of immune responses. In their pioneer experiments, increases in peritoneal leukocytes similar to those observed after an antigenic challenge were reported as a result of behavioral conditioning (Metalnikov and Chorine, 1926; Metalnikov and Chorine, 1928). Guinea pigs were given daily association trials with contingent pairing of scratching or heating of the skin as a CS, and with an immune challenge as US (i.p. injection of a small dose of tapioca, Bacillus anthrax, or a Staphylococcus filtrate). The conditioning protocols consisted of 15 to 20 association trials, and after 12 to 15 days recall took place. The CR was an increase in peritoneal leukocyte numbers that was weaker and more transitory than the UR. Followup experiments examined the survival of guinea pigs after a normally lethal injection of Vibrio cholera bacteria. Conditioning was essentially carried out as before. Association was performed by a daily presentation of the CS (scratch) contingently paired with a US (injection of either Staphylococcus filtrate or B. anthrax). The number of association trials varied greatly among these experiments, and different resting intervals between association and recall were reported. In each experiment, there were two conditioned animals and two control animals, which received neither the CS nor the US in the association phase. One day post-recall, all animals were given an injection of V. cholera. The unconditioned control animals died after 7–8 hours, whereas the conditioned animals died after 36 hours. Some conditioned “survivors” were tested once more a month later, receiving an injection of Streptococcus. One animal was evoked (CS exposure) before such injection, whereas another was not. The re-evoked individual survived, whereas the non–re-evoked and two controls died. These initial results were rapidly replicated (Nicolau and Antinescu-Dimitriu, 1929a; Nicolau and Antinescu-Dimitriu, 1929b; Ostravskaya, 1930), and in the following years considerable attention was given to the question of conditioned immune effects by

Soviet investigators (Ader, 1981; Luk’ianenko, 1961). Many of these experiments were basically similar to those performed by Metalnikov and Chorine (1926, 1928), and apparently there was some controversy over the reproducibility of some experiments. In parallel to the Soviet experiments, but not as well known, investigations were performed in Romania by I. Baciu (Baciu et al., 1965; Benetato, 1955; Benetato et al., 1952) demonstrating that the phagocytic activity of polymorphonuclear cells in the peripheral blood of dogs could be markedly increased by behavioral conditioning. In 1975, a new interest in the behavioral conditioning of immunity was generated by the publication of R. Ader and N. Cohen on conditioned immunosuppression (Ader and Cohen, 1975), which formally is the beginning of psychoneuroimmunology as a modern (inter)discipline.

II. THE PHENOMENON OF BEHAVIORALLY CONDITIONED ENHANCEMENT OF IMMUNE RESPONSES There are several ways to arrange the data reporting on the behavioral conditioning of immune functions. One possibility is to organize the data according to the nature of the CS (e.g., gustatory, olfactory, visual, auditory, touch) or the type of US (e.g., stress, immunosuppressive, or immunoenhancing drugs; antigens; cytokines). However, regardless of the experimental protocol, we decided to classify the existing data according to the conditioned immune response, with the aim of giving relevance to the possible biological and clinical significance of immunoconditioning. In general, the conditioned enhancement of immune responses can be classified into two main categories: (a) the innate immune responses and (b) the adaptive immune responses. Furthermore, a nomenclature will specify the conditioning protocol employed in each experiment. For example, a putative conditioning protocol with 5 association and 11 recall trials will be described as 5/11 conditioning.

A. Innate vs. Adaptive Immune Responses The immune system can be roughly classified into two subsystems: the innate and the adaptive immune system. In general, innate immunity is considered a non-specific response, whereas the adaptive immune system is thought to be very specific. The main distinctive features of both innate and adaptive immune responses are depicted in Table 1 and have been reviewed elsewhere (Janeway et al., 2005).

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30. Conditioned Immune Enhancement TABLE 1

Summary of Innate and Adaptive Immune Responses

Innate immune responses

Adaptive immune responses

Receptors

Germline-encoded

Distribution Repertoire

Subset-specific but non-clonal Limited Selected in groups of individuals within a given species No Phagocytic cells (e.g., Neutrophils, Macrophages, Monocytes), as well as NK cells, eosinophils, dendritic cells Complement, certain cytokines (e.g., IFN, TNF-α), enzymes (e.g., lyzozime), nitric oxide There is immediate maximal response

Immune memory Cellular component Humoral component Speed of the response

B. Stress Induces Enhancement of Immune and Cognitive Functions The impact of stress on both immune and cognitive functions (e.g., learning and memory) has already been documented (Dhabhar and Viswanathan, 2005; Engler et al., 2004; Shors, 2004). For example, acute stressors have been shown to enhance certain immune functions such as mitogen-induced cell proliferation, phagocytosis, and NK cell activity (Lyte et al., 1990; Millar et al., 1993; Shurin et al., 1994a; Shurin et al., 1994b). In addition, it has been reported that acute stress also enhances associative neural capacities (Shors et al., 2000). In several immunoconditioning protocols, either at association or at recall time, the experimental subjects experienced stressful conditions, thus affecting the immune and/or cognitive functions. In particular, activation of the HPA axis is often conditioned as a result of the behavioral protocols that also produce a CR in the immune system (Buske-Kirschbaum et al., 1996; Pacheco-López et al., 2004; Shurin et al., 1995). The signaling of an unconditioned immune stimulus to the CNS at association time can differ considerably between animals that are stressed and those that are not. Thus, the associative strength between the CS and the US may differ, and the CR magnitude as well. Moreover, the fact that different strains of animals displayed diverse CR magnitude, in corticosterone plasma levels, for example, in the same conditioning protocol (Shurin et al., 1995) may further complicate this issue.

C. Conditioned Enhancement of Innate Immune Responses An overview of studies reporting the conditioned enhancement of innate immune responses is shown in

Antigen receptors are products of site-specific somatic recombination Antigen receptors are clonally distributed Immense Selected in each individual within a given species Yes Lymphocytes (e.g., T- and B-cells, NK cells) Immunoglobulins, certain cytokines There is a lag time between exposure and maximal response

Table 2. In order to be able to objectively compare such data, important methodological details are included, such as the nature, duration, and intensity of CS and US; the specific conditioning protocol (number of association trials, rest interval, number of recall trials); the type of experimental subjects (species, strain, and gender); and the conditioned responses (immune and others). 1. Natural Killer Cell Activity Natural killer (NK) cells are important in innate immunity with regard to viruses and other intracellular pathogens. In addition, NK cells have a significant role within antibody-dependent cellular toxicity, which is an adaptive immune response (Janeway et al., 2005). One of the best-described immune response that can be enhanced by behavioral conditioning is the cytotoxic activity of NK cells. The US employed is polyinosinic : polycytidylic acid (poly I : C), a doublestranded synthetic RNA that mimics a viral infection. The advantages of using poly I : C over live viruses include safety, convenience, and, more importantly, reproducibility and control over dose and time of administration of the immunological challenge. Systemic administration of poly I : C elicits an acute-phase reaction, fever, and sickness behavior in a variety of species including mice, rabbits, guinea pigs, and rhesus monkeys. Poly I : C is also a potent inducer of alpha and beta-interferon (IFN) which, in turn, stimulate NK-cell–mediated cytotoxicity (Magee and Griffith, 1972; Manetti et al., 1995). Conditioning protocol: It is necessary to indicate some key features of the conditioned enhancement of NK cell activity. All experiments were performed in colonyhoused BALB/c mice, mostly in females and just once in males (Spector et al., 1994), and with the condition-

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TABLE 2 Conditioned stimulus Nature Duration Description

Unconditioned stimulus Nature Dose Administration route

Overview of the Conditioning Protocols Enhancing Innate Immune Responses Conditioning protocol Association # trials

Rest interval # days

Recall # trials

Subjects Species Strain Gender

Conditioned responses Immune Others

Ref.

poly I : C 20 μg/mouse i.p.

9

3

1

Mouse BALB/c Female

Increased NK-cell activity 1 day after each evocation trial NR

(Ghanta et al., 1985; Ghanta et al., 1987)

Odor 60–90 min Camphor


9

3

2

Mouse BALB/c Male

Increased NK-cell activity 1 day after each evocation trial NR

(Spector et al., 1994)

Taste & Visceral 60 min 0.1% saccharin + i.p. LiCl 125 mg/kg


1

2 or 5 or 6

1&2

Mouse BALB/c Female

Increased NK-cell activity 1 day after each evocation trial. Additive CR: i.e., CR after 2nd evocation trial >1st evocation trial. Rest interval of 6 days induces stronger CR than rest interval of 2 days NR

(Ghanta et al., 1987; Hiramoto et al., 1987)

Odor 60 min Camphor

LPS 100 μg/mouse i.p.

1

2

1

Mouse BALB/c Female

Increased splenic NK cells activity 1 day post evocation (in which a low-dose activating injection of 2.5 μg of poly I : C was i.p. administered) NR

(Demissie et al., 1997)

Odor 60 min Camphor

poly I : C 1.5 mg/kg i.p.

1

2

1

Mouse BALB/c Female

Increased splenic NK cells activity 1 day post evocation NR

(Hsueh et al., 1995; Hsueh et al., 1996; Kuo et al., 2001; Hsueh et al., 2002)

Odor 60 min Camphor

poly I : C 1.5 mg/kg i.p.

1

2

1

Mouse BALB/c Female

Increased splenic neutrophil activity 1 day post evocation NR

(Chao et al., 2005)

NR: Not reported; Poly I : C: polyinosinic : polycytidylic acid; LPS: lipopoly saccblaride


Odor 240 min (after US) Camphor


ing procedure taking place at the beginning of the inactive phase (light phase). The conditioned immune response occurred during one or more association trials (Ghanta et al., 1985; Solvason et al., 1992). Solvason et al. (1988) reported that, using camphor odor (240 minutes) as the CS and poly I : C as the US, more than six association trials were required to elicit a statistically significant enhancement of NK cell activity. For conditioning protocols in which either camphor odor (60 minutes), citronella odor (60 minutes), or saccharin + LiCl were employed as conditioned stimuli, the effect of increasing the number of association trials on the magnitude of the CR has not been systematically analyzed. CS neutrality: Using saccharin + LiCl as CS induced a significant decrease in NK cell activity (Solvason et al., 1988); therefore, this CS cannot be considered as neutral. In contrast, the neutrality of camphor odor as CS for the conditioned enhancement of NK cell activity has been proved (Hiramoto et al., 1992). The neutrality of citronella odor on NK cell activity has been suggested but not completely proved (Solvason et al., 1991). The CS duration varied from 4 hours to 30 minutes of odor exposure; however, the number of association trials (9 to 1) was modified at the same time, which makes a systematic analysis of which of these two factors is responsible for the CR magnitude impossible. The most common CS exposition length is 1 hour for camphor odor. In the first reports on conditioned enhancement of NK cell activity, the exact procedure for preparing the camphor odor (CS) was not clearly reported (Ghanta et al., 1985; Ghanta et al., 1987a; Hiramoto et al., 1987). However, in order to optimize the experimental protocol there is a citation from a later report by the same group “. . . we modified our earlier experiments using camphor odor as the CS to a onetrial conditioning event by intensifying the camphor smell during the CS exposure” (Solvason et al., 1988). The inter-stimuli interval at association time has been systematically tested within the 1/1 conditioning protocol, and it is documented that the optimum CR is achieved when the CS precedes the US by 24 hours. Intervals of 15 minutes and 48 hours yielded a CR of similar magnitude. In the backward association scheme (US→CS), intervals of 96 hours and 24 hours resulted in a similar CR magnitude (Hiramoto et al., 1992; Solvason et al., 1992). However, the fact that the association between the CS and US is still possible with intervals as long as 48 hours is striking. The rapid habituation to neural (cortical and mitral) responses seems to allow filtering of background odors, enhancing the ability of the subject to discriminate new odors (Wilson and Stevenson, 2003). This neurobiological

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feature of the olfactory sense itself rules out the possibility that such long inter-stimuli intervals are related to a possible residual camphor odor on the fur of the mice that is still present at US administration. The authors mention that the CS trace memory (camphor odor) must be latent for such a long time in order to be able to make an association with the US. This seems to be supported by the long latency (maximum 12 hours) reported for the association of the stimuli in conditioned taste aversion paradigms (Schafe et al., 1995). However, to our knowledge there is no other paradigm showing such long inter-stimulus interval. Actually, conditioned odor aversion in rats occurs only if the time interval separating the odor (CS) from the subsequent intoxication (US) is very short (less than 30 minutes), suggesting that the memory trace of the odor is subject to rapid decay (faster than taste), at least for associative processes (Ferry et al., 1999). Since it has been reported that the inter-stimulus interval (for conditioned taste aversion avoidance) is modulated by the US intensity, subject’s age, and body temperature (Hinderliter et al., 2002; Misanin et al., 2002), it will be necessary to systematically analyze such unusually long inter-stimuli intervals under more specific conditions (i.e., gender, age, temperature), before discarding this possibility. The rest interval before recall phase varied within the 9/1 conditioning paradigm from 1 to 6 days, producing unexpected results, since the longer intervals resulted in a stronger CR (Hiramoto et al., 1987). When saccharin + LiCl was used as a CS, varying the rest interval from 3 to 7 days did not influence the magnitude of the CR (Solvason et al., 1988). For the 1/1 conditioning protocols where camphor or citronella odor were employed as a CS, the rest interval reported was always 2 days. However, a systematic approach testing several rest intervals is necessary to elucidate how this factor affects the CR. Recall: The conditioned enhancement of NK cell activity has repeatedly been demonstrated 24 hours after a single recall trial and the extinction of the CR has partly been tested. The data of a report where a second recall trial was implemented (9/2 conditioning) indicate an additive effect for the CR; i.e., the magnitude of the CR after the second recall trial was larger than after the first (Hiramoto et al., 1987). In parallel, 1/2 conditioning produced a stronger CR than a 1/1 conditioning protocol (Solvason et al., 1991). Here, the authors argue that due to the kinetics of the augmentation of NK cell activity, they believe that each recall trial boosts NK cell activity in an additive manner. The fact that the same experimental subjects cannot be tested during extinction processes complicates the experimental design. Consequently, more

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recall trials should be included in future experimental designs to evaluate this unexpected result. The specificity of the CS to recall the CR has already been demonstrated (Solvason et al., 1991). Conditioned animals displayed an increase in NK cell activity only in response to the specific CS (camphor or citronella) employed at association time. No CR was evoked in animals conditioned with one CS (e.g., citronella) but evoked with another stimulus (e.g., camphor). The CR kinetics have been described for 1/1 conditioning, showing that adrenocorticotropic hormone (ACTH) was elevated in the serum within 30 minutes post-recall, followed by an increase in IFN-α mRNA expression (4–6 hours) and NK cell activity (24 hours) (Hsueh et al., 1994a). However, in a previous report IFN levels were not detectable when assayed at 6 hours post-recall in a 9/1 conditioning protocol (Ghanta et al., 1987a). In order to be able to compare the CR and the UR, it is necessary to assay the NK cell activity at other time-points after recall (e.g., 2 hours, 12 hours, 48 hours) for each conditioning protocol (1/1 vs. 9/1), and to determine the expression and production of several cytokines at different time-points post-recall. In addition to the conditioned immune effects, fever response has been reported as an additional CR elicited by 1/1 conditioning (Hiramoto et al., 1991a; Rogers et al., 1992). And, it is possible that a broader repertory of physiological and behavioral responses, not tested, have been as well conditioned, e.g., aversive/avoidance behaviors. The impact of aging on the conditioned enhancement of NK cell activity has also been suggested (Spector et al., 1994). Three-month-old and 24-monthold BALB/c male mice were submitted to a 9/2 conditioning protocol with camphor odor paired with poly I : C. Although young animals displayed a stronger immune response to poly I : C than older ones, both displayed a conditioned immune response (enhanced NK cell activity), indicating that even old animals are able to associate olfactory and immune stimuli, and evoke such association. However, old animals showed a lower CR magnitude than young ones. This is in parallel with a report showing that aged mice have diminished conditioned immunosuppression (Gorczynski, 1987; Gorczynski, 1991). Underlying mechanism: Regarding the CS (camphor odor), the neurobiology of olfactory perceptual learning has been reviewed elsewhere (Wilson and Stevenson, 2003) and is out of the scope of the present chapter. We shall, therefore, concentrate on analyzing the US within this conditioning paradigm. There is experimental evidence that one important genuine US for this conditioning protocol is the cytokine IFN-β. Interferon-β, but not IFN-α, can replace poly I : C as the US in order

to acquire a CR with enhancing effects on NK cell activity (Solvason et al., 1988). In this regard, several doses of the US (poly I : C or IFN-β) were tested, indicating that 0.5 mg/kg of poly I:C is the threshold which induces sufficient signaling as a US (Hiramoto et al., 1993a). Interestingly, lower doses of poly I : C can still raise the NK cell activity in the periphery, but apparently do not induce sufficient signaling to reach the CNS. This indicates that the level of IFN-β produced in the periphery is important, as this mediator may penetrate the CNS. It has been reported that association can be achieved by i.v. administration of IFN-β at a dose of 10,000 IU, but not at 1,000 IU. However, if IFN-β was directly delivered into the CNS via the cisterna magna, a smaller dose of IFN-β (100 IU) was enough to be associated with the CS. Supportive evidence about the preponderant role of IFN-β as a genuine US also comes from experiments in which anti–IFN-β (100 neutralizing units) was injected into the cisterna magna 24 hours before association, blocking the CR when poly I : C (1 mg/kg) was used as a US (Solvason et al., 1993). The afferent mechanism responsible for the associative learning process supporting the CR that enhances NK activity seems to be distinguishable from that by which conditioned fever is induced. Intraperitoneal injection of sodium carbonate blocked the CS-US association responsible for the enhancement of NK cell activity, but left the fever response intact. In contrast, indomethacin treatment, which blocks prostaglandin synthesis, abolished the fever response but had no adverse effect on the association of NK cell response (Rogers et al., 1992). Recently, it has been shown that interleukin (IL)-1 is the main peripheral messenger responsible for fever induction after injecting poly I : C (Fortier et al., 2004). Central processing: In order to differentiate learning from memory processes, a series of experiments was designed in which the 1/1 conditioning protocol was analyzed using a neuropharmacological approach. In these experiments different drugs were administered peripherally or centrally before association (learning) or before recall (memory). Moreover, since a long inter-stimuli interval at association time had been demonstrated for this conditioning protocol (Hiramoto et al., 1992), it was possible to administer the drugs (peripherally or centrally) after the CS but shortly before the US exposure, with the aim of distinguishing the sensory processes of the CS from those of the US. In summary, central but not peripheral lidocaine administration blocked the association as well as the recall of the CR (Rogers et al., 1994a). Additionally, it has been reported that central administration of lidocaine blocked the CNS sensory processes of the CS, but not those of the US. Following the same rationale,


peripheral or central administration of monosodium glutamate (Ghanta et al., 1994) or sodium carbonate (Rogers et al., 1994b) seems to block the US association with the CS, without affecting the CS perception. Furthermore, some data demonstrated that the hypothalamic arcuate nucleus is needed at association but not at recall time (Ghanta et al., 1994). Recall of the conditioned enhancement of NK cell activity seems to be dependent on opioid signaling. Central but not peripheral opioid pathways are thought to be involved in this associative learning process, since injection of the less potent opioid receptor antagonist quaternary naltrexone, which does not penetrate the CNS, did not affect the CR. In contrast, the association process does not seem to require central opioid pathways, since naltrexone given prior to association did not interfere with the formation of this conditioning (Solvason et al., 1989). In an initial attempt to determine the role of catecholamines within this model, reserpine treatment was administered before recall. Results showed that this treatment, unspecifically depleting central and peripheral catecholamine contents, blocked the CR (Hiramoto et al., 1990). More recently a relevant role for central catecholamines at recall time has been confirmed. α- and β-adrenoceptor antagonists or dopamine (DA)1– and DA-2–receptor antagonists administered immediately before recall also blocked the effects on the conditioned immune response (Hsueh et al., 1999). In addition, glutamate but not gamma-aminobutyric acid (GABA) was required at recall time (Kuo et al., 2001). In the last of this series of experiments, the role of cholinergic and serotonergic central systems at both association and recall times was assessed. At association, acetylcholine seems to act through the nicotinic, M2-, and M3-muscarinic receptors, whereas at recall neither the latter receptors nor the M1 receptors appear to affect the CR. In both association and recall phases, serotonin acts through the 5-HT1 and 5-HT2 receptors to modulate the CR (Hsueh et al., 2002). Determining neurotransmitter contents in brain structures during recall has produced a set of interesting results. Immediately after recall the contents of several neurotransmitters in the cerebellum, hippocampus, striatum, cortex, and amygdala were determined. Results showed that the norepinephrine content in the cerebellum and dopamine contents in the striatum and hippocampus were significantly higher in conditioned animals than in controls (Hsueh et al., 1999). In contrast, glutamate contents did not differ among the groups in any of the analyzed brain structures at recall time (Kuo et al., 2001). Neural efferent pathway: The possibility that the sympathetic innervation of the spleen is responsible for the

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conditioned increase in NK cell activity has been partially discarded. Peripheral sympathectomy (using 6OHDA) performed after association but before recall did not prevent the expression of the conditioned immune response (Hiramoto et al., 1990). However, conclusive evidence can only be obtained after splenic denervation. The humoral efferent pathway was also explored within this conditioning protocol. In conditioned animals, plasma ACTH and β-endorphin levels and splenic IFN-α expression were determined at recall time (Hsueh et al., 1994a) showing that plasma ACTH and splenic IFN-α expression were elevated after recall. These data indicate a sequence in the appearance of each signal: plasma ACTH (30 minutes) → IFN-α mRNA expression (4–6 hours) → NK cell activity (24 hours). Peripheral administration of the synthetic glucocorticoid dexamethasone blocked the recall but not the association of the CR, presumably by negative feedback inhibition of HPA axis activity (Hsueh et al., 1994b). However, control experiments are needed in which the effects of blocking the proposed signals on NK cell activity are analyzed. Recently, it has been shown that the CR may be independent of the nature of the US and that it is basically directed by the immune status of the individual at recall time (Demissie et al., 2000). In a set of experiments, two different US (poly I : C or arecoline) were paired with camphor odor as CS. At recall time, it was observed that both NK cell activity and cytotoxic T lymphocyte (CTL) activity were enhanced. Pairing only one of the two US (arecoline or poly I : C) with the CS (camphor odor) at association time resulted in similar associative learning, as shown by a similar CR at recall time. However, when the conditioned animals received an antigen challenge before association, thereby priming the CTL immune response, the resulting CR was enhanced CTL activity. More importantly, it was possible to induce both conditioned immune responses, the CTL and the NK cell activity, in animals receiving the antigen challenge before association and a suboptimal poly I : C dose immediately post-recall. It is also the case that lipopolysaccharide employed as US induces enhanced NK cell activity as CR when a suboptimal poly I : C dose was applied post-recall (Demissie et al., 1997). These data indicate that following the same conditioning protocol the immune response observed as a CR may vary among subjects, depending on their immune history and/or status at association and recall time. This phenomenon should be characterized in more detail with a view to the possible application of conditioning protocols to human subjects, who may show larger variability in their immune history and immune status at a given time-point.

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2. Neutrophil Activity Neutrophils play a key role in the cellular defense against various pathogens. The functions of neutrophils include phagocytosis, chemotaxis, and the release of inflammatory factors such as granzyme, perforin, chemokines, reactive oxygen species, and reactive nitrogen substance (Segal, 2005). Conditioning protocol: In a 1/1 conditioning protocol, using camphor odor as a CS and poly I : C as a US, splenic neutrophil activity was enhanced 24 hours after recall (Chao et al., 2005). The main control group chosen for comparison with conditioned animals was a group of animals that received association trials in a backward scheme, in which the US preceded the CS by some minutes (personal communication, C.-H. Hsueh). It has been argued that poly I : C by itself does not directly signal to the CNS. Thus, the immune consequences (e.g., cytokine production) are thought to be the genuine US that in fact reaches the CNS (Solvason et al., 1993). This process may take some minutes to be induced; therefore, it is surprising that animals submitted to a backward association scheme did not associate the CS (camphor odor) with the genuine US (cytokines) which may occur in parallel. In order to better control for conditioning, it will be necessary in future experiments to include a group of animals conditioned but not evoked, or to increase the inter-stimuli interval in the backward association. CS neutrality was analyzed in this paradigm by testing the effects of a single exposure to camphor on neutrophil activity. Nitrite levels remained the same at 0, 4, 12, 24, 48, and 72 hours after a single camphor exposure. Additionally, data from animals exposed twice to the CS but not conditioned further support the neutrality of the camphor stimulus on neutrophil activity. Chao et al. (2005) also reported the UR kinetics. Animals receiving a poly I : C injection showed that splenic neutrophil activity was significantly increased 4 hours post-stimulation. In another set of experiments, neutrophil activation markers, such as myeloperoxidase activity and adherence activity, were also determined in the splenic neutrophils isolated 4 hours after poly I : C treatment. Results showed that in addition to the release of nitrite, myeloperoxidase activity and cellular adhesiveness of the splenic neutrophils were also significantly increased. Regarding the CR kinetics, the only time-point in which the neutrophil activity was measured was 24 hours post-recall. Thus, it would be interesting to delineate the kinetics of this CR. Underlying mechanisms: Since the association protocol is identical to that used for the conditioned enhance-

ment of NK cell activity, it is likely that the afferent pathway is similar (see Section II in this chapter). However, this does not mean that the neural principles apply equally to every conditioned immune response at recall time. Central catecholamines seem to be relevant for evoking the conditioned enhancement of neutrophil activity (Chao et al., 2005). With the aim of localizing the action sites of catecholamines, the same authors analyzed the expression levels of tyrosine hydroxylase (TH) in various brain structures 24 hours post-recall. Although the results showed that conditioned animals had significantly more neurons expressing tyrosine hydroxylase in the locus coeruleus, hypothalamus, and cortex than control animals, this result does not necessarily indicate a neural memory process. It is unlikely that the memory trace lasts for 24 hours after presentation of the CS. Appropriate controls are needed in order to refute the view that a possible immunoception process (feedback) at recall phase might be responsible for the observed effects. Additionally, it is not clear whether the enhanced expression of tyrosine hydroxylase in the brain is linked to the memory process. Regarding the efferent pathway responsible for this CR (neutrophil activity), the data are different from that reported for NK cell conditioning. Peripheral sympathectomy before recall completely blocked the CR, indicating that sympathetic innervation of the spleen might be one efferent pathway. However, the main efferent pathway for the conditioned enhancement of NK cell activity seems to be independent of the peripheral sympathetic nervous system (see above). Dexamethasone treatment before recall resulted in higher neutrophil activity in conditioned animals than in controls, indicating that this specific CR is basically independent of hypothalamicpituitary-adrenocortical activity. NK cells vs. neutrophils vs. CTL activity as CR: The published data indicate that the immune consequences of poly I : C and an olfactory stimulus can be rapidly associated. The recall of such associative learning may modulate several immune functions by activation of neural and humoral (neuroendocrine) efferent pathways. However, these facts do not prove that NK cell, neutrophil, and CTL activity are truly behaviorally conditioned. Moreover, the data indicate that some immune responses are enhanced at recall time depending on the immunological history of the particular subject (Demissie et al., 1997; Demissie et al., 2000). 3. Humoral Innate Immunity Little attention has been given to humoral defense in the context of behavioral immunoconditioning,


However, several experiments indicated conditioned changes in complement activity (Dolin et al., 1960; Reidler, 1956) and lysozyme (Gasanov, 1953, cited in Ader, 1981). It is likely that humoral factors in immunity become enhanced as a CR, but to our knowledge no research program has attempted to continue this research line, nor even to confirm initial reports.

D. Conditioned Enhancement of Adaptive Immune Responses Tables 3, 4, and 5 summarize data reporting the conditioned enhancement of adaptive immune responses. In addition, they include important methodological details such as the nature, duration, and intensity of the CS and US; the conditioning protocol (i.e., number of associations trials, rest interval days, and recalls trials); the experimental subjects (species, strain, and gender); and the conditioned responses (immune and others) observed. 1. Antibody Production A central question is whether the immune response to a specific antigen can be sensed by the CNS, enabling a US to induce an associative learning process within behavioral conditioning settings. In this case, the CNS should be capable of inducing antibody production at recall time. During association, a complex cascade of immune responses (e.g., phagocytosis → antigen processing-presentation process → cytokines release → recognition of antigen by T- and/or B-cells → clonal expansion) is induced after the exposure to the US (immunization), ultimately resulting in the production of immunoglobulins (UR). The genuine US that signals to the CNS is a key feature in this process. Thus, the research reports are grouped according to the specific antigen (US) that was employed to elicit conditioned antibody production (see Table 3). In the mid-1950s Soviet researchers reported the first controlled conditioning studies in which antibody production was elicited as a CR (Zeitlenok and Bychkova, 1954). Albino rats were submitted to daily association trials, pairing a short tactile stimulus (CS) with either simultaneous or subsequent injection of influenza A virus (US). After 30–45 days, animals were exposed to the CS, and daily blood samples were taken over the next 10–12 days. When antibody titers had again stabilized at a sufficiently low level, recall was repeated for a second and a third time. Conditioned animals elicited an antibody production as a CR. However, the CR magnitude and kinetics differ to those from the UR. Interestingly, the conditioned increase in antibody production was not extinguished

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over the three recall trials and was even more pronounced after the second and the third recall trial. Several other studies followed this initial attempt to demonstrate the capabilities of the CNS to elicit antibody production as a CR. A detailed review of these pioneer reports can be found elsewhere (Ader, 1981; Luk’ianenko, 1961). Jenkins and colleagues reported elicited antibody production as a CR following a 1/2 conditioning protocol (Jenkins et al., 1983). In this report intraperitoneal immunization with sheep red blood cells (SRBC) was employed as the US, and was paired with a complex visceral-gustatory CS (LiCl intraperitoneally and saccharin orally). Seven days later, the animals were exposed to the CS and for the second time 2 days later. Anti-SRBC antibody titers were significantly elevated in conditioned animals compared to control groups 4 days after the second recall trial. A third recall trial took place 16 days postassociation, and antibodies were determined 4 days later. However, a meta-analysis of the original data reported that there was no conditioned effect (Ader and Cohen, 1991). Keyhole limpet hemocyanin (KLH): Conditioned antibody production employing KLH as the US was reported by Ader et al. (1993). In a 3/1 conditioning protocol, male BALB/c mice were repeatedly injected with a low dose of KLH as US. By its nature this protein is highly immunogenic, inducing a strong antibody response with high and long-lasting immune memory. Association was initially implemented by three trials (at 3-week intervals), in each of which a complex gustatory stimulus (chocolate milk drinking solution) as a CS was followed by an immunization with KLH (100 ng). A single recall trial was conducted 3 weeks after the last association trial, and serum antibody titers were determined at 5-day intervals. The conditioned animals showed no conditioned taste avoidance at recall time. However, the same animals, boosted with a small dose of antigen (2.5 ng) at recall phase, elicited a relatively modest but significant enhancement of anti–KLH-IgG antibody production (observed at day 10 post-recall). In order to increase the CR magnitude, a 5/1 conditioning protocol with lower US intensity (50 ng of KLH) and higher booster antigen dose (5 ng) was implemented. Results showed that the magnitude of the CR was increased, almost reaching the same magnitude and kinetics as the UR. However, with these data it is not possible to distinguish which of the parameters (i.e., number of association trials, US intensity, or antigen booster dose) mainly affects CR magnitude. In order to narrow down the genuine US, a backward association scheme was implemented in which the inter-stimuli interval was systematically varied in a backward association manner. In

TABLE 3 Unconditioned stimulus Nature Dose Administration route

Taste & Visceral 60 min 0.1% saccharin + i.p LiCl 128 mg/kg

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Conditioned stimulus Nature Duration Description

Overview of the Conditioning Protocols Enhancing Antibody Production Conditioning protocol Subjects Species Strain Gender


2

Rats Hooded & CD Male

Increased anti-SRBC antibody titers 4 days post last evocation NR

(Jenkins et al., 1983)

21

1

Mouse BALB/c Male

Increased anti-KLH IgG antibody titers, peaking at 10 days post evocation (in which a low-dose booster injection of 2.5 ng of KLH was i.p. administered) NR

(Ader et al., 1993)

1

14

1

Rat Wistar NR

Increased anti-OVA antibody titers and OVA-induced T-cell proliferative response in vitro at 4 days post evocation Conditioned taste aversion

(Husband et al., 1993)

Ovalbumin (OVA) 80 μg i.p.

1

30

1

Rats Wistar Male

Increased anti-Ova −IgG antibody titers, peaking at 25 days post evocation NR

(Chen et al., 2004)

“Touch” 15 min Electro-stimulation (2 ms/2 Hz/2 or 4 V)


1

30

1

Rats Wistar Male

Increased anti-Ova −IgG antibody titers, peaking at 17 days post evocation. This CR was also obtained when acquisition was performed under anesthesia NR

(Huang et al., 2004)

Taste 10 min 0.1% saccharin

Hen egg-white lisozyme (HEL) 0.5 mg/Rat i.p.

1

30

1

Rat Wistar Male

Increased anti HEL −IgG and −IgM antibody titers, peaking at 8 days post evocation NR

(Alvarez-Borda et al., 1995; Ramérez-Amaya & Bermúdez-Rattoni, 1999)

Odor 10 min Almond essence

Hen egg-white lisozyme (HEL) 0.5 mg i.p.

1

30

1

Rat Wistar Male

Increased anti HEL −IgG antibody titers, peaking at 8 days post evocation NR

(Ramírez-Amaya & Bermúdez-Rattoni, 1999)



1

45

1

Rat Wistar Male

Increased anti-HEL −IgG antibody titers, peaking at 8 days post evocation NR

(Madden et al., 2001)



1

37

1

Rat Wistar Male

Increased anti-HEL −IgG antibody titers, peaking at 8 days post evocation NR

(Espinosa et al., 2004)


Recall # trials

Sheep red blood cells (SRBC) 2 ml/kg (1% thrice washed SRBC suspension) i.p.

1

7

Taste & Odor 60 min Chocolate milk

Keyhole limpet hemocyanin (KLH) 100 ng/Mouse i.p.

3




NR: Not Reported

Ref.


Association # trials


this experiment, the CS was presented 3, 6, 9, or 15 days after the US during the five association trials. After a single recall trial, no conditioned effects were found at any time. These results suggest that the genuine US is one or more of the relatively early events in the sequence of immune responses that are triggered by the antigen. Recently, it has been reported that following immunization with KLH, rapid and transient neural activity (c-Fos expression) can be detected within specific brain structures (PachecoLópez et al., 2002). These data support the hypothesis that early immune effects of KLH are sensed by the CNS, possibly resulting in associative learning when conditioning is applied. However, no investigations have been undertaken so far to elucidate the mechanisms behind this specific behavioral conditioning of antibody production. Ovalbumin (OVA): In the early 1990s an Australian group reported conditioned enhancement of antibody production following a 1/1 conditioning protocol (Husband et al., 1993). During the association trial, male rats received an intraperitoneal injection of OVA (200 μg) as a US, after presenting saccharin solution as a CS. Recall was implemented 14 days later, in which the conditioned animals were exposed to the CS, and 4 days later the conditioned immune response was assayed. Since a further US exposition may induce a different UR (secondary antibody immune response; IgM), the positive control group was not exposed to the US at recall. Results indicated that anti-OVA IgG antibody titers (CR) were higher in conditioned animals than in controls. Importantly, this CR was achieved without the necessity of booster antigen administration at recall time. Additionally, the conditioned animals displayed a conditioned taste avoidance at recall time which might have been induced by the administration of Freund’s adjuvant with OVA at association. Although the attempt to exactly replicate these results failed (Boehm et al., 1998) unless a booster non-immunogenic antigen was administered at recall (Li et al., 1997), a recent follow-up report successfully reproduced the original 1/1 conditioning protocol (Chen et al., 2004). Importantly, in this follow-up report most appropriate controls were included to assure the associative learning process and CS neutrality on the CR. The major variations in the conditioning protocol were a lower antigen dose (80 μg) and a longer rest interval between association and recall (30 days); this may have resulted in a lower primary antibody response, which hence increased the CR. The anti-OVA IgG antibody titers were measured in 5-day intervals after the recall trial. Although anti-OVA IgG antibody titers were still above baseline values, the results showed a

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clear increment of antibody production as a CR, compared to the appropriate control groups. Interestingly, the CR kinetics differed from the UR, showing a smooth slope and peaking at 25 days post-recall. In addition, pre-exposure to saccharin prevented the conditioned antibody response (i.e., latent inhibition), suggesting that the conditioned immune response was neither due to the effect of stress from the aversive behavioral re-sponse nor to the physiological effects of saccharin. In order to further investigate the neural mechanisms underlying this specific conditioning protocol, neural activity was quantified within the insular cortex after recall (Chen et al., 2004). The study revealed that reexposure to the CS resulted in a significant increase in c-Fos immunoreactivity in all insular areas (granular, dysgranular, and agranular) 120 minutes postrecall. Accordingly, several studies have demonstrated that the insular cortex subserves the association, retrieval, retention, and extinction of long-term visceral-gustatory memory (Bermúdez-Rattoni, 2004; Bermúdez-Rattoni et al., 1997; Nerad et al., 1996). Additionally, recent data support the idea that immune-gustatory associative learning may be stored in this neo-cortex (Pacheco-López et al., 2005; RamírezAmaya and Bermúdez-Rattoni, 1999; Ramírez-Amaya et al., 1996). In an interesting follow-up report, it was demonstrated that antibody response can be conditioned using electro-acupuncture as a CS (Huang et al., 2004). Following a 1/1 conditioning protocol, OVA (80 μg) was employed as a US, paired with electroacupuncture (at acupoint Zusanli = ST36) as a CS. Thirty days after conditioning, animals were reexposed to the CS and anti-OVA IgG antibody titers were analyzed on days 10, 17, 24, and 31 post-recall. On days 10 and 17 post-recall, a significant augmentation of antibody production was observed as a CR. In order to minimize background responses during conditioning, e.g., stress due to the restraint procedure as a part of the CS, a second experiment was implemented in which association and recall trials were performed under anesthesia. The neutrality of the CS on the CR was assured by a non-contingent conditioned but evoked group. Results clearly indicated that electroacupuncture (CS) and OVA (US) can be associated and recalled under anesthesia. Furthermore, the exclusion of a stress response and other possible associative factors blocked by the anesthesia resulted in a clearer conditioned effect. In this regard, electro-acupuncture effects have also been behaviorally conditioned in humans using imagery as the CS (Ulett, 1996). It is tempting to speculate whether some of the documented effects of acupuncture on the immune system (Joos

646


et al., 2000; Karst et al., 2003; Kou et al., 2005; Ye et al., 2002) could be behaviorally conditioned. Hen egg lysozyme (HEL): A Mexican research group developed a 1/1 conditioning protocol in male Wistar rats, pairing saccharin solution (CS) with an intraperitoneal injection of HEL (0.5 mg) as a US (AlvarezBorda et al., 1995). Recall was implemented on day 39 post-association, and blood samples were collected on days 4, 8, 12, and 16 after recall. The rats did not show avoidance behavior to the saccharin. However, the levels of anti-HEL IgM and IgG were significantly enhanced as the CR. Importantly, these conditioned effects did not require further antigen immunization at recall time. Surprisingly, the magnitude of the CR for both IgM and IgG was comparable to the UR after the second antigen administration at peaking timepoints, but the CR kinetics differed from those of the UR. A conditioned but not evoked group was implemented to assure for associative learning and another group to assure CS neutrality. These findings were confirmed by an independent group. However, a much lower magnitude of the conditioned immune response (antibody production) was found, and significant effects were observed for IgG, but not IgM (Madden et al., 2001). With the aim of investigating the neural mechanisms behind this immunoconditioning, brain lesions were performed in the insular cortex, the amygdala, or the dorsal hippocampus before association (RamírezAmaya and Bermúdez-Rattoni, 1999). Results indicate that the insular cortex and the amygdala are brain structures indispensable for associating the CS with the US. Although the hippocampus plasticity is modulated by neuroimmune interactions (Avital et al., 2003; Balschun et al., 2004; Ikegaya et al., 2003), this limbic structure does not seem to be necessary for this associative learning process. Additionally, it was demonstrated that an odor (almond odor) can be successfully employed as a CS and paired with HEL as a US, resulting in a conditioned immune response of similar magnitude at recall time (Ramírez-Amaya and Bermúdez-Rattoni, 1999). The insular cortex lesion blocked the association of this odor-antigen conditioning. Furthermore, data showing that this neo-cortex is not necessary for the development of odor-conditioned avoidance behavior (Kiefer et al., 1982) indicate that this brain structure may be more related to an immune associative process, or at least to immune-visceral sensory processes, rather than to pure aversive/avoidance learning, as has traditionally been assumed. Conversely, a number of studies have demonstrated that amygdala lesion disrupts conditioning when using an odor, but not when using a taste as a CS (Ferry et al., 1995; Hatfield and

Gallagher, 1995). However, conditioned antibody production was blocked in animals with amygdala lesions when using either taste or odor as a CS, indicating that the amygdala may be related to an immune sensory process. Common mechanism for conditioned enhancement of antibody production: Although it has been demonstrated that the CNS is able to detect or sense certain immune signals, such as cytokines or neurotransmitters released by immune cells, it is unlikely that the CNS is able to distinguish specific antigens or epitopes. Rather, the current data indicate that the CNS may be able to codify the immune responses according to their profile, i.e., inflammatory versus non-inflammatory reactions, T-dependent versus B-dependent antigens, as well as the magnitude and localization of the immune response occurring in the periphery (Blalock, 2005; Goehler et al., 2000). However, at recall time the efferent pathways (endocrine/neural) that are involved in the conditioned enhancement of antibody production have not been investigated. Surgical denervations or pharmacological blockades in the periphery may result in a basic delineation of the efferent pathway used by the CNS to promote antibody production. In addition, the specificity of the CR has not been tested yet. It is not known whether the CNS enhances antibody production of all clones activated at recall time, or whether it is able to specify which clone will proliferate. The use of different antigens at association time has been tested in order to analyze the generality of conditioning of antibody production (Espinosa et al., 2004). Two different antigens (KLH or HEL) were employed as a US in a 1/1 conditioning protocol in rats. It seems that one-trial association to induce conditioned enhancement of antibody production is not a general protocol that produces the optimum response for all antigens. For instance, KLH-immunized animals did not show enhancement of KLH-antibody production at recall time, whereas HEL-immunized animals did, indicating that the immune signaling to the CNS might differ from antigen to antigen, resulting in different associative strengths. In fact, Soviet researchers in the early 1960s already reported more rapid association with SRBC as a US than with influenza virus (Il’enko and Kovaleva, 1960). Thus, for some antigens at certain doses, it may be necessary to carry out more association trials in order to induce a stable association with the CS. 2. Anaphylactic Response First indications about the role of associative learning in allergic reactions date back to the nineteenth century (MacKenzie, 1886). An overview of condition-


ing protocols eliciting an anaphylactic response is shown in Table 4. The first experiments demonstrating an asthma-like response after behavioral conditioning were performed in guinea pigs, when an allergic attack was paired with an auditory stimulus (Noelpp and Noelpp-Eschenhagen, 1951a; Noelpp and NoelppEschenhagen, 1951b; Noelpp and Noelpp-Eschenhagen, 1951c; Noelpp and Noelpp-Eschenhagen, 1952a; Noelpp and Noelpp-Eschenhagen, 1952b; Noelpp and Noelpp-Eschenhagen, 1952c). After five association trials, one out of eight animals displayed the CR, i.e., asthmatic respiration in response to the CS. Additional association trials resulted in a conditioned asthmatic response in three more animals. These initial findings were extrapolated to humans in the mid-1950s, suggesting behavioral conditioning as one of the causes of asthmatic attacks (Dekker et al., 1957). In this case report, two asthmatic patients suffering from skin sensitivities to house-dust extract and grass pollen were exposed to these allergens by inhalation. After a series of conditioning trials, they experienced allergic attacks after inhalation of the neutral solvent used to deliver the allergens. This work showed not only fast conditioning of the asthmatic attack, but also tenacious retention, i.e., low rate extinction. Ottenberg and colleagues demonstrated that administration of a sprayed egg-white solution (US) in a certain context (CS) to immune-sensitized guinea pigs on several daily association trials provoked a UR consisting of respiratory distress (Ottenberg et al., 1958). When the animals were returned to the environmental chamber in which the association had occurred and sprayed with vehicle rather than antigen, a similar response was observed. Furthermore, the CR was completely extinguished in all animals after 13 daily recall trials, supporting the concept of associative learning. These data resulted in the early hypothesis that asthma could be conceived of as a learned response (Turnbull, 1962). Few attempts to elucidate the principles and underlying mechanisms behind this phenomenon were performed until the beginning of the 1980s. Several experiments demonstrated that a conditioned anaphylactic response can be obtained using olfactory stimuli as a CS (Dark et al., 1987; Peeke et al., 1987a; Peeke et al., 1987b; Russell et al., 1984). The UR measured in these studies was an antigen-induced increase in blood histamine levels in animals immunized with bovine serum albumin. A classical discrimination conditioning protocol was employed, in which two distinct olfactory stimuli were used: a positive olfactory stimulus (CS+), which signaled the administration of antigen (US); and a negative olfactory stimulus (CS−), which was not paired with the presentation of the antigen. In this specific protocol, each animal served as its own control,

647

since at recall time it was possible to determine the CR elicited after CS+ or CS− presentation. An increase in blood histamine levels in the absence of antigen was observed after presentation of the CS+, but not after the CS−. Importantly, the conditioned histamine release showed a typical extinction process, since the CR magnitude was lower after the second recall trial than after the first. Additionally, it has been shown that mild stress, induced by handling, administered weeks prior to association, predisposes animals to learn the association between the antigen (US) and the odor (CS) (Dark et al., 1987; Peeke et al., 1987b). Furthermore, HPA axis activity also seems to be conditioned during this paradigm; and similar enhancing effects of prior stress were found with circulating cortisol levels as a CR (Peeke et al., 1987a). Interestingly, the conditioning of a cough response in awake guinea pigs was demonstrated following a similar discriminative conditioning protocol (CS+: camphor odor/US: capsaicin), indicating that the CR elicited by the CNS is a complex repertoire of physiological responses orchestrated to protect the organism (Pinto et al., 1995). These results have been replicated employing a 5/1 conditioning protocol, also demonstrating that the release of histamine cannot be recalled under anesthesia (Irie et al., 2001). Furthermore, it has been shown that stress (fasting) immediately after association exacerbates the CR at recall time (Irie et al., 2002). This indicates the sensitivity of this specific CR to stress before and/or after association. Finally, it has been reported that diazepam treatment before recall prevented the conditioned histamine release (Irie et al., 2004), which indicates that this sedative treatment prevents the recall or at least the manifestation of the associative learning process. This finding may have important clinical implications in the management of allergic disease, particularly bronchial asthma, by helping to prevent the stress-anxiety–related airway responses. It has been demonstrated that auditory and visual stimuli (CS) can be associated with the presentation of a specific antigen (US). For instance, MacQueen and colleagues showed that rats were sensitized with a subcutaneous injection of aluminium precipitated ovalbumin (OVA) and an intraperitoneal injection of Bordetella pertussis (MacQueen et al., 1989). Two weeks later, the animals additionally received a larval infection with the nematode Nippostrongylus brasiliensis, in order to produce higher levels of IgE after subsequent antigen injections and to increase the number of mast cells in their intestine. Subsequently, subcutaneous injections of OVA (US) were paired with an auditory/ visual clue (CS) in three association trials. One hour after a single recall trial (CS alone), rat mast cell protease (RMCP) II levels in the serum were enhanced,

648

TABLE 4 Unconditioned stimulus Nature Dose Administration route

Odor 3 sec 1% dimethylsulfide or triethylamine

Conditioning protocol Subjects Species Strain Gender


Association # trials


Recall # trials

Bovine serum albumin 0.5 mg inhalated

5

14

1&2

Guinea pig Hartley Male

Increased plasma histamine concentration. Extinguishable CR: i.e., CR after 1st evocation trial > 2nd evocation trial Conditioned increases in plasma cortisol. Additive CR: i.e., CR after 2nd evocation trial > 1st evocation trial

(Russell et al., 1984; Dark et al., 1987; Peeke et al., 1987)

Odor ≈6 min 1% dimethylsulfide

Bovine serum albumin 1.0–3.0 mg/ml inhalated

5

7

1

Guinea pig Hartley Male

Increased plasma histamine concentration 8–10 min post evocation. This CR could not be evoked under anesthesia NR

(Irie et al., 2001; Irie et al., 2002, 2004)

Audio-visual 15 min Light flash (300 ms alternation rate) and continuous sound (ventilation fan)

Ovalbumin (OVA) 300 mg s.c.

3

11

1

Rat SpragueDawley Male

Increased levels of rat mast cell protease II in plasma 1 h after evocation NR

(MacQueen et al., 1989)

Audio-visual 15 min Light flash (10 W green lamp) and continuous sound (ventilation fan)

Ovalbumin (OVA) 40 mg/kg inhalated

3

7

1

Rat Wistar Male

High levels of lung anaphylactic response immediately post evocation Conditioned increases in plasma corticosterone

(Palermo-Neto & Guimarães, 2000)

NR: Not Reported

Ref.


Conditioned stimulus Nature Duration Description

Overview of the Conditioning Protocols Enhancing Anaphylactic Response


suggesting that the degranulation of mucosal mast cells was the conditioned immune response. Five hours after recall, serum RMCP II levels did not significantly differ from control groups. A strong anaphylactic response in the lung as conditioned response was recently reported following the same conditioning protocol (Palermo-Neto and Guimarães, 2000). In addition, they reported high levels of stress and anxiety post-recall, and demonstrated that the audio-visual stimulus (CS) was stressful and anxiogenic, per se. Another set of animal studies supports the associability of environmental stimuli (CS) with anaphylactic shock reaction (Djuric et al., 1987; Djuric et al., 1988; Markovic et al., 1988; Markovic et al., 1992). Rats sensitized with OVA were given an injection of the same antigen to elicit a second anaphylactic shock response in a context different from that in which the first anaphylactic shock was induced. Animals experiencing both anaphylactic shocks in the same context displayed a much smaller shock than rats subjected to the second shock in a different context. Repeated, unreinforced presentation of the antigen in the new context prior to the CS and the induction of the first anaphylactic shock prevented the increased resistance to the induction of a second anaphylactic shock reaction. These findings were ascribed to the CR which attenuated the UR (anaphylactic shock). Additionally, it has been demonstrated that rats immunized with a shock-inducing dose of OVA exhibit a conditioned aversion to the taste (CS) with which it was previously associated (Djuric et al., 1987). Conditioned taste avoidance was found 8 weeks after the association trial, and pre-exposure to the CS weakened the CR (i.e., latent inhibition). Interestingly, this avoidance behavioral CR was obtained following three different modes of CS-US presentation at association time: (1) CS orally/US i.v., (2) CS orally/ US i.p., and (3) CS i.v./US i.v. (Markovic et al., 1988). Furthermore, a dose-response relationship between the amount of antigen used for the induction of anaphylactic shock and the conditioned taste avoidance was demonstrated: the higher the antigen dose, the more pronounced the conditioned taste avoidance (Djuric et al., 1988). Accordingly, it has recently been shown that OVA-immunized mice avoid the context previously associated with presentation of the allergen against which they have been immunized (Costa-Pinto et al., 2005). In a modified classical passive avoidance test, OVA aerosol was employed as an aversive stimulus (US); albeit attracted by the supposedly safer, dark compartment of the apparatus (CS), OVA-immunized mice avoided entering the dark side, preferring the bright (usually aversive) side of the box. When CNS activity was tracked using c-Fos expression as a neuronal metabolic marker, allergic animals showed

649

enhanced c-Fos immunoreactivity in the hypothalamic paraventricular nucleus and central nucleus of the amygdala after airway OVA challenge. These brain structures are commonly linked to emotional and affective behavioral patterns that are important components in the development of learned aversive behavior, such as conditioned taste aversion/ avoidance (Bermúdez-Rattoni, 2004). Allergic asthma proceeds through two distinct phases: an immediate response mediated by IgE and mast cells; and a late phase, during which the presence of Th2 cells, eosinophils, and other immune cells is evident (Maddox and Schwartz, 2002). It will, therefore, be relevant to determine the messenger(s) and afferent pathway(s) by which the immune system signals the CNS. Since activation of the hypothalamic paraventricular nucleus and central nucleus of the amygdala has been detected 90 minutes after nasal OVA challenge, it is likely that the immediate hypersensitivity reaction, which is dependent on IgE-mediated mastcell deregulation with the concomitant histamine release, plays a major role in the brain activation reported (Costa-Pinto et al., 2005). However, both the early and late phases of the allergic responses may mediate the learned avoidance behavior to the dark side of the cage. This behavioral CR was displayed in animals receiving multiple aerosol OVA challenges, and consequently there was an ongoing inflammatory lung reaction, as well as high levels of IgE. 3. Delayed-type Hypersensitivity Cell-mediated immunity is defined as a beneficial host response characterized by an expanded population of specific T-cells, which, in the presence of antigens, produce cytokines locally. The activation and recruitment of cells into an area of inflammation are a crucial step in the development of certain cellular immune responses, e.g., delayed-type hypersensitivity (DTH). The data summarizing these conditioning protocols are shown in Table 5. It has been demonstrated that, depending on the concentration and the timing, cyclophosphamide (CY) can suppress an initial DTH response and enhance the response to subsequent antigenic challenge (Bovbjerg et al., 1986). Based on such a phenomenon, DTH response has been reported to be enhanced as a CR, when CY is employed as US (Bovbjerg et al., 1987). In this conditioning paradigm, three daily association trials were employed in which a saccharin solution (CS) was paired with an intraperitoneal injection of CY (50 mg/kg) as the US. Three weeks later, all animals were sensitized to SRBC, and 2 days later, conditioned animals were re-exposed to the CS (sac-

650

TABLE 5 Conditioned stimulus Nature Duration Description

Overview of the Conditioning Protocols Enhancing Adaptive Cellular Immune Responses

Unconditioned stimulus Nature Dose Administration route

Conditioning protocol Association # trials


Recall # trials

Subjects Species Strain Gender


Ref.

Alloantigen (skin graft) from C57BL/6 mice 1.0 cm2 Abdominal dermis

3

40

1, 2, 3 &4

Mouse CBA/J Male

Increased in cytotoxic T lymphocyte precursors in blood 2 days post evocation. Extinguishable CR: i.e., CR after 1st evocation trial > 2nd evocation trial > 3rd evocation trial NR

(Gorczynski et al., 1982)

Odor 60 min Camphor

Alloantigen (spleen cells) from C57BL/6 mice 1.5 × 107 i.p.

1

5

1

Mouse BALB/c Female

Increased splenic CTL cells activity 1 day post evocation NR

(Hiramoto et al., 1993)

Taste 60 min + 0.1–0.2 ml 0.1% saccharin

Cyclophosphamide 50 mg/kg i.p.

3

23

3

Mouse CD-1 Male

Increased in delayed-type hypersensitivity peaking at 7 days post last evocation Conditioned taste aversion

(Bovbjerg et al., 1987)


Levamisole 3 mg/rat s.c.

1

14

1

Rat Wistar NR

Increased T helper: T suppressor subset ratio 24 h post evocation Conditioned taste aversion

(Husband et al., 1987)


Staphylococcal enterotoxin B (SEB) 2.0 mg/kg i.p.

1

6

1

Rat Dark agouti Male

Increased IL-2 and INF-γ plasma levels at 2 h post evocation Conditioned taste aversion and corticosterone plasma increments

(Pacheco-López et al., 2004)

NR: Not Reported


Multi-sensory ≈91/2 days Grafting procedure


charin), alone, on 3 consecutive days. On the second recall trial, conditioned animals and controls received another CY injection. Later, three SRBC-challenges were employed to detect the DTH response. The results indicate that the DTH response was enhanced in conditioned animals after the second and third challenges. However, this specific CR may actually represent a conditioned immunosuppressive effect (Ader and Cohen, 2001), since CY applied on the second recall trial may depress suppressor cell function (Gill and Liew, 1978). 4. T-Cell Function Cytotoxic T lymphocyte (CTL) numbers were reported to be increased as a result of a 3/3 conditioning protocol (Gorczynski et al., 1982). CBA/J mice were repeatedly grafted with skin from C57BL/6 mice. Association was achieved by pairing the CS (the surgical procedure: shaving, anesthetizing, grafting, banding) and the US (the skin graft). This procedure was repeated three times at an interval of 40 days. At recall, all groups received a sham graft, i.e., the CS without the US, and conditioned animals displayed an increase in CTL precursor numbers as a CR. However, only 50–60% of the conditioned animals showed this conditioned immune response. Those animals that displayed the CR (responders) were subdivided into two groups that were subsequently given two further recall trials in order to develop extinction, or two further association trials in order to reinforce the CR. After 40 days of rest, both groups were submitted to a further recall trial. The results showed that the CR was either extinguished or reinforced after repeated extinction or reinforcement trials, respectively, clearly indicating an associative learning phenomenon. Following a 1/1 conditioning protocol, CTL activity has been reported to be enhanced as a CR (Demissie et al., 2000; Hiramoto et al., 1993b). At association, BALB/c mice were immunized with splenocytes from C57BL/6 mice (US), and this procedure was paired with an exposure to camphor odor (CS). A recall trial was implemented 6 days later, in which conditioned animals were exposed to the CS. Enhanced CTL activity was observed 24 hours post-recall. In a next step, naltrexone, a central and peripheral opioid receptor blocker, was administered before recall. This treatment blocked the recall of the CR (CTL activity) due to a central opioid blockade, since quaternary naltrexone, a peripheral opioid receptor blocker, did not affect the conditioned increase in CTL activity. In rats, levamisole injection results in an elevation of the T helper : T suppressor subset ratio due to a selective depression in the cytotoxic/suppressor

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subset. This response was shown to be elicited as a CR through a 1/1 conditioning protocol (Husband et al., 1987). Saccharin solution (CS) was paired with levamisole (3 mg) administration (US), and 14 days later a single recall trial (CS only) was employed to recall this associative learning. One day after recall, T-cell subsets were assessed, indicating that the T helper : T suppressor ratio was enhanced. 5. Cytokines Cytokines are soluble glycoproteins released by cells of the immune system, which act non-enzymatically through specific receptors to regulate immune responses. Cytokines resemble hormones in that they act at low concentrations bound with high affinity to a specific receptor. Pro-inflammatory cytokines such as IL-1, IL-6, and tumor necrosis factor (TNF)-α are produced predominantly by activated immune cells, such as macrophages, and are involved in the amplification of inflammatory reactions. Anti-inflammatory cytokines such as IL-10, IL-4, and IL-12 are involved in the reduction of inflammatory reactions (Janeway et al., 2005). Due to the immunoregulatory properties of cytokines, the possibility of enhancing the production of cytokines by behavioral conditioning has been repeatedly addressed but mainly without success (Goebel et al., 2005; Mormède et al., 2003). It is likely that one of the major issues in those experiments was the nature of the US. If the immune response induced by the US is weak, slow, and long-lasting, then the CNS may not be able to detect these changes in the periphery, resulting in a weak association with the CS. Another key factor may be the number of association trials employed. The use of superantigens as the US in a conditioning paradigm has several advantages, since it induces strong, rapid, and transient immune stimulation (Proft and Fraser, 2003). It has been demonstrated that, when superantigen administration was preceded by exposure to a novel taste, the animals were able to associate those stimuli and later elicit a complex CR (PachecoLópez et al., 2004). At association time, the taste of saccharin (CS) was paired with an intraperitoneal injection of staphylococcal enterotoxin B (SEB) as the US. SEB is a superantigen that induces strong and transient T-cell activation, followed by increased cytokine production, mainly IL-2, IFN-γ, and TNF-α. In addition to these immune effects, SEB immunization induces significant changes in endocrine, neurobiological, and behavioral functions (Gonzalo et al., 1993; Kusnecov and Goldfarb, 2005; Shurin et al., 1997). At recall time, conditioned animals were exposed to the

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CS, resulting in enhanced IL-2 plasma levels as a conditioned immune response. The HPA axis also seemed to be conditioned, since plasma corticosterone levels were enhanced as well after recall (conditioned endocrine response). Furthermore, conditioned animals demonstrated a behavioral CR, since a strong taste avoidance to the CS was observed. The mechanisms of behavioral conditioning using SEB as the US are still not known. However, at association, it may involve T-cell–derived cytokines signaling to the brain. This hypothesis is supported by previous work demonstrating that central monoamine activity is modified by systemic IL-2 administration (Lacosta et al., 2000). Similar results were reported after SEB immunization, since striatum catecholamine levels followed a dose-response curve in reaction to increased amounts of peripheral SEB immunization (PachecoLópez et al., 2004). At recall time the efferent pathway has not been investigated, and the cellular origin of the IL-2 is not known.

III. CLINICAL AND ADAPTIVE RELEVANCE OF CONDITIONED IMMUNE ENHANCEMENT Conditioning experiments performed in humans have revealed the possibility of enhancing immune activity, indicating the feasibility of modulating immune reactivity through behavioral approaches. One study has shown that NK cell activity may be enhanced by behavioral conditioning, after pairing a subcutaneous injection of epinephrine as a US with a novel taste as a CS (Buske-Kirschbaum et al., 1992). However, the results could not be confirmed in a follow-up study (Kirschbaum et al., 1992). Although not conclusive—and still controversial (see Ader and Cohen, 2001), interesting results suggest that behavioral conditioning regulates tumor growth (Ghanta et al., 1987b; Ghanta et al., 1988; Ghanta et al., 1990; Hiramoto et al., 1991b), as well as reverses the immune senescence process (Spector, 1996; Spector, 1999; Spector et al., 1994).

A. Conditioned Immune Enhancement as Supportive Therapy It may be advisable to treat several idiopathic illnesses or syndromes with various therapeutic approaches in order to optimize the outcome. In this context, behavioral conditioning regimens should be employed as complementary therapy supporting the pharmacological treatment and ameliorating undesired pharmacological side effects.

1. Cancer: Tumor Progression Behavioral conditioning as supportive therapy has been delineated in the context of cancer treatment (Bovbjerg, 2003; Hiramoto et al., 1991b; Spector, 1996). Pioneer experiments performed at the University of Alabama reported that female BALB/c mice inoculated with a transplanted MOPC 104E myeloma were able to reverse the growth of the tumor and enhance survival as a function of the conditioned immune response (Ghanta et al., 1987a). The conditioning protocol was composed of 10 association trials (CS: camphor, US: poly I : C) at 3-day intervals. One day after the last association trial, animals were inoculated with the tumor. One day post-inoculation, animals were re-exposed to the camphor odor and subsequently every third day. The findings indicate that for the conditioned group there was an increase in median survival on day 43, compared to days 34, 37, and 38 of various control groups. Actually, two of these conditioned mice lived more than 120 days; they had shown early tumor growth but were free of disease on day 97. Although the group differences could be explained by the differences due to the outliers, an alternative hypothesis is that through behavioral conditioning, immune tolerance was induced in some animals by unknown mechanisms. The neutrality of 1-hour (Ghanta et al., 1988) and 4hours (Ghanta et al., 1987a) exposure to camphor odor (CS) on the tumor growth was documented. In summary, these results indicate that conditioning has beneficial effects on tumor control. However, tumor inoculation took place after the association phase, complicating further applications in clinical settings. A second series of experiments was performed in the following order: tumor inoculation → conditioning. Results showed conditioned resistance against the YC8 lymphoma in female BALB/c mice (Ghanta et al., 1990). YC8-specific resistance seems to be conferred by immunizing BALB/c mice with DBA/2 splenocytes (Parmiani et al., 1982; Sensi et al., 1986); therefore, DBA/2 spleen cells were used as a US (alloantigen) paired with a CS (camphor odor 1 h). Several conditioning protocols were implemented (2/6, 3/5, 4/4), with the first association trial always taking place 5 days after tumor implantation. Association trials were separated by 2 or 3 days, and recall trials by 2 or 7 days. The rest interval between association and recall varied from 2 days (3/5 conditioning) or 3 days (2/6 conditioning), to 7 days (4/4 conditioning). The tumor size was measured, and the mortality rate was recorded to evaluate the effects of conditioning and immunotherapy. Results indicate that tumor growth and sur-


vival are improved by conditioning protocols in the following odd order: 3/5 > 4/4 > 2/6. Astonishingly, the results also indicated that regulation of tumor growth (as measured by growth delay) through behavioral conditioning was more effective than the immunotherapy treatment itself. Finally, it was shown that camphor odor exposure by itself does not trigger tumoricidal activity (i.e. CS neutrality). In a follow-up report, similar results were observed following a 4/4 conditioning protocol, pairing camphor odor (CS) with DBA/2 splenocytes (US) (Hiramoto et al., 1991b). Mice were then subcutaneously injected with YC8 tumor cells. Five days post-implantation, the animals were submitted to the association phase (four trials, each separated by 2 or 3 days), and 14 days afterwards the animals were evoked. Three further recall trials were employed on days 26, 33, and 40 post-tumor implantation. Results indicate that the conditioned group which was re-exposed to the CS at weekly intervals appeared to resist tumor growth, as evidenced by the slower growth of the tumor and by an increase in the survival of this group versus controls. Although these studies suggest that behavioral conditioning improves the survival rate of experimental animals after tumor inoculation, this effect seems to be modest. Moreover, a number of questions must be addressed in order to improve the conditioning process and the effects on immune functions. For instance, it is not known how long the CR can be maintained in each animal after recall. Since it may be necessary to apply reinforcement at appropriate intervals, is reconditioning possible? As tumors appear frequently in elderly life stages, how do gender and age affect this conditioned immune response?

B. Conditioned Immune Enhancement as an Adaptive Process Neuroimmune interactions appear to bring several adaptive advantages to those organisms that acquired and further developed during ontogeny and phylogeny (Ottaviani et al., 1998; Tada, 1997). A complex repertoire of physiological responses, including immune, endocrine, neural, and behavioral responses, may be orchestrated to achieve a better adaptation of the organism to a constantly challenging environment. For example, one interesting hypothesis regarding psychoneuroimmunological interactions has been delineated: It associates the adolescent’s acne, timed to cortical development of the brain, as an adaptive evolutionary strategy of Homo sapiens; delaying the mate choice (although reproductively mature) until the indi-

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viduals are emotionally, intellectually, and physically fit to be parents (Bloom, 2004). In vertebrates, it is well established that there are many intricate interactions between the immune and nervous systems, and vice versa (Ottaviani and Franceschi, 1996; Salzet et al., 2000; Straub and Besedovsky, 2003; Vishwanath, 1996). Recently, it has been demonstrated that invertebrate biology has also evolved around acquiring and developing complex neuroimmune communication. For example, interaction between neurons and immune cells has been demonstrated in the mollusk Aplysia californica (Clatworthy, 1998). Furthermore, invertebrates also express neuropeptides (e.g., opioids) in the neural and immune tissues, which play a key role as neuroimmune messengers during their evolution (Salzet et al., 2000). Neuroimmune complexity appears as well in the behavior of insects, for example, in the linkage between the immune system and the nervous system of the bee (Mallon et al., 2003; Schedlowski, 2006). Non-infected honey bees whose immune systems were challenged by a non-pathogenic immunogenic elicitor (lipopolysaccharide) displayed a reduced ability to associate an odor with sugar reward in a classical conditioning paradigm. Classical conditioning can be understood as learning about the temporal or causal relationships between external and internal stimuli to allow for the appropriate preparatory set of responses before biologically significant events occur (Rescorla, 1988; Rescorla, 2003). In this regard, the capacity to associate a certain immune response or status (e.g., allergens, toxins, antigens) with a specific stimulus (e.g., environments or flavors) is of high adaptive value; we consider that it was acquired during evolution as an adaptive strategy in order to protect the organism and/or prepare it for danger. Furthermore, such associative learning is typically acquired under certain stressful conditions. For example, the exposure to a specific antigen (and its categorization as an allergen) might be associated (learning) with a specific environment or food. An adaptive response is then elicited (memory), consisting first of a behavioral modification, in order to avoid the place or food associated with the antigen (Costa-Pinto et al., 2005; Djuric et al., 1987). If this is not possible, the organism will try to reduce the contact with the allergen, i.e., by coughing or sneezing (Pinto et al., 1995), and, at the same time, its immune system may prepare the body for interaction with the antigen, for instance: mast cell degranulation (Irie et al., 2001; MacQueen et al., 1989; Palermo-Neto and Guimarães, 2000; Russell et al., 1984) or antibody production (Ader et al., 1993; Alvarez-Borda et al., 1995; Chen et al., 2004; Husband et al., 1993). Although under experimental conditions such associative learning can be extin-

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guished, it is likely that it will last for a long time, since, in natural situations, the organism will try to avoid contact with the environmental clues that signal the CS. 1. Perspectives and Summary Conceptualizing Pavlovian conditioning as a mechanism by which an organism anticipates the onset of a biologically important event (the US) and initiates preparatory responses (CR) to allow the organism to deal better with the US effects invites the hypothesis that one reason for the neural control of immunity lies in accommodating the adaptive value of classical conditioning. In its natural environment, an animal with a cut or a scratch has to build up immunological defense against micro-organisms. In the laboratory or a clinical setting, an antigen is reliably preceded by an injection. Therefore, conditioned immune effects may, in fact, be very common. The difficulty for the investigator lies not so much in inducing such responses, but in employing the proper controls, both immunological and psychological, in order to demonstrate that these responses exist and to expose the underlying mechanism.

C. Expectations Regarding Immunoconditioning Due to the physiological basis of the conditioned effects, the magnitude of the conditioned immune response should not be expected to override the homeostatic balance of the organism. However, this does not mean that conditioned effects on immune functions are not of biological/clinical significance, as has been reviewed here and in previous work (Ader, 2003). A very small increase in the potential of the immune system may be of great value in the fight against pathogens when the system reaches an allostatic load (McEwen, 1998; McEwen and Lasley, 2003), but it may increase the occurrence and severity of allergies and autoimmune disorders in other conditions. It is important to emphasize that several immune responses may be affected by behavioral conditioning protocols, but this does not necessarily imply that such immune responses were conditioned. Due to the complexity of neuroimmune interaction, such differentiation is not easy to establish. As has been reviewed in this chapter, the use of a US with immune consequences, such as immunomodulating drugs or antigens, is not the only requirement for genuinely conditioning an immune response through a behavioral protocol. Experimental data reflect a dichotomy

that is possibly supported by different mechanisms and therefore might follow different rules. In our own experience and after reviewing the available literature, we conclude that a key feature for associative learning is when the direction of the CR is maintained during conditioning; i.e., the direction of this response should be independent of the immune, endocrine, and circadian status of the subject at association and recall time. Before behavioral immunoconditioning is implemented as supportive therapy together with traditional pharmacological regimens, it is essential to describe some of its features. For example, it is not known how long conditioned immune responses last and how immune specific they are. Since it may be necessary to apply reinforcement at appropriate intervals, the question arises as to whether re-conditioning is possible. Since at some time the therapy will stop, what is the forgetting pattern of conditioned immune responses? How predictable is the conditioned immune response in a human population with different immune and psychological histories? What is the impact of age and gender on immunoconditioning? When using immunomodulating drugs as the US, are some side effects also conditioned? To date, experimental evidence indicates that behavioral conditioning may have practical implications in a clinical setting and be of use as supportive therapy, with the aim of reducing undesired side effects and maximizing the effects of pharmacological therapies. For instance, immunosuppressive status in humans could be achieved by combining the pharmacological effects of cyclosporine A with intermittent recall trials of its association with a specific exteroceptive clue (e.g., gustative stimulus) (Goebel et al., 2002).

IV. SUMMARY In this chapter we have reviewed and summarized the current data indicating that both innate and adaptive immune responses are affected by behavioral conditioning protocols. An understanding of the effects of behavioral conditioning on immune functions is beginning to emerge, with potential clinical applications. In future studies it will be essential to analyze the afferent and efferent pathways in brain-to-immune communication before behavioral conditioning paradigms can be employed as beneficial tools in a clinical setting. Finally, the research on behavioral immunoconditioning has revealed that the organism has important adaptive psychoneuroimmune strategies, acquired to deal with the constantly changing and challenging environment in a better way.


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Solvason, H., Ghanta, V., and Hiramoto, R. (1988). Conditioned augmentation of natural killer cell activity. Independence from nociceptive effects and dependence on interferon-beta. J. Immunol., 140, 661–665. Solvason, H., Ghanta, V., and Hiramoto, R. (1993). The identity of the unconditioned stimulus to the central nervous system is interferon-beta. J. Neuroimmunol., 45, 75–81. Solvason, H., Ghanta, V., Lorden, J., Soong, S., and Hiramoto, R. (1991). A behavioral augmentation of natural immunity: odor specificity supports a Pavlovian conditioning model. Int. J. Neurosci., 61, 277–288. Solvason, H., Ghanta, V., Soong, S., Rogers, C., Hsueh, C., Hiramoto, N., and Hiramoto, R. (1992). A simple, single, trial-learning paradigm for conditioned increase in natural killer cell activity. Proc. Soc. Exp. Biol. Med., 199, 199–203. Solvason, H., Hiramoto, R., and Ghanta, V. (1989). Naltrexone blocks the expression of the conditioned elevation of natural killer cell activity in BALB/c mice. Brain Behav. Immun., 3, 247–262. Spector, N. (1996). Neuroimmunomodulation: a brief review. Can conditioning of natural killer cell activity reverse cancer and/or aging? Regul. Toxicol. Pharmacol., 24, S32–38. Spector, N. (1999). The NIM revolution. Rom. J. Physiol., 36, 127–143. Spector, N., Provinciali, M., di Stefano, G., Muzzioli, M., Bulian, D., Viticchi, C., Rossano, F., and Fabris, N. (1994). Immune enhancement by conditioning of senescent mice. Comparison of old and young mice in learning ability and in ability to increase natural killer cell activity and other host defense reactions in response to a conditioned stimulus. Ann. N.Y. Acad. Sci., 741, 283–291. Straub, R., and Besedovsky, H. (2003). Integrated evolutionary, immunological, and neuroendocrine framework for the pathogenesis of chronic disabling inflammatory diseases. FASEB J., 17, 2176–2183. Tada, T. (1997). The immune system as a supersystem. Annu. Rev. Immunol., 15, 1–13. Tracey, K. (2002). The inflammatory reflex. Nature, 420, 853–859. Tully, T., Boynton, S., Brandes, C., Dura, J., Mihalek, R., Preat, T., and Villella, A. (1990). Genetic dissection of memory formation in Drosophila melanogaster. Cold Spring Harb. Symp. Quant. Biol., 55, 203–211. Turnbull, J. (1962). Asthma conceived as a learned response. J. Psychosom. Res., 6, 59–70. Ulett, G. (1996). Conditioned healing with electroacupuncture. Altern. Ther. Health Med., 2, 56–60. Vishwanath, R. (1996). The psychoneuroimmunological system: a recently evolved networking organ system. Med. Hypotheses, 47, 265–268. Wilson, D., and Stevenson, R. (2003). Olfactory perceptual learning: the critical role of memory in odor discrimination. Neurosci. Biobehav. Rev., 27, 307–328. Ye, F., Chen, S., and Liu, W. (2002). Effects of electro-acupuncture on immune function after chemotherapy in 28 cases. J. Tradit. Chin. Med., 22, 21–23. Zeitlenok, N., and Bychkova, E. (1954). Role of the higher nervous function in infection and immunity. Zh. Vyssh. Nerv. Deiat. Im. I. P. Pavlova, 4, 267–281.


C H A P T E R

31 Exercise and Immunity: Clinical Studies DAVID C. NIEMAN

I. II. III. IV.

I. INTRODUCTION

INTRODUCTION 661 EXERCISE, IMMUNITY, AND URTI RISK 662 ATHLETIC ENDEAVOR AND URTI 668 CONCLUSIONS 670

Exercise immunology is a relatively new area of scientific endeavor, with 80% of articles published during the past decade (Nieman, 2003). Growing evidence indicates that physical activity does influence immune function and as a consequence risk of certain types of infection, in particular the most common of all, upper respiratory tract infections (URTI). This chapter will summarize recent investigations showing that in contrast to moderate physical activity, prolonged and intensive exertion causes numerous negative changes in immunity and an increased risk of URTI.

ABSTRACT Moderate to heavy exercise workloads have contrasting influences on immune function. Moderate exercise training has been associated with favorable perturbations in immunity and a reduction in incidence of upper respiratory tract infection (URTI). More research is needed to establish whether or not a link exists between moderate exercise-induced perturbations in immunity and improvements in other clinical outcomes such as cancer, heart disease, type 2 diabetes, arthritis, and aging. Epidemiological data indicate that endurance athletes are at increased URTI risk during periods of heavy training and the 1–2 week period following race events. For several hours subsequent to heavy exertion, components of both the innate (e.g., natural killer cell activity and neutrophil oxidative burst activity) and adaptive (e.g., T- and B-cell function) immune system exhibit suppressed function. Some attempts have been made through nutritional means (e.g., glutamine, vitamins C and E, and carbohydrate supplementation) to attenuate immune changes following intensive exercise and thus lower URTI risk. Carbohydrate supplementation during heavy exercise has emerged as the most effective countermeasure, and attenuates increases in blood neutrophil counts, stress hormones, and inflammatory cytokines. PSYCHONEUROIMMUNOLOGY, 4E VOLUME I

A. Exercise, Immune Changes, and Clinical Outcomes The link between exercise-induced perturbations in immunity and improvements in other clinical outcomes such as cancer, heart disease, type 2 diabetes, arthritis, and aging has not been investigated as thoroughly as with URTI. More research is needed to determine if immune alterations during exercise training help explain the lowered risk of chronic disease. For example, although epidemiological and experimental studies with animal models suggest that physical activity may protect against several forms of cancer, evidence linking this to enhanced immunity is limited and controversial (Fairey et al., 2005). Hoffman-Goetz and Husted (1995) more than a decade ago proposed that although various exercise-induced mechanical and hormonal changes best explain the relationship to reduced risk of colon, breast, and prostate cancer in

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physically active individuals, several potential immunological effects may be contributing factors. These include exercise-induced immune modulation of cytokines; activation and changes in signal transduction of natural killer (NK) cells, macrophages, neutrophils, and tumor-infiltrating lymphocytes; changes in the expression of cell adhesion molecules; and alterations in prostaglandins. Accumulating evidence indicates that exercise training causes changes in pro- and antiinflammatory cytokine levels and activity that may influence both the prevention and treatment of certain types of carcinomas (Allgayer et al., 2004). This area of research endeavor is highly complex, and more research is needed. Immune senescence or age-associated immune deficiency appears to be partly responsible for the afflictions of old age. Elderly persons are more susceptible to many infections, autoimmune disorders, and cancers when compared with younger adults. A new and growing area of research endeavor is the study of the relationship between physical activity and immune senescence. One study of adults over the age of 65 years showed that a 10-month exercise program enhanced the antibody titer response to an influenza immunization (Kohut et al., 2004), thus showing that exercise can have some immediate benefits for the elderly. Regular physical activity may also attenuate the age-related decrease in T lymphocyte function (Smith et al., 2004). Very few studies have been conducted in this area, but the available data taken together suggest that exercise training may need to be long term and of sufficient volume to induce changes in body weight and fitness before any change in immunity can be expected in old age (Kohut and Senchina, 2004; Nieman et al., 1993). In other words, because the aging process is so dominant in old age, long-term physical activity combined with leanness and other positive lifestyle habits may be necessary before immune function is enhanced. Type 2 diabetes and cardiovascular disease are associated with chronic low-grade systemic inflammation. During exercise, IL-6 is produced by muscle fibers and stimulates the appearance in the circulation of other anti-inflammatory cytokines such as IL-1ra and IL-10 (Petersen and Pedersen, 2005). IL-6 also inhibits the production of the pro-inflammatory cytokine TNF-alpha and stimulates lipolysis and fat oxidation. With weight loss from energy restriction and exercise, plasma levels of IL-6 fall, skeletal muscle TNF-alpha decreases, and insulin sensitivity improves (Ferrier et al., 2004; Ryan and Nicklas, 2004). Thus, IL-6 release from the exercising muscle may help mediate some of the health benefits of exercise including metabolic control of type 2 diabetes (Petersen and Pedersen,

2005). The exercise-induced cytokine links between adipose and muscle tissues clearly warrant further study (Tomas et al., 2004).

II. EXERCISE, IMMUNITY, AND URTI RISK Upper respiratory tract infections are the most frequently occurring illnesses in humans worldwide. More than 200 different viruses cause the common cold, and rhinoviruses and coronaviruses are the culprits 25–60% of the time. The U.S. Centers for Disease Control and Prevention has estimated that more than one billion URTIs occur annually in the United States, a leading cause of lost school and work days. The average person has two or three respiratory infections each year, with young children suffering six to seven. Among elite athletes and their coaches, a common perception is that heavy exertion lowers resistance and is a predisposing factor to URTI. In a 1996 survey by the Gatorade Sports Science Institute of 2,700 high school and college coaches and athletic trainers, 89% checked “yes” to the question, “Do you believe overtraining can compromise the immune system and make athletes sick?” (personal communication, Gatorade Sports Science Institute, Barrington, IL). Conversely, there is also a common belief among fitness enthusiasts that regular exercise confers resistance against infection. In a survey of 170 non-elite marathon runners (personal best time, average of 3 hours 25 minutes) who had been training for and participating in marathons for an average of 12 years, 90% reported that they definitely or mostly agreed with the statement that they “rarely get sick” (unpublished observations). A survey of 750 masters athletes (ranging in age from 40 to 81 years) showed that 76% perceived themselves as less vulnerable to viral illnesses than their sedentary peers (Shephard et al., 1995). Although the relationship between exercise and URTI and other types of infections has been explored since early in the twentieth century (Baetjer, 1932), the number of well-designed epidemiological and exercise training experimental trials on humans is still small, limiting our understanding of this important topic (Nieman, 1997a, 2003). Data from animal studies have been difficult to apply to the human condition but in general have supported the finding that one or two periods of exhaustive exercise following inoculation lead to a more frequent appearance of infection and a higher fatality rate (but results differ depending on the pathogen, with some more affected by exercise than others) (Friman et al., 1995; Pedersen and Bruunsgaard, 1995).

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Davis et al. (1997), for example, exposed mice to rest, 30 minutes of moderate exercise, or 2.5–3 hours of exhaustive exercise following intranasal infection with the herpes simplex virus (HSV-1). Mice exercised to fatigue had a greater overall mortality during a 21-day period than did controls or moderately exercised mice. In humans, it is well established that various measures of physical performance capability are reduced during an infectious episode (Friman et al., 1995). Several case histories have been published demonstrating that sudden and unexplained deterioration in athletic performance can be traced to either recent URTI or subclinical viral infections that run a protracted course. In some athletes, a viral infection may lead to a debilitating state known as “post-viral fatigue syndrome” (Maffulli et al., 1993; Parker et al., 1996). The symptoms include lethargy, easy fatigability, and myalgia, and can persist for several months.

A. Exercise-Induced Changes in Immune Function Together, these data imply that there is a relationship between exercise and infection, and that heavy exertion may suppress various components of immunity. Research data on the resting immunity of athletes and non-athletes, however, are limited and present a confusing picture at present (Nieman, 1997b). For example, the few studies available suggest that the innate immune system responds differentially to the chronic stress of intensive exercise, with natural killer cell activity tending to be enhanced while neutrophil function is suppressed (Nieman et al., 1995b, 1999; Pyne et al., 1995; Smith and Pyne, 1997). The adaptive immune system (resting state) in general seems to be largely unaffected by athletic endeavor. Each acute bout of cardiorespiratory endurance exercise leads to transient but significant changes in immunity and host defense (Gabriel and Kindermann, 1997; Hoffman-Goetz and Pedersen, 1994; Nieman et al., 1997b, 2002a, 2003a, 2004; Pedersen and Brunsgaard, 1995). Natural killer cell activity, various measures of T- and B-cell function, upper airway neutrophil function, and salivary IgA concentration have all been reported to be suppressed for at least several hours during recovery from prolonged, intense endurance exercise (Bruunsgaard et al., 1997; Gabriel and Kindermann, 1997; Mackinnon and Hooper, 1994; Müns, 1993; Nieman et al., 1995a, 1995c, 2001, 2003b; Shinkai et al., 1993). During this “open window” of decreased host protection, viruses and bacteria may gain a foothold, increasing the risk of subclinical and clinical infection (Figure 1).

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FIGURE 1 The “open window theory”: Moderate exercise causes mild immune changes; in contrast, prolonged, intensive exercise (90 minutes or longer) leads to a downturn in immunosurveillance that increases the likelihood for opportunistic upper respiratory tract infections.

Although this is an attractive hypothesis, no one has yet demonstrated conclusively that athletes showing the most extreme immunosuppression are those that contract an infection (Lee et al., 1992; Mackinnon et al., 1993). In one study, salivary IgA secretion rate decreased by nearly half in a group of 155 ultramarathon runners following a 160 km race (Nieman et al., 2006). Nearly one in four runners reported an URTI episode during the 2-week period following the race, and the decrease in sIgA secretion rate was significantly greater in these runners (54%) compared to those not reporting URTI (31%). It is doubtful, however, that sIgA output alone can be used to predict URTI at the individual athlete level. In this study, the overall predictive value for URTI was 55%, indicating that sIgA output was more useful at the group compared to the individual level, and that other factors need to be discovered and combined with sIgA before URTI risk can be predicted for individual athletes.

B. Heavy Exertion and URTI: Epidemiological Evidence Understanding the relationship between exercise and infection has potential implications for public health, and for the athlete, it may mean the difference between being able to compete or performing at a subpar level or missing the event altogether because of illness. It has been proposed that the relationship between exercise and URTI may be modeled in the form of a “J” curve (Nieman, 1997a) (Figure 2). This model suggests that although the risk of URTI may decrease below that of a sedentary individual when

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FIGURE 2 Model on the relationship between exercise workload, risk of upper respiratory tract infection (URTI), and immunosurveillance.

one engages in moderate exercise training, risk may rise above average during periods of excessive amounts of high-intensity exercise. The model in Figure 2 also suggests that immunosurveillance mirrors that relationship between infection risk and exercise workload. In other words, it makes sense that if regular moderate exercise lowers infection risk, it should be accompanied by enhanced immunosurveillance. On the other hand, when an athlete engages in unusually heavy exercise workloads (e.g., overtraining or a competitive endurance race event), infection risk should be related to diminished immunosurveillance. At present, there is more evidence, primarily epidemiological in nature, exploring the relationship between heavy exertion and infection, and these data will be reviewed first followed by a section on moderate exercise training and infection (Table 1). Much more research using larger subject pools and improved research designs is necessary before this model can be wholly accepted or rejected. The epidemiological studies summarized in Table 1 all used self-reported URTI data (primarily retrospective, with two studies using 1-year daily logs). None have attempted to verify symptomatology using viral identification or verification by physicians. There is some concern that the symptoms reported by endurance athletes following competitive race events may reflect those associated with an inflammatory response rather than URTI (Castell et al., 1997; Drenth et al., 1995; NehlsenCannarella et al., 1997). Nonetheless, the data are consistent in supporting the viewpoint that heavy exertion increases the risk of URTI, while moderate exercise training is associated with a decreased risk.

Several epidemiological reports suggest that athletes engaging in marathon-type events and/or very heavy training are at increased risk of URTI (Table 1). Nieman et al. (1990a) researched the incidence of URTI in a group of 2,311 marathon runners who varied widely in running ability and training habits. Runners retrospectively self-reported demographic, training, and URTI episode and symptom data for the 2-month period (January, February) prior to and the 1-week period immediately following the 1987 Los Angeles Marathon race. During the week following the race, 12.9% of the marathoners reported an URTI compared to only 2.2% of control runners who did not participate (odds ratio, 5.9, using a logistic regression model that controlled for various training and demographic variables). Forty percent of the runners reported at least one URTI episode during the 2-month winter period prior to the marathon race. Controlling for various confounders, it was determined that runners training more than 96 km/wk doubled their odds for sickness compared to those training less than 32 km/wk. Linde (1987) studied URTI in a group of 44 elite orienteers and 44 non-athletes of the same age, sex, and occupational distribution during a 1-year period. Athletes and controls recorded symptoms of sickness using daily logs for 1 year. The orienteers experienced significantly more URTI episodes during the year in comparison to the control group (2.5 vs. 1.7 episodes, respectively). While one-third of the controls reported no URTI during the year-long study period, this applied to only 10% of the orienteers. The average duration of symptoms in the group of orienteers was 7.9 days compared to 6.4 days in the control group (NS). The control group had the expected seasonal variation with the peak incidence in winter and relatively few cases in summer, while the orienteers tended to show a more even distribution. Heath et al. (1991) also followed a cohort of runners (N = 530) who self-reported URTI symptoms daily for 1 year. The average runner in the study was about 40 years of age, ran 32 km/wk, and experienced a rate of 1.2 URTI per year. Controlling for various confounding variables using logistic regression, the lowest odds ratio for URTI was found in those running less than 16 km/wk. The odds ratio more than doubled for those running more than 27 km/wk. This study demonstrated that total running distance for a year is a significant risk factor for URTI, with risk increasing as the running distance rises. Peters and Bateman (1983) studied the incidence of URTI in 150 randomly selected runners who took part in a 56 km Cape Town race in comparison to matched controls who did not run. Symptoms of URTI occurred in 33.3% of runners compared with 15.3% of controls

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31. Exercise and Immunity: Clinical Studies TABLE 1

Epidemiological Research on the Relationship Between Intense, Prolonged Exercise and Upper Respiratory Tract Infection (URTI)

Investigators Peters and Bateman (1982) Linde (1987) Nieman et al. (1989)

Peters (1990)

Nieman et al. (1990a)

Subjects

Method of determining URTI

141 South African marathon runners vs. 124 live-in controls 44 Danish elite orienteers vs. 44 matched non-athletes 294 California runners training for race

2-week recall of URTI incidence and duration after 56 km race URTI symptoms self-recorded in daily log for 1 year 2-month recall of URTI incidence; 1 week recall after winter 5, 10, 21 km races 2-week recall of URTI incidence and duration after 56 km race 2-month recall of URTI incidence during training for marathon; 1-week recall after winter race 1-year daily log using selfreported, pre-coded, symptoms 2-week recall of URTI incidence and duration after 90 km race

108 South African marathon runners vs. 108 live-in controls 2,311 Los Angeles marathon runners

Heath et al. (1991)

530 runners, South Carolina

Peters et al. (1993)

84 South African marathon runners vs. 73 non-runner controls

Nieman (1993) (unpublished data)

170 North Carolina marathon runners

Peters et al. (1996)

178 South African runners vs. 162 controls

Castell et al. (1996)

151 endurance athletes in the United Kingdom

1-week recall of URTI incidence after heavy exertion

Nieman et al. (2002a)

91 marathon runners, North Carolina

15-day recall of URTI incidence after marathon race event

Nieman et al. (2006)

155 ultramarathoners, Western U.S. 160 km

2-week recall of URTI incidence after 160 km race event

1-week recall of URTI incidence after summer marathon race 2-week recall of URTI incidence and duration after 90 km race

during the 2-week period following the race, and were most common in those who achieved the faster race times. The most prevalent symptoms after the race were reported to be sore throats and nasal symptoms. Of the total number of symptoms reported by the runners, 80% lasted for longer than 3 days, suggesting an infective origin. Several subsequent studies from this group of researchers have confirmed this finding (Peters, 1990; Peters et al., 1993, 1996). During the 2-week period following the 56 km Milo Korkie Ultramarathon in Pretoria, South Africa, 28.7% of the 108 subjects who completed the race reported non-allergy–derived URTI symptoms as compared to 12.9% of controls (Peters, 1990). In another study, 68% of runners reported the

Major finding URTI incidence twice as high in runners after 56 km race vs. controls (33.3% vs. 15.3%) Orienteers vs. controls had 2.5 vs. 1.7 URTIs during year Training 42 vs. 12 km/wk associated with lower URTI; no effect of race participation on URTI URTI incidence 28.7% in runners vs. 12.9% in controls after 56 km race Runners training ≥97 vs. 30 days) space missions. However, it is difficult to determine the medical significance of these immunological PSYCHONEUROIMMUNOLOGY, 4E VOLUME II

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lunar orbits, exploration will begin with a return to the moon for stays on the surface of approximately 180 days. An exploratory mission to Mars will follow this phase. The first human exploration mission to Mars will probably consist of a detailed study of the surface from orbit. This approximately 400-day Mars orbital mission will be followed by missions in which astronauts will descend to the Martian surface for closer study. The transit time for a round trip of nearly 90 million miles to Mars with current propulsion systems is expected to be about 12 months, and astronauts will explore the surface for about 12 to 18 months, making the total mission duration 24 to 30 months. The estimated date for the beginning of the Mars missions is no earlier than 2025. Exploration of Mars will be a singular accomplishment in human history.

efficiency particulate air (HEPA) filters to remove microbial contaminants and particulates. The levels of bacterial and fungal contaminants in the air and on surfaces of the ISS are lower than in most homes and offices. The most commonly isolated bacterial species found in air and on surfaces of the ISS, Mir, and space shuttle are Staphylococcus spp., Micrococcus spp., and Bacillus spp. The fungal species most commonly isolated are Aspergillus spp. and Penicillium spp. The microbiological risks associated with space flight and characterization of microbial content of spacecraft have been reported (Castro et al., 2004; Pierson, 2001). Mars exploration spacecraft will provide similar internal environments for crew habitation. However, because of vast distances, the Mars spacecraft must be much less dependent upon Earth for resupplies (e.g., water and food) and technical support.

III. SPACECRAFT ENVIRONMENT The environment typically provided by spacecraft for astronauts to live and work in is relatively small, confined, and closed, yet complex with respect to technology and contaminants. Major challenges for exploration missions will include maintenance of basic environmental parameters. The spacecraft environment must provide appropriate temperature, humidity, and breathing air with generally low levels of chemical and biological contaminants. Biologically and chemically safe drinking water and a nutritious and varied food supply are also essential. For exploration missions, the environmental systems providing and maintaining the air and water must function without failure for about 3 years in a space environment. The environment on board the space shuttle, the Russian Soyuz spacecraft, and the International Space Station (ISS) includes an atmosphere of 20% oxygen and 80% nitrogen at 14.7 psi. A temperature range from 70 to 75°F (21 to 24°C) and a relative humidity of 35 to 45% provide a shirt-sleeve environment. Drinking water on board the ISS is currently provided by a Russian system that reclaims humidity condensate (and in the future will reclaim urine). It provides drinking water and hot and warm water for rehydration of foods and drinks. The U.S. will provide a waterprocessing assembly that will produce additional potable water. Ground-supplied water is also transported to the ISS and, along with shuttle-produced water, is distributed for use. Gram-negative bacteria of low pathogenic potential are the major microbiological contaminants of water on the ISS (Pierson, 2001). Breathing air is reconditioned by removal of trace chemical contaminants and recycled through high-

IV. LIMITATIONS OF SPACE FLIGHT INVESTIGATIONS The space flight environment puts many constraints on science investigations, and performing experiments can itself be a source of stress for astronauts. All human space flight investigations require approval from the Johnson Space Center Committee for the Protection of Human Subjects, and astronauts may select investigations in which to participate as a human subject. Access of researchers to astronauts is always limited, but is more likely to be available before and after space flight than during flight. The earliest time that post-flight samples can be obtained is several hours after landing. During space shuttle missions, collection of blood, urine, saliva, and other specimens is very limited because of the great demands on astronauts’ time and the limited equipment available. Equipment needed to support the in-flight phase of science investigations must meet stringent limitations on its volume, mass, power requirements, calibration needs, maintenance, consumables (shelf life must be considered), waste products, and expertise required for operation. In addition, many instruments commonly used in biomedical experiments do not operate properly in microgravity. Few shuttle flights are dedicated to life sciences investigations. Typically, a crew of seven astronauts is divided into two teams. Work proceeds throughout a 24-hour period; one team is working while the other team sleeps. The workload is very heavy for the flight crew, who conduct many science investigations and carry out other spacecraft duties. An investigation that meets all of the science requirements and equipment

40. Reactivation of Latent Herpes Viruses in Astronauts

constraints, and in which crew members are willing to participate, may still not be included because space available for stowing the investigation’s support equipment is insufficient. Most studies are limited to small numbers of human or animal subjects.

V. STRESS ASSOCIATED WITH SPACE FLIGHT Astronauts experience uniquely stressful environments during space flight. Potential stressors include confinement in an unfamiliar, crowded environment, isolation, separation from family, anxiety, fear, sleep deprivation, psychosocial issues, physical exertion, noise, variable acceleration forces, increased radiation, and others. Many of these are intermittent and variable in duration and intensity, but variable gravity forces (including transitions from launch acceleration to microgravity and from microgravity to planetary gravity) and variable radiation levels are part of each mission and contribute to a stressful environment that cannot be duplicated on Earth. Radiation outside the Earth’s magnetosphere is particularly worrisome because it includes ionizing radiation from cosmic galactic radiation. Increased stress levels appear even before flight, presumably from the rigors of pre-flight training and the anticipation of the mission.

VI. NEUROENDOCRINE AND IMMUNE RESPONSES A. Stress Hormones The main neuroendocrine responses to stress are mediated through the hypothalamic-pituitaryadrenocortical (HPA) and sympathetic-adrenalmedullary axes. Both of these systems are active in the human response to space flight, but they seem to be activated by different stresses. Because of constraints on sampling and stowage during space flight, most of the data pertaining to changes in stress hormones and immune studies have been derived from samples taken before launch and after landing. Cortisol is the stress hormone that has been measured most often in samples from astronauts. Even before launch, stress-related changes in cortisol concentrations can occur. Serum cortisol levels of two cosmonauts was greater 15 days before launch than at 2 months before launch (Caillot-Augusseau et al., 1998). Plasma cortisol levels of shuttle astronauts was also greater before flight (10 days before launch) than at their annual medical exams, a period well removed

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from launch and generally considered to be a period of low stress (Stowe et al., 2000). These results indicate that the time when pre-flight samples are obtained must be considered in interpreting results for analytes that could be affected by stress. For example, L − 10 (10 days before launch) has been used in many studies as a baseline value. More recent studies have demonstrated that most astronauts are experiencing considerable stress by L − 10, leading to the conclusion that the annual medical exam is a more suitable baseline than a time shortly before flight (Mehta et al., 2000b; Mehta et al., 2001; Pierson et al., 2005). Although in-flight data are very limited, a pattern has emerged for the early phase of flight. One astronaut had a significant elevation in urinary cortisol immediately after launch (Leach, 1987). For both crews of the Spacelab Life Sciences (SLS) missions, urinary cortisol was also significantly increased immediately after launch, 128% in the SLS-1 crew and 146% in the SLS-2 crew (Leach et al., 1996). During flight days 2–9 of SLS-1, urinary cortisol returned to pre-flight levels, whereas during SLS-2 it was significantly elevated throughout most of the flight. In the STS-95 mission, plasma cortisol of both participating crew members was significantly elevated after launch (Stowe et al., 2001a). In the later “adaptive” phase of space flight, cortisol levels have been reported to either not change or increase. Throughout the Skylab missions (28 to 84 days duration), plasma and urinary cortisol concentrations were generally greater than they were before flight (Leach and Rambaut, 1977). During the Salyut-7 mission, on flight days 43–45 urinary excretion of cortisol was less than before flight, but on flight day 88 it was greater than pre-flight values (Vorobyev et al., 1986). On days 216–219 of the 237-day Salyut flight, plasma cortisol was increased; urinary cortisol was not observed to change (Gazenko et al., 1988). Toward the end of a 241-day Mir mission, plasma cortisol was unchanged from pre-flight levels (Grigoriev et al., 1990). Urinary cortisol levels of two subjects on a later Mir mission were greater between flight days 88 and 186 than before flight, but urinary cortisol of four other subjects was less during flight (Stein et al., 1999). It has been proposed that the variability in cortisol levels during long-duration missions may be caused by mission-specific stress (Stein et al., 1999), changes in the steroidogenesis pathway (Vorobyev et al., 1986), disruption of circadian rhythms (Leach Huntoon and Cintron, 1996), and the operation of negative feedback loops (Gauquelin et al., 1990; Leach Huntoon and Cintron, 1996). Plasma cortisol concentrations have generally been lower after short-term space flight than before flight:

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They were 27% less in 30 Apollo astronauts at landing than before flight (Leach et al., 1975), and 3% less in 133 Shuttle astronauts at landing (Leach, 1992). In contrast, measurement of 24-hour urine pools has generally revealed a greater concentration of cortisol after landing than before flight (Crucian, 2000; Mehta et al., 2000b; Stowe et al., 1999; Stowe et al., 2000). Plasma cortisol has a short half-life in the circulation (13 months previously). Clearly, data from some of the individuals in the former cohort are likely to have reflected antibody maintenance rather than formation. It is evident that prospective studies, in which repeated assessments of both stress and immune outcomes are required if the impact of stress on antibody formation versus maintenance is to be disentangled. One study to have clearly distinguished between stress effects on antibody maintenance and formation was conducted by Glaser and colleagues (2000). This group assessed the responses of 11 current and 13 former caregivers to a pneumococcal pneumonia vaccination, in relation to 28 healthy controls. Unlike the previously discussed vaccines, pneumococcal is a bacterial vaccine that elicits a thymus-independent response, and therefore no T-helper cells are generated. Although not significant, current caregivers reported greater levels of stress than did former caregivers or controls. Furthermore, current caregivers reported significantly less social support than both former caregivers and control participants. Unlike their previous studies of caregivers, however, there were no differences in pneumococcal antibody titers between the groups for several weeks post-inoculation. However, the groups did differ in the maintenance of their

response over a 6-month period. That is, while antibody levels in controls and former caregivers were stable over 6 months, current caregivers demonstrated reduced antibody levels over the same time period. This study therefore provided preliminary evidence that the ongoing chronic stress associated with caregiving impacts differently on the antibody response to a bacterial vaccine than a viral vaccine and may be related to the requirement for a thymus-dependent role in processing the antigen in the case of a viral vaccine. The data suggest that chronic stress may impact on the maintenance of the response and, therefore, on ongoing levels of protection. Finally, other factors have been suggested to have a buffering effect on the deterioration of immune protection over time. Costanzo et al. (2004) assessed the influence of mood and optimism on the in vitro cellular immune response to an influenza virus vaccine using cells from 18 healthy older adults reporting low to moderate levels of life stress. Greater cytokine responses were observed in those individuals who reported greater levels of vigor and optimism. However, the relationships between these factors and humoral responses are unknown. As immune outcomes were assessed 1 year after vaccination, Costanzo and colleagues suggest that the observed beneficial effects on cytokine levels may reflect a buffering by “vigor” and “optimism” of the deterioration of memory T-cells. Such a proposal further supports the view that psychological factors can also affect the maintenance of immune responses as well as initial responses. 2. Studies Reporting Contradictory Relationships between Psychological Factors and Immune Responses to Vaccination Despite the presence of a large body of evidence suggesting that stress and other psychological factors can have deleterious effects on vaccination responses, three studies report findings to the contrary. That is, these studies have observed greater antibody responses in individuals reporting higher levels of stress. Petry et al. (1991) assessed relationships between life stress and responses to Hep B vaccination in a sample of 85 healthy students. Vaccinations were administered at 0, 1, and 6 months to those individuals who had no previous exposure to hepatitis B. All participants completed questionnaires regarding their perceptions (either positive, negative, or neutral) of life experiences on the final day of vaccinations and again 3 months post-vaccine, at which time a final assessment of antibody levels was obtained. Individuals with higher levels of perceived stress, as measured at 6 months, demonstrated the highest peak responses to

42. Stress-associated Immune Dysregulation Can Effect Antibody and T-cell Responses to Vaccines

the vaccine 3 months later. That is, high stress in the 6 months of the vaccination program was indicative of greater antibody responses. Similar findings have also been reported following influenza virus vaccination. Larson et al. (2002) reported that greater numbers of stressful life events were related to increased antibody titers 2 weeks postvaccination in 149 healthy young adults. Similarly, in a study of healthy students, Burns et al. (2002c) observed better rates of protection 5 weeks postvaccination in those individuals reporting higher life events stress. Although positive relationships were observed in the short term, the latter study also reported that reduced protection was associated with increased life events and perceived stress 5 months following vaccination. This subsequent finding is, therefore, more in line with the majority of studies demonstrating relationships between lowered immunity and negative psychological factors. Furthermore, this finding indicates that, in the longer term, negative factors (e.g., increased life stress) can have deleterious effects on the maintenance of antibody responses, supporting previously reported findings (e.g., Burns et al., 2002b; Glaser et al., 2000). While these results may initially appear to be anomalous, there is some support for relationships in this direction in the literature. For example, previous work has demonstrated that modest levels of stress can actually result in enhancements in immunity (e.g., Jemmott and Locke, 1984). However, as there are no recognized thresholds according to which we can distinguish between levels of stress that may have neutral or beneficial effects (sometimes referred to as “eustress”) (Selye, 1976) and those that may have harmful consequences, such an explanation can provide only a limited account of these data. Instead, we must consider that methodological differences in the choice of population, the measurement of stress, etc., may have contributed to these apparently contradictory findings. 3. No Relationships between Psychological Factors and Immune Responses to Vaccination Several studies have failed to find any significant relationships between psychological factors and vaccine responses. One of the earliest studies in this area (Locke et al., 1979) assessed responses to an influenza virus vaccination in 124 healthy individuals. Antibody titers were assessed before and 2 weeks after receiving an influenza virus vaccination and compared with measures of anxiety, depression, hostility, and immediate and long-term changes in life stress. Postvaccination HAI titers were unrelated to any of the

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psychological variables. Although these findings suggest that the chosen psychological parameters have no relationship to vaccination responses, it is possible that the 2-week follow-up period was not long enough for a relationship to be observed. Indeed, most studies in this area have observed relationships between psychological factors and vaccination responses approximately 1 month following vaccination (e.g., Kiecolt-Glaser et al., 1996, Vedhara et al., 1999). In another study by Jabaaij et al. (1996), the effects of self-reported stress on a standard Hep B vaccination were explored in a sample of 68 healthy students, with extreme stress scores (i.e., low or high). In keeping with the Hep B vaccine schedule, students were administered the vaccine at 0, 1, and 6 months, and antibody levels were determined at 0, 2, 6, and 7 months. In addition, levels of self-reported psychological stress were recorded prior to the first vaccine and again at the 6-month follow-up. Unlike their previous study, no relationships were observed between psychological stress at 0 and 6 months and antibody levels at any of the follow-ups. The authors suggested that the antigenic doses of the vaccines used in the two studies may have accounted for the differences in their results (they had adopted a reduced dose vaccine in their earlier study). However, the studies also differed in other aspects of their methodology. In particular, the first study observed relationships between stress at 2 months and antibody levels at 7 months. However, there was no 2-month assessment of stress in the second study. If, as the authors suggested, stress is most damaging during this 2-month period, the absence of a stress measurement at 2 months may account for the reported null effects. A third study assessed whether a naturally occurring stressful event could disrupt vaccination responses. Boyce et al. (1995) administered a pneumococcal vaccination to a group of 39 pre-school children (aged 5 years) 1 week before the stressful experience of commencing kindergarten. Subsequent antibody responses were assessed 1 week later and related to reported incidences of problem behavior, an indirect measure of stress. The authors speculated that if stress had adverse effects on vaccine responses, the children for whom starting school was the most stressful would demonstrate the poorest vaccination responses. Although aspects of cellular immunity (e.g., CD4 counts) increased in response to the vaccine, no relationships between problem behavior and antibody responses were observed. There are at least two possible explanations for these findings. First, the chosen measure of stress (reports of problem behavior) was not sufficiently sensitive to measure the stress of starting kindergarten. Thus, any stress effects on immunity

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may have been overlooked by this methodology. Second, as has been suggested by Glaser and colleagues (2000), stress may exert effects on the maintenance of pneumococcal vaccine responses, not the initial formation of antibodies. Although this suggestion was based on data from older adults, who are already immuno-compromised, subsequent assessments of antibody responses in the months following the starting of kindergarten may have revealed differences in the maintenance of antibody responses to the vaccine. Finally, Vedhara et al. (2002) observed no relationships between distress and responses to an influenza virus vaccination in younger caregivers of patients with multiple sclerosis. The caregivers assessed in previous studies of this kind were all elderly (e.g., KiecoltGlaser et al., 1996, mean age = 73.1). It is possible, therefore, that the immune deficits observed in these studies were exacerbated by a natural age-related decline in their immune defenses and/or a chronic stress/aging interaction. To examine this issue, Vedhara et al. (2002) compared influenza virus vaccine responses in a younger population (mean age = 45.7 years) with age-matched controls. Unlike previous studies, no differences in vaccine responses were observed between caregivers and healthy controls. Furthermore, these younger caregivers did not differ from healthy controls in terms of psychological morbidity (e.g., anxiety and depression). This finding suggests that age-related decline in immune system efficacy might be responsible for the stress-related reductions in vaccine responses observed in previous caregiver studies. However, other differences between younger and older caregivers might account for the differences in vaccine response. For example, younger caregivers differ in terms of their levels of psychological morbidity. That is, despite providing continual care for a spouse, younger carers were considerably less distressed than their elderly counterparts. This may be socially mediated—e.g., younger carers have better social networks and therefore greater levels of support—or financial—i.e., the younger carers were more likely to be employed and therefore more financially secure. Further, these differences may be related to the illness of the spouse; e.g., caregivers of dementia patients report a greater burden of care giving than other caregiver groups (Ory et al., 1999). The differences in levels of distress between the older and younger cohorts obviously hinder any definitive conclusions regarding the effects of age-related decline on vaccine responses. Taken together, these studies suggest that there is no simple association between psychological factors and vaccination responses. However, clear method-

ological differences do differentiate between studies that have observed detrimental effects of stress on vaccine responses and studies that have not, and these differences are likely to account for many of the apparently contradictory findings. Trying to define the complex interactions that are taking place in the HPA and SAM axes with immune response to various types of vaccines is a complex challenge to the investigator interested in this area of research.

B. Intervention Studies Observational studies can provide information regarding how individuals respond to vaccination in “normal” compared with “stressed” conditions and how “stressed” individuals respond in relation to “normal, non-stressed” controls. It therefore follows that if stress leads to a reduced vaccination response, it should be possible to maximize immune responses to vaccinations if levels of stress are reduced. Relatively few studies have assessed the impact of interventions on responses to vaccine. This section details three such studies. The first study assessed the effects of an emotional disclosure intervention on responses to a Hep B vaccination program (Petrie et al., 1995). Emotional disclosure involves writing about a personally traumatic or stressful event for short periods of time (e.g., 20 minutes) over several consecutive days (e.g., 3–4 days). It is suggested that inhibition of stress or trauma can cause states of psychological and biological tension leading to a compromised immune system and subsequent ill health (Pennebaker, 1986). A controlled release of this tension, through written emotional disclosure can, therefore, lead to a reduction in stress and a subsequent improvement in immune function. Petrie and colleagues assessed potential improvements on immune function through responses to a Hep B vaccination program. Potential participants were screened for previous exposure to Hep B. Only those who demonstrated a negative Hep B antibody response were recruited into the study, and as such, it could be assumed that only primary immune responses to the vaccine were subsequently observed. A group of 40 healthy students were randomly assigned to either the emotional disclosure condition (i.e., writing about a traumatic or upsetting experience for 4 consecutive days) or a control condition in which they were asked to write in a descriptive manner about how they had spent the previous day, what they were intending to do the following day, week, and year. The Hep B vaccination was then administered to both groups on the final day of the writing task, and subsequent booster shots were given 1 and 4 months later. Blood samples


were obtained for the measurement of Hep B antibody and a measure of cellular immunity (NK cells) immediately before each of the vaccinations and again 6 months after the initial vaccination. Although there was very little difference between the groups 1 month post-vaccination, levels of Hep B antibody in disclosure participants were greater than those in control participants 4 and 6 months later. However, as there were no assessments of mood at each of the follow-up sessions in this study, it is not possible to determine whether the intervention resulted in a longer-term reduction in stress and whether this “stress-reduction” was responsible for the beneficial immune outcomes in the disclosure group. An important caveat to this study involved the measurement of primary and secondary antibody responses. Initial screening of potential participants resulted in only those participants with no Hep B antibody at baseline being entered into the study. However, more sensitive immune assays of the samples obtained immediately pre-vaccine revealed Hep B antibody to be present in five participants. Although the levels of antibody were low, detection indicated that these individuals had, at some stage, been previously exposed to the Hep B virus. The authors therefore employed specific statistical techniques to account for the presence of individuals who were expressing a secondary rather than primary antibody response. Although the presence of these individuals seemed to have very little impact on the overall findings, the subsequent identification of seropositive individuals highlights the importance of rigorous assay techniques. As previously demonstrated (Vedhara et al., 1999), elderly caregivers report greater levels of stress and have reduced responses to vaccination compared with controls. Vedhara et al. (2003) therefore implemented an intervention aimed at reducing the levels of distress in this population. Of a sample of 43 spousal caregivers of dementia patients, 16 were allocated to a stress-management intervention (SMI) and 27 to a control group. These groups were subsequently compared with 27 sociodemographically matched noncarer controls. Levels of distress (combined levels of anxiety, depression, and perceived stress) were measured at the baseline, halfway through, and at the end of the SMI. The SMI consisted of 8 weeks of 1-hour group cognitive-behavioral sessions facilitated by a clinical psychologist. The sessions focused on dealing with stressors specific to the role of being a caregiver, as well as the development of relaxation and cognitive restructuring techniques aimed at reducing the overall distress levels associated with care giving. At the end of the SMI, IgG antibody levels were assessed immediately prior to receiving a standard influenza virus

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vaccination. Subsequent IgG levels were then assessed 2, 4, and 6 weeks after vaccination. The results revealed that both carer groups reported significantly higher levels of distress than control participants before, during, and after the SMI. However, despite the absence of significant changes in distress, significantly more of the carers in the SMI group (50%) generated a clinically appropriate (four-fold increase in antibody levels) response to the vaccine compared with carers not in the intervention (7%). Indeed, the proportion of intervention carers achieving this fourfold increase in antibody levels exceeded that observed in the control group (29%), although this difference between the intervention carers and the control groups was not statistically significant. This study provided clear evidence that participation in an intervention aimed at reducing levels of stress can have remarkable effects on immune system efficacy, as measured by responses to an influenza virus vaccination. However, there were no changes in levels of distress following the intervention. That is, despite the intervention having a substantial effect on immune system efficacy, the intervention had no apparent effect on self-reported levels of distress. The authors suggested that a response shift could account for the absence of change in levels of stress. That is, the SMI encouraged participants to evaluate their lives in terms of stressors and responses to these stressors. This process of cognitive restructuring allowed participants to deal more effectively with the stressors in their lives; however, it may also have resulted in an increasing number of factors being relabeled as stressful. Finally, Davidson and colleagues assessed the effects of a meditation intervention on responses to an influenza virus vaccination (Davidson et al., 2003). Forty-eight healthy adults were randomly assigned to either a mindful meditation intervention or a waitinglist control group. The intervention lasted for a period of 8 weeks and involved weekly meditation meetings, home practice of the meditation techniques, and a single meditation retreat in the sixth week of the intervention. At the end of the 8-week period, all participants were administered an influenza virus vaccine and assessed for antibody levels 3–5 and 8–9 weeks post-vaccine. Those individuals who had participated in the meditation intervention demonstrated the greatest increases in influenza virus antibodies between the two follow-up periods. In addition, at the end of the intervention period, these individuals also demonstrated significant reductions in state anxiety when compared with controls. This study demonstrated that a psychological intervention—in this case, meditation—resulted in reduced levels of distress

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and increased antibody responses to an influenza virus vaccine. The efficacy of this intervention has, however, been brought into question. It has been suggested that the observed enhancement in immune response may not be unique to the implemented intervention (Smith, 2004), and that any prolonged activity that involves periods of quiet and pleasurable activity could evoke changes in affect and brain and immune system activity. This, of course, is not a criticism of the mindful meditation intervention in particular, but moreover, an important consideration for any studies using an intervention that involves activities that would not ordinarily be encountered. This is especially important when the implementation of an intervention involves the removal of an individual from the source of the stress (e.g., time away from work or time away from his/her caregiving responsibilities). Vedhara et al. (2003) attempted to control for this effect through the provision of respite to non-intervention carers. However, a reluctance of the carers to accept this offer led the authors to suggest that respite from the chronic stressor could also account for immune enhancement in elderly caregivers. Further, Davidson et al. (2003) acknowledged that the observed effects on immunity are probably not unique to their chosen intervention. Conclusions regarding the “active ingredients” of these interventions can, therefore, only be drawn following simultaneous comparison of the components of the interventions alongside respite controls. Despite potential methodological flaws, the implications of studies that assess the impact of stressreduction interventions on vaccination responses are far reaching. Such studies demonstrate that interventions can improve responses to vaccinations; however, it remains unclear as to whether these increases occur as a direct result of a reduction or removal of stress. These studies also provide essential information that could aid the effectiveness of vaccination programs. Further intervention work is clearly needed in this area, including studies which explore whether postintervention improvements in immune response are related to clinical improvements in health.

C. Experimental Studies The previously described observational and intervention studies have in common that the vaccine challenge is central to the aims of the study, and subsequent immune responses to the vaccine are the primary outcome. There is, however, another group of studies evident in the literature in which the vaccination and immune responses are incidental to the main aims of the study. Indeed, in many of these studies the immune

response data are not measured or reported. We describe these studies as being “experimental,” and although they are diverse in their design and objectives, they will all be reviewed here. One unique study assessed psychological, but not immune, responses to a vaccination. In order to assess the effects of trait negative affect on symptom attribution, Petrie et al. (2004) recorded a range of physiological symptoms 20 minutes and 7 days following a range of vaccinations typically offered prior to overseas travel (e.g., typhoid, hepatitis, and yellow fever). Individuals with higher levels of negative affect were more likely to attribute their symptoms to the vaccination despite the fact that they were not directly related to vaccination. Negative mood has also been assessed in relation to responses to a typhoid vaccination. Strike et al. (2004) were interested in sickness behavior, that is, common symptoms exhibited by individuals during times of infection or inflammation (e.g., lethargy and malaise). In a double-blind study, a sample of 26 healthy participants was administered either a placebo or typhoid injection. Mood and body temperature were assessed for 8 hours post-vaccination. No measures of humoral or cellular immune responses were reported; instead, the vaccination was used to model the physiological responses associated with sickness behavior. No differences were observed between the vaccination and placebo group in relation to reported symptoms or bodily temperature post-vaccination; however, significant differences in mood did occur between the groups. That is, while facets of negative mood generally improved in those who had received the placebo injection, negative mood deteriorated in the vaccination group. Furthermore, post-vaccination changes in negative mood were greater in those individuals who reported higher levels of chronic stress. In addition, the time course of the changes in negative mood in the current study was consistent with the changes that might be expected in cytokine levels following challenge (e.g., within 8 hours). It is therefore suggested that the changes in mood may represent changes in cytokine levels elicited by the vaccination. While cytokine activity does provide a plausible pathway through which infection and inflammation lead to sickness behaviors, the direct role of cellular responses was not assessed in this study. Further support for this hypothesis is provided by Glaser et al. (2003). They examined levels of depression and cytokines (IL-6) in a sample of 119 older adults before and 2 weeks after an annual influenza virus vaccination. As predicted, individuals with higher levels of depressive symptoms demonstrated greater IL-6 levels both before and after vaccination.


Increased depressive symptoms were also associated with the greatest post-vaccination increase in IL-6. Although increased depression is also associated with increases in health-compromising behaviors (e.g., smoking), controlling for these factors did not alter the relationship between depression and levels of IL-6. Cytokines, in part, are responsible for alerting other immune cells to the site of infection or the presence of an antigen and for regulating the immune response. Increased sensitivity of cytokines, such as that observed in individuals with higher levels of depression, may therefore lead to dysregulation of the immune system as a whole. Another study utilized a vaccine in an attempt to understand the possible etiology of chronic fatigue syndrome (CFS) (Vedhara et al., 1997). Viral infection has been implicated as one potential cause of CFS. In order to assess this assertion, 14 CFS patients were administered either a placebo or a live poliovirus, and aspects of their immune responses were compared with the responses of matched controls who had also received the vaccine. The vaccine did not significantly increase the incidence of CFS-related symptoms, psychological symptoms (e.g., anxiety), or affect aspects of cognitive performance. However, peak responses in T-cell proliferation were found to be greater in CFS patients than controls, although there was no difference between the groups in terms of the magnitude of these responses. Similarly, no significant differences were observed between the groups with regard to cytokine activity, mucosal immunity, or specific antibody responses to the vaccine. There were, however, some emerging differences in the rate of virus shedding, with some CFS patients demonstrating increased shedding compared with controls. Given the absence of consistent differences between the groups, the authors concluded that it was unlikely that enterovirus responses could be implicated in the etiology of CFS. Marsland et al. (2001) adopted an experimental design to assess whether responses to Hep B vaccination were related to immune reactivity to an acute stressor. A sample of 84 healthy students with no previous exposure to Hep B were administered a routine course of three Hep B vaccinations at 0, 6 weeks, and a final booster at 6 months. Following the second vaccination, participants were classified as either low or high responders on the basis of their antibody responses to the vaccine. These groups did not differ in terms of age, sex, alcohol, or tobacco use, but lower antibody responders reported greater levels of trait negative affect. Participants were then administered a laboratory-based stressor 2 to 4 months following their final vaccination. The stressor comprised a 2-minute preparation period followed by a 3-minute period of

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simulated public speaking in which participants were asked to defend themselves against a hypothetical shoplifting charge while being videotaped. Blood samples were taken immediately before and after the stressor task for measurement of Hep B antibody levels and various aspects of cellular immunity (e.g., lymphocyte proliferation, number of circulating T-cells and NK cells). Individuals classified as low antibody responders also demonstrated reduced T-cell proliferative responses following the stressor. This study supported previous findings suggesting that adverse psychological factors can have deleterious effects on vaccination responses. Furthermore, this study demonstrated that reactivity to a vaccine may be analogous to acute stress reactivity. That is, individuals classified as mounting a low antibody response to vaccination also demonstrated reduced cellular responses to an acute stress task.

D. Studies Examining Mechanisms The preceding sections have sought to distinguish vaccination studies into three main categories according to their objectives. However, many of the studies reviewed here have also sought to address the issue of potential mechanisms as part of their inquiry. Mechanisms implicated include both direct biological pathways linking stress and immune responses to vaccinations and indirect pathways whereby stress alters behavior, which in turn affects the response. In this section, we review the evidence on the mechanisms underlying psychological factors and immune responses to vaccination. 1. Biological Mechanisms There now exists a considerable literature indicating that stress can lead to dysregulation of the HPA axis, resulting in blunted immune responses (Sapolsky, 1996). Several studies have, therefore, examined the role of cortisol as a mediating mechanism between stress and vaccination responses. Two different assessments of HPA functioning have been assessed in relation to vaccination responses: Several studies have examined basal levels of cortisol, whereas other studies have assessed cortisol responses to a stressor (i.e., HPA reactivity). Vedhara et al. (1999) observed inverse relationships between basal levels of cortisol and antibody responses to influenza virus vaccine in elderly caregivers. However, this finding was not replicated in their later study assessing the efficacy of a stress management intervention in reducing stress levels in spousal care (Vedhara et al., 2003). That is, there were no

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differences in cortisol levels between those carers who did and did not partake in a stress management intervention and, furthermore, no differences between these groups and healthy controls. Other studies have also failed to find a relationship between levels of cortisol and vaccination responses. For example, Miller et al. (2004) and Pressman et al. (2005) observed negative associations between stress and loneliness and vaccine responses, respectively. However, no associations were observed between aggregated levels of stress or loneliness and cortisol. Moreover, no associations were observed between levels of cortisol and vaccine responses. The potential mediating role of cortisol cannot be discounted, however. Miller et al. (2004) suggest that an absence of a relationship may be due to the timing of the cortisol samples in relation to the self-report measures of stress. That is, the strongest associations between stress and antibody responses were only observed after the saliva-sampling period was completed. Other studies have assessed the relationship between vaccination responses and activation of the HPA through the measurement of cortisol in response to a stressor. Boyce et al. (1995) assessed cortisol responses in children following the stress of starting kindergarten. The lowest antibody responses to the pneumococcal vaccine were observed in those children who demonstrated the greatest cortisol increases to the stress of starting kindergarten. Other studies have assessed vaccination responses in relation to HPA reactivity following an acute laboratory stressor. Cacciopo (1994) assessed cellular immune responses (IL-2) in healthy older adults 3 months following an influenza virus vaccine in response to an evaluative speech task. Individuals who demonstrated the greatest cortisol reactivity to the task had lower levels of IL-2. Contrasting effects on immune outcomes have been observed in healthy young adults (Burns et al., 2002d). A sample of 30 healthy students completed an evaluative mental arithmetic stressor following completion of a routine series of Hep B vaccinations. Although there was some evidence that antibody titers were lower in individuals who demonstrated a diminished post-task reduction in cortisol, in the main, relationships between cortisol and antibody levels were not in the expected direction. That is, individuals with the highest antibody levels consistently demonstrated higher levels of cortisol (i.e., prior to and following the stressor). The findings regarding the role of cortisol as a mediating mechanism between stress and responses to vaccination are, therefore, mixed. However, the role of the HPA axis in immune dysregulation should not be discounted. Activation of the HPA axis in humans is typi-

cally assessed using salivary cortisol. Although there are obvious methodological benefits in utilizing noninvasive measurements of HPA activation, there are some limitations associated with the measurement of cortisol in saliva. For example, as there is considerable inter-individual variation in salivary cortisol, large sample sizes are required in order to observe intergroup differences. Yet, many of the studies published in this area have relatively small sample sizes. This variation may also lead to differences between studies. For example, Burns et al. (2002d) reported mean cortisol levels in their cohort that were lower than those observed in other similar studies (e.g., Clow et al., 1997). Individuals with low or high levels of cortisol may not, therefore, be comparable between studies, which can, as a consequence, influence the direction of relationships with other psychological variables and vaccination responses. Second, as human cortisol secretion is subject to diurnal variation, specific aspects of the diurnal cycle may be more sensitive to the effects of stress than others, for example, the waking response (Kunz-ebrecht et al., 2004). Finally, studies that have examined the role of cortisol as a mediating mechanism all differ in terms of their methodology—with differences evident not only in approaches to the quantification of cortisol (e.g., area under the curve, early morning peak, slopes, etc.), but also in the aspects of immunity under investigation (e.g., cellular and humoral) in response to a variety of vaccinations, over different time periods, in populations differing in terms of age and stressor type, duration, and frequency. Careful consideration must be given to these methodological variations when attempting to elucidate the role of the HPA axis as a mediating mechanism between stress and the immune system. Another key system involved in the human stress response is the SAM system. Activation of the SAM system is typically measured through cardiovascular parameters (e.g., blood pressure and heart rate) and levels of catecholamines. Cacioppo (1994) assessed the relationships between the reactivity of these parameters to acute stress and in response to cellular immune responses (IL-2) to influenza virus vaccination. Individuals who demonstrated the greatest sympathetic cardiac reactivity, as measured by pre-ejection period (PEP), to the stressor exhibited the greatest decline in immune responses 3 months after vaccination. Catecholamine levels (epinephrine) and heart rate were, however, unrelated to immune responses during the same period. Burns et al. (2002d) have reported similar findings following Hep B vaccination. Greater sympathetic activation (cardiac output and cardiac contractility) following an acute stressor task was observed in those individuals with low antibody titers. Marsland


et al. (2001) observed a relationship between another marker of sympathetic activation (increased heart rate in response to acute stress) and decreases in lymphocyte proliferative responses. However, no relationships were observed between heart rate reactivity and antibody responses to influenza virus vaccination. Like the studies assessing HPA reactivity, studies assessing the potential role of sympathetic activation and vaccination responses are mixed. Furthermore, like the HPA studies, these studies differ in their methodologies. Taken together, however, these studies indicate that it is cellular rather than humoral immune responses that are most associated with sympathetic parameters. This may reflect the greater individual differences in HPA reactivity. That is, while most stressors elicit a sympathetic response of some kind, HPA responses have a higher threshold for activation. Furthermore, HPA responses may be more determined by individual differences in the interpretation of a stressor than the more immediate, fight-flight responses elicited via the SAM system. The stress response is characterized by the activation of both the HPA and SAM systems, and as such, poor vaccine response may not occur as a direct result of dysregulation of either system but, moreover, an interaction of the two. Further studies attempting to elucidate these mechanisms could, therefore, usefully employ assessment of both HPA and sympathetic responses (e.g., Burns et al., 2002d). Specific brain areas have also been implicated as potential pathways by which psychological factors can influence immune system efficacy. Davidson et al. (2003) assessed activity in the left anterior regions of the brain following periods of meditation. This area is associated with emotion (Davidson and Irwin, 1999) and also enhanced cellular immunity (Kang et al., 1991), and as such may mediate between stress and immune dysregulation. Meditation resulted in increased activation of these brain areas and was also related to the greatest antibody responses. Although the authors suggested that their measures of brain activity were crude, this study provided further evidence that a stress-reduction intervention can increase immune system efficacy. In addition, this intervention also reduced perceived reports of distress and highlighted a potential mediating pathway. Evidence also implicates the prefrontal cortex as a potential pathway. Rosenkranz et al. (2003) assessed activation of the prefrontal cortex via EEG and eyeblink magnitude during an emotional recall task. These neurobehavioral responses were subsequently compared with influenza virus antibody titers. Increased activation of the right prefrontal cortex and larger eyeblink magnitude were associated with lower antibody

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titers. This brain region has been previously associated with higher levels of depression (Henriques and Davidson, 1991) and therefore provides a potential pathway by which negative emotions can lead to suppressed immunity. 2. Psychosocial and Behavioral Mechanisms Stress not only influences biological systems, but also can affect behavior. For example, periods of examination stress have been associated with increases in smoking and reductions in exercise and food intake (Ogden and Mtandabari, 1997). In turn, these behaviors can directly or indirectly compromise the immune system, for example, disruption of antibody formation (Kiecolt-Glaser and Glaser, 1988). One study reported earlier (Burns et al., 2002b) suggests that the way an individual copes with stressful situations can affect immune responses. That is, coping with a stressful situation by acceptance was associated with better responses to Hep B vaccination. In contrast, coping by substance abuse was related to a reduced antibody response. Coping by substance abuse implies engaging in health behaviors that may independently compromise immunity (e.g., alcohol consumption); however, controlling for alcohol intake did not reduce the strength of the observed association. Like alcohol, a whole range of other health behaviors could affect immune status; however, very few studies have provided significant evidence of the mediating role of health behaviors. Two studies have suggested that sleep may mediate between levels of stress and antibody responses. Miller et al. (2004) observed no mediating effect of a range of health behaviors, for example, smoking behavior, alcohol consumption, and levels of physical activity. However, sleep quality was associated with both levels of stress and antibody responses to influenza virus vaccination. That is, individuals with higher levels of cumulative stress reported fewer hours sleep, levels of which were subsequently associated with poorer antibody responses. Further evidence regarding the role of sleep comes from an experimental study. Lange et al. (2003) compared two groups of healthy young adults following a primary Hep A vaccination. All participants were vaccinated in the morning and then allocated to either a normal-sleep or a sleep-deprived group. Individuals who had a normal night of sleep following vaccination demonstrated a two-fold higher increase in Hep A antibody titers 1 month following vaccination. Interestingly, apart from increased tiredness the following morning, the sleepdeprived individuals did not report greater incidences of other negative states, suggesting that it is lack of sleep rather than a state of stress created by lack of

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sleep that is responsible for the observed immune suppression. Although this study provided support for the role of sleep as a unique mediating variable, the authors also reported differences between the groups with regard to endocrine responses following vaccination. Specifically, individuals in the normal sleep group demonstrated higher levels of prolactin and growth hormone. These hormones have been previously demonstrated to exert stimulatory effects on several aspects of the immune system (Gala, 1991). This study highlighted the complexity of the interactions between these proposed mediators. That is, the precise impact of behavioral factors on vaccination responses is unclear and could also be mediated by other pathways (e.g., the endocrine system). The observed associations, therefore, indicate that, at the very least, behavioral factors should be controlled in future studies evaluating the effects of psychological factors on immune responses. It would, however, be beneficial not only to control these factors, but also to examine them as potential mediating mechanisms. Furthermore, the inter-relationships observed between behavioral and biological pathways highlight the importance of their simultaneous measurement.

IV. CONCLUSIONS AND FUTURE DIRECTIONS Several issues are apparent from this overview of the literature regarding the use of vaccinations in PNI studies. These issues can be broadly distinguished into those concerning methodology and those concerning research findings. With regard to methodology, two main observations can be made. First, a variety of vaccines have been assessed in this body of literature. These vaccines all vary in terms of the expected time course of responses and also in the likelihood of previous exposure. This is especially pertinent for studies that have examined responses to influenza virus vaccines, where previous exposure seems to influence subsequent relationships between psychological variables and vaccination response. For example, Miller et al. (2004), Vedhara et al. (1999), and Moynihan et al. (2004) all observed differential effects on each of the strains; that is, increases in stress were associated with one strain but not the other. Although there appears to be no consistent explanation regarding the causes of the differential effects of stress on differing viral strains, prior exposure to these strains is likely to alter the relationship between psychological variables and antibody responses to vaccination. These vaccines do, however, have one thing in common. With the excep-

tion of Jabaaij et al. (1993), all the studies described in this overview utilized standardized vaccinations. These vaccines are not intended for experimental use but instead are designed to create maximal responses in a maximum number of people, thus creating widespread immunity to infectious diseases. For example, most Hep B vaccinations elicit responses in 90% of individuals (Petrie et al., 1995). This issue may account for the fact that the most consistent results are observed in elderly individuals, who, as a result of age-related declines in immune system efficacy, are less likely to mount an adequate response to vaccination. The second methodological issue concerns the considerable variation in the psychological factors under investigation. For example, stress has been defined and measured in many ways. Studies have assessed the absence or presence of stress (e.g., Vedhara et al., 1999), individual perceptions of stress (e.g., Miller et al., 2004), numbers of life events (Burns et al., 2002c), and even proxy measures of stress (incidences of problem behavior, e.g., Boyce et al., 1995). With such a range of definitions and, therefore, assessment tools, it is unsurprising that a mixed pattern of results has been observed. The predominant theme, however, is that adverse psychological events can lead to poorer vaccine responses, whereas other factors (e.g., positive mood, increased social support) can have a protective role, resulting in enhanced cellular responses and maintenance of humoral immune responses to vaccination. These studies do suggest, therefore, that increasing positive factors (e.g., well-being, optimism, quality of relationships and social support), possibly through interventions, can enhance immune responses to vaccination. Among the salient issues regarding research findings, perhaps one of the most prominent concerns the fact that, upon initial inspection, the findings regarding the effects of psychological factors upon vaccination responses appear to be mixed. Some of these inconsistencies may be due to the methodological issues described earlier. However, lack of clarity regarding whether the study is designed to explore antibody formation or antibody maintenance may also contribute to this inconsistent literature as psychological factors may affect each of these processes differently. Future research may benefit from longitudinal designs in which both antibody formation and maintenance are explored. A second issue concerns the fact that, while several studies have attempted to examine potential mechanisms, few have explored these mechanisms directly, as they are inherently correlational in their design. Thus, while a factor may be statistically correlated, a full examination of its influence as a moderator or


mediator would require some degree of manipulation. For example, if the HPA axis is believed to moderate responses to influenza virus vaccines as suggested by Vedhara et al. (1999), then one test of that mediating relationship would be to modify levels of the hormone cortisol (e.g., pharmacologically) and examine the effects on immune responses to vaccination. This may prove to be a fruitful area for future enquiry. A third, and related, issue concerns the fact that only three studies have examined the effects of interventions upon vaccination responses. Although they consistently demonstrated post-intervention improvements in vaccination responses, the underlying mechanisms of this process remain unclear. That is, is it respite from the source of stress or a specific element of these interventions that is responsible for improvements in immunity? A fourth issue concerns the studies described in the experimental section. The range and diversity of these studies indicate that the vaccination paradigm can be usefully employed to examine a range of research questions, other than those that explicitly focus on psychoneuroimmunological pathways between stress and disease. This may prove to a fruitful area of inquiry. In conclusion, despite differences in methods and, on occasion, contradictory findings, in the main these studies demonstrate that adverse psychological factors have deleterious effects on vaccination responses. However, what remains unclear is whether these deficits result in increased susceptibility to disease. That is, if an individual fails to mount a clinically significant antibody response to a vaccine, is that individual more likely to experience disease or illness at a later date? Furthermore, intervention studies demonstrate that reducing adverse psychological factors can improve responses to vaccination. Yet it remains unclear as to whether observed improvements in immunity have actual implications for the health status of the individual. Future studies should, therefore, focus on clinical indications of health and disease in order to examine whether the levels of immune deficits and enhancements observed in experimental studies increase or decrease the likelihood of subsequent episodes of illness or disease.

References Bovberg, D. H., Manne, S. L., and Gross, P. A. (1990). Immune responses to influenza vaccine is related to psychological state following exams. Psychosomatic Medicine, 52, 229 [Abstract]. Boyce, W. T., Adams, S., Tschann, J. M., Cohen, F., Wara, D., and Gunnar, M. R. (1995). Adrenocortical and behavioural predictors of immune responses to starting school. Pediatric Research, 38, 1009–1016.

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Burns, V. E., Carroll, D., Drayson, M., Whitham, M., and Ring, C. (2002a). Psychological stress, cortisol and antibody response to influenza vaccination in a young, healthy cohort. Brain, Behavior and Immunity, 16, 173 [Abstract]. Burns, V. E., Carroll, D., Ring, C., Harrison, L. K., and Drayson, M. (2002b). Stress, coping and hepatitis B antibody status. Psychosomatic Medicine, 64, 287–293. Burns, V. E., Drayson, M., Ring, C., and Carroll, D. (2002c). Perceived stress and psychological well-being are associated with antibody status after meningitis C conjugate vaccination. Psychosomatic Medicine, 64, 963–970. Burns, V. E., Ring, C., Drayson, M., and Carroll, D. (2002d). Cortisol and cardiovascular reactions to mental stress and antibody status following hepatitis B vaccination: a preliminary study. Psychophysiology, 39, 361–368. Cacioppo, J. T. (1994). Social neuroscience: autonomic, neuroendocrine and immune responses to stress. Psychophysiology, 31, 113–128. Clow, A., Patel, S., Najafi, M., Evans, P. D., and Hucklebridge, F. (1997). The cortisol response to psychological challenge is preceded by a transient rise in endogenous inhibitor of monoamine oxidase. Life Sciences, 61, 567–575. Cohen, S., Miller, G. E., Rabin, B. S. (2001). Psychological stress and antibody response to immunization: a critical review of the human literature. Psychosomatic Medicine, 63, 7–18. Costanzo, E. S., Lutgendorf, S. K., Kohut, M. L., Nisly, N., Rozeboom, K., Spooner, S., Benda, J., and McElhaney, J. E. (2004). Mood and cytokine responses to influenza virus in older adults. Journal of Gerontology: Medical Sciences, 59, 1328–1333. Davidson, R. J., and Irwin, W. (1999). The functional neuroanatomy of emotion and affective style. Trends in Cognitive Science, 3, 25–39. Davidson, R. J., Kabat-Zinn, J., Schumacher, J., Rosenkranz, M., Muller, D., Santorelli, S. K., Urbanowski, F., Harrington, A., Bonus, K., and Sheridan, J. F. (2003). Alterations in brain and immune function produced by mindfulness meditation. Psychosomatic Medicine, 65, 564–570. Gala, R. R. (1991). Prolactin and growth hormone in the regulation of the immune system. Proceedings of the Society of Experimental Biology and Medicine, 198, 513–527. Glaser, R., Kiecolt-Glaser, J. K., Bonneau, R. H., Malarkey, W., Kennedy, S., and Hughes, J. (1992). Stress-induced modulation of the immune response to recombinant hepatitis B vaccine. Psychosomatic Medicine, 54, 22–29. Glaser, R., Kiecolt-Glaser, J. K., Malarkey, W., and Sheridan, J. F. (1998). The influence of psychological stress on the immune response to vaccines. Annals of the New York Academy of Science, 840, 649–655. Glaser, R., Robles, T. F., Sheridan, J., Malarkey, W. B., and KiecoltGlaser, J. K. (2003). Mild depressive symptoms are associated with amplified and prolonged inflammatory responses after influenza virus vaccination in older adults. Archives of General Psychiatry, 60, 1009–1014. Glaser, R., Sheridan, J., Malarkey, W. B., MacCallum, R. C., and Kiecolt-Glaser, J. K. (2000). Chronic stress modulates the immune response to a pneumococcal pneumonia vaccine. Psychosomatic Medicine, 62, 804–807. Hayney, M. S., Dienberg Love, G., Buck, J. M., Ryff, C. D., Singer, B., and Muller, D. (2003). The association between psychosocial factors and vaccine-induced cytokine production. Vaccine, 21, 2428–2432. Henriques, J. B., and Davidson, R. J. (1991). Left frontal hypoactivation in depression. Journal of Abnormal Psychology, 100, 535–545.

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Jabaaij, L., Grosheide, P. M., Heijtink, R. A., Duivenvoorden, H. J., Ballieux, R. E., and Vingerhoets, A. J. J. M. (1993). Influence of perceived psychological stress and distress on antibody response to low dose rDNA hepatitis B vaccine. Journal of Psychosomatic Research, 37, 361–369. Jabaaij, L., vanHattum, J., Vingerhoets, A. J. J. M., Oostveen, F. G., Duivenvoorden, H. J., and Ballieux, R. E. (1996). Modulation of immune response to rDNA hepatitis B vaccination by psychological stress. Journal of Psychosomatic Research, 41, 129–137. Jemmott, J. B. III, and Locke, S. E. (1984). Psychosocial factors, immunologic mediation and human susceptibility to infectious disease: how much do we know? Psychological Bulletin, 95, 78–108. Kang, D. H., Davidson, R. J., Coe, C. C., Wheeler, R. E., Tomarken, A. J., and Ershler, W. B. (1991). Frontal brain asymmetry and immune function. Behavioral Neuroscience, 105, 860–869. Kiecolt-Glaser, J. K., and Glaser, R. (1988). Methodological issues in behavioural immunology research with humans. Brain, Behavior and Immunity, 2, 67–78. Kiecolt-Glaser, J. K., Glaser, R., Gravenstein, S., Malarkey, W. B., and Sheridan, J. (1996). Chronic stress alters the immune response to influenza virus vaccine in older adults. Proceedings of the National Academy of Sciences of the United States of America, 93, 3043–3047. Kunz-ebrecht, S. R., Kirschboum, C., Marued, M., Steptoe, A. (2004). Differences in cortrsol certaining response on work days and weekends in women and men from Whitehall II cohort. Psychoneuroendocrinology, 29, 516–528. Lange, T., Perras, B., Fehm, H. L., and Born, J. (2003). Sleep enhances the human antibody response to hepatitis A vaccination. Psychosomatic Medicine, 65, 831–835. Larson, M. R., Treanor, J. J., and Ader, R. (2002). Psychosocial influences on responses to reduced and full-dose trivalent inactivated influenza vaccine. Psychosomatic Medicine, 64, 113 [Abstract]. Locke, S. E., Hurst, M. W., Heisel, S. J., Kraus, L., and Williams, M. (1979). The influence of stress and other psychosocial factors on human immunity. In Proceedings of the 36th Annual Meeting of the Psychosomatic Society. Marsland, A. L., Cohen, S., Rabin, B. S., and Manuck, S. B. (2001). Associations between stress, trait negative affect, acute immune reactivity, and antibody response to hepatitis B injection in healthy young adults. Health Psychology, 20, 4–11. Miller, G. E., Cohen, S., Pressman, S., Barkin, A., Rabin, B. S., and Traenor, J. J. (2004). Psychological stress and antibody response to influenza vaccination: when is the critical period for stress, and how does it get inside the body? Psychosomatic Medicine, 66, 215–223. Morag, M., Morag, A., Reichenberg, A., Lerer, B., and Yirmiya, R. (1999). Psychological variables as predictors of rubella antibody titers and fatigue—a prospective double blind study. Journal of Psychiatric Research, 33, 389–395. Moynihan, J. A., Larson, M. R., Treanor, J., Duberstein, P. R., Power, A., Shore, B., and Ader, R. (2004). Psychosocial factors and the response to influenza vaccination in older adults. Psychosomatic Medicine, 66, 950–953. Ogden, J., and Mtandabari, T. (1997). Examination stress and changes in mood and health related behaviours. Psychology and Health, 12, 289–299. Ory, M., Hoffman, R. R., Yee, J. L., Tennstedt, S., and Schulz, R. (1999). Prevalence and impact of caregiving: a detailed comparison between dementia and non-dementia caregivers. Gerontologist, 39, 177–185. Pennebaker, J. W., and Beall, S. (1986). Confronting a traumatic event: toward an understanding of inhibition and disease. Journal of Abnormal Psychology, 95, 274–281.

Petrie, K. J., Booth, R. J., Pennebaker, J. W., Davison, K. P., and Thomas, M. G. (1995). Disclosure of trauma and immune responses to a hepatitis B vaccination programme. Journal of Consulting and Clinical Psychology, 63, 787–792. Petrie, K. J., Moss-Moriss, R., Grey, C., and Shaw, M. (2004). The relationship of negative affect and perceived sensitivity to symptom reporting following vaccination. British Journal of Health Psychology, 9, 101–111. Petry, J., Weems, L. B., and Livingstone, J. N. (1991). Relationship of stress, distress and the immunologic response to a recombinant hepatitis B vaccine. Journal of Family Practice, 32, 481–486. Pressman, S. D., Cohen, S., Miller, G. E., Barkin, A., Rabin, B. S., Treanor, J. J. (2005). Loneliness, social network size, and immune responses to influenza vaccination in college freshmen. Health Psychology, 24 (3), 297–306. Rabin, B. S. (2005). Introduction to immunology and immuneendocrine interactions. In K. Vedhara and M. E. Irwin (Eds.), Human psychoneuroimmunology. (pp. 1–25) Oxford: Oxford University Press. Rosenkranz, M. A., Jackson, D. C., Dalton, K. M., Dolski, I., Ryff, C. D., Singer, B. H., Muller, D., Kalin, N. H., and Davidson, R. J. (2003). Affective style and in vivo immune response: neurobehavioral mechanisms. Proceedings of the National Academy of Sciences of the United States of America, 100, 11148–11152. Sapolsky, R. M. (1996). Why stress is bad for your brain. Science, 273, 749–750. Selye, H. (1976). The stress of life (rev. ed.). New York: McGraw-Hill. Smith, J. C. (2004). Alterations in brain immune function produced by mindfulness meditation: three caveats. Psychosomatic Medicine, 66, 148–152. Strike, P. C., Wardle, J., and Steptoe, A. (2004). Mild acute inflammatory stimulation induces transient negative mood. Journal of Psychosomatic Research, 57, 189–194. Vedhara, K., Bennett, P. D., Clark, S., Lightman, S. L., Shaw, S., Perks, P., Hunt, M. A., Philip, J. M. D., Tallon, D., Murphy, P. J., Jones, R. W., Wilcock, G. K., and Shanks, N. M. (2003). Enhancement of antibody responses to influenza vaccination in the elderly following a cognitive-behavioural stress management intervention. Psychotherapy and Psychosomatics, 72, 245–252. Vedhara, K., Cox, N. K. M., Wilcock, G. K., Perks, P., Hunt, M., Anderson, S., Lightman, S. L., and Shanks, N. M. (1999). Chronic stress in elderly carers of dementia patients and antibody response to influenza vaccination. Lancet, 353 (9153), 627–631. Vedhara, K., Llewelyn, M. B., Fox, J. D., Jones, M., Jones, R., Clements, G. B., Wang, E. C. Y., Smith, A. P., and Borysiewicz, L. K. (1997). Consequences of live poliovirus vaccine administration in chronic fatigue syndrome. Journal of Neuroimmunology, 75, 183–195. Vedhara, K., McDermott, M. P., Evans, T. G., Treanor, J. J., Plummer, S., Tallon, D., Cruttenden, K. A., and Schifitto, G. (2002). Chronic stress in non-elderly caregivers—Psychological, endocrine and immune implications. Journal of Psychosomatic Research, 53, 1153–1161. Vedhara, K., Miles, J., Bennett, P. D., Plummer, S., Tallon, D., Brooks, E., Munnoch, K., Schreiber-Kounine, C., Fowler, C., Lightman, S. L., Sammon, A., Rayter, Z., and Fardon, J. (2003). Salivary cortisol, stress, anxiety and depression. Biological Psychology, 62, 89–96. Wallsten, S. M. (1993). Comparing patterns of stress in daily experiences of elderly caregivers and non-caregivers. International Journal of Aging and Human Development, 37, 55–68.

P A R T

V PSYCHONEUROIMMUNOLOGY AND PATHOPHYSIOLOGY DAVID A. PADGETT AND JOHN F. SHERIDAN

MIND BODY INTERACTIONS AND THEIR INFLUENCE ON THE PATHOPHYSIOLOGY OF DISEASE Now that we have made it to the fourth edition of Psychoneuroimmunology, many of us who work in the field take for granted that mind-body interactions influence immune function. As the previous Parts in this compendium have noted, we toil with the understanding that cells of the immune system have receptors for neurotransmitters, neuropeptides, and hormones, and we embrace the notion that primary and secondary lymphoid tissues are innervated by the sympathetic nervous system. Today, in 2006, we have a solid working knowledge of how stressors activate the hypothalamicpituitary-adrenal axis and the sympathetic nervous system; we also have an appreciable grasp of how products of these systems affect molecular and cellular changes in the immune system. In an attempt to capture the breadth of the extant literature in PNI and disease pathophysiology, the chapters included in this Part were solicited to provide an in-depth understanding of psychoneuroimmunological influences on disease. As far back as the early twentieth century, investigators who were undertaking hypothesis-driven research in psychosomatic medicine demonstrated the importance of mind-body interactions in the pathophysiology of disease. In the 1930s Walter Cannon was one of the first to note that fear and stress contributed to the development of physical symptoms akin to heart disease (Cannon, 1939). In the first chapter in this Part, Kop and Cohen discuss how mind-body interactions directly and indirectly influence the development of cardiovascular disease. They note multiple psychological risk factors for heart disease. These can be categorized as chronic personality traits, such as

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V. Psychoneuroimmunology and Pathophysiology

hostility; they can be episodic factors, which can include depression and exhaustion; or risk factors can manifest in the form of something like anger, which they classify as an acute psychological trigger. As the years passed since Cannon’s observations, the results of an increasing number of studies suggest that the immune system plays an important role in the relationship between these psychological risk factors and future coronary syndromes. In Chapter 43, Kop and Cohen review the potential interrelationships between the myriad of psychological factors and immune system mediators and their influence on pathophysiology of coronary artery disease progression. The role of psychoneuroimmunological processes in coronary heart disease is continued in the second chapter, prepared by Steptoe and Brydon. According to the authors, “the role of psychoneuroimmunology in coronary heart disease (CHD) has only become widely recognized in the last decade, and research is rapidly evolving as the contribution of immunological processes to disease becomes better understood (Steptoe and Brydon, 2005)”. Chapter 44 summarizes current understanding of this field and addresses two major issues. The first is whether emotional and behavioral factors stimulate the inflammatory and immunological processes that contribute to coronary atherosclerosis and CHD morbidity, while the second asks whether psychoneuroimmunological pathways mediate links between psychosocial risk factors and CHD. Psychological factors have been implicated in the onset and exacerbation of various skin disorders. Chapter 45, by Buske-Kirschbaum, addresses the influence of immunological and psychological factors on various skin diseases with a focus on atopic dermatitis (AD). Dysregulation of the hypothalamic-pituitary-adrenal axis and sympathetic-adreno-medullary system are proposed to aggravate the allergic inflammatory process in atopic dermatitis. Buske-Kirschbaum states . . . “although it is well accepted that exacerbation of skin condition in AD is closely linked to emotional stress, the underlying mechanisms of stress-induced exacerbation of AD are rarely understood.” She goes on to propose a psycho-neuro-endocrine-immunological model of AD in an attempt to integrate the existing data and to highlight potential future research strategies in the field. While the influence of stress on the development of cardiovascular and inflammatory diseases was among the first areas in psychoneuroimmunology to receive attention, the field has broadened significantly as the role of inflammation in numerous central and peripheral processes became evident. The next chapter in this Part, by Guest et al., summarizes evidence relating obesity and immunity with an emphasis on the mechanism associated with the underlying pathologies. Obesity has become a significant health problem in the United States, and the view of adipose tissue as an inert depot for excess energy has shifted. In Chapter 46, the authors state that “adipose tissue is now recognized as a dynamic organ with important roles in modulating satiety, reproduction, immunity and metabolism.” And importantly, it has been reported that the obese exhibit increased markers of inflammation including macrophage infiltration. Studies on the relationship between inflammation and obesity may provide information on the pathologies associated with obesity.


The remaining chapters in this Part focus on psychoneuroimmunology and host defense. Pathogen recognition has been a major focus of immunological research for the past decade. Studies on innate resistance and the mechanism by which a mammal initially recognizes a viral or bacterial challenge have provided insight into basic host resistance. The long-held dogma of “self versus non-self” discrimination as the capstone of the immune response has been modified to include the concept of “danger signals” (Matzinger, 1994). Danger signals are elicited in damaged tissues in response to insult/ injury, and they provide information through primitive receptor–ligand interactions as part of innate resistance. In Chapter 47, by Fleshner et al., the authors address the concept that exposure to mental or physical stressors stimulates a cascade of behavioral and physiological responses that are directed at improving an organism’s chance of survival. They put a novel twist on this idea and focus on danger signals. Fleshner and colleagues review a collection of data showing that, at the cellular level, the induction of heat shock proteins—specifically heat-shock protein 72 (Hsp72)—may be a previously unrecognized feature of an acute stress response. Their chapter advances the hypothesis that Hsp72 may function as an endogenous “danger signal,” leading to the facilitation of immune responses during times of acute stress. In sum, their chapter focuses on how even the sub-cellular response to a stressor is not intended to be immunosuppressive, but instead is aimed at improving an organism’s survival opportunities. Various infectious diseases commonly befall the average person, and diseases caused by bacteria and viruses continue to be a significant cause of morbidity and mortality worldwide. The remaining chapters in this Part focus upon the underlying mechanisms of psychoneuroimmune interactions that influence microbial pathogenesis and individual susceptibility to infection. Chapter 48, by Emeny and Lawrence, focuses on the consequences of short-term restraint or cold-restraint treatment on the pathophysiology of infection with the bacterium Listeria monocytogenes (LM). The authors initially focus attention on host defense mechanisms that are known to contribute to a successful anti-LM response. This is followed by an in-depth analysis of the neuroimmune interactions that likely influence the host’s immune response during LM infection. In Chapter 49, Sloan et al. point out that a “large body of research has linked psychosocial factors to variations in HIV disease progression. . . . However, the biological processes mediating those effects remain poorly understood.” The chapter reviews the biological signaling pathways that “convey the effects of psychosocial factors to the cellular and molecular processes involved in HIV pathogenesis.” The chapter concludes with a consideration of the broader relationship between psychosocial characteristics and clinical disease outcomes in an effort to understand the behavioral ecology of those relationships. The next chapter, written by Bonneau and Hunzeker, examines another latent viral infection (herpes simplex virus, HSV) that is associated with stress-induced reactivation. A review of HSV biology is followed by studies that probe the relationship between psychological stress and the immune response to, and pathogenesis of, an HSV

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infection. Studies are included that examine the stress-immune-HSV relationship at both the cellular and molecular levels. Chapter 50 concludes with a discussion of “. . . how stress-induced modulation of immunity alters the pathogenesis of HSV infection and possibly plays a role in current and yet-to-be-developed strategies to prevent HSV infection. . . .” The next chapter in this Part, by Bailey et al., delineates the influence of experimental laboratory stressors on the immune response to viral infection. The authors focus attention on the immune response to influenza virus starting with innate immunity and continuing through the development of immunological memory. In sum, Chapter 51 outlines the delicate balance that occurs between nervous, endocrine, and immune system functioning during stressful and quiescent periods. They suggest that if that balance is disturbed by a stressor, the implications for the health of the host can be dire. The final chapter in this Part of Psychoneuroimmunology, Chapter 52, looks at how stress-induced neuroendocrine interactions influence the immune system during a viral infection that leads to an autoimmune disease. Meagher et al., address the effects of stress on the pathophysiology of a Theiler’s virus infection of the central nervous system. Using this experimental model for multiple sclerosis (MS), the authors review the basic pathology of the disease and present evidence supporting a role for viral infection in the etiology of MS. Meagher and colleagues conclude with a review of the extant literature and focus on the influences of stress on the development of MS, paying particular regard to anti-viral immune responses in the development of disease. The chapters in this Part are not intended to be an exhaustive review of the influence of psychoneuroimmune interactions on the pathophysiology of disease. These chapters were solicited to provide a broad overview of PNI and disease. The goal is to provide an idea of where the field is and where work needs to be done to define the mechanisms linking social and behavioral processes to peripheral physiological responses.

References Cannon, W. B. (1939). The wisdom of the body (2nd ed.). New York: WW Norton and Company. Matzinger, P. (1994). Tolerance, danger, and the extended family. Annu. Rev. Immunol., 12, 991–1045. Steptoe, A., and Brydon, L. (2005). Psychoneuroimmunology and coronary heart disease. In K. Vedhara and M. R. Irwin (Eds.), Human psychoneuroimmunology (pp. 107–135). Oxford: Oxford University Press.

C H A P T E R

43 Psychoneuroimmunological Pathways Involved in Acute Coronary Syndromes WILLEM J. KOP, AND NICHOLAS COHEN

I. IMMUNE SYSTEM INVOLVEMENT IN CORONARY DISEASE PROGRESSION 921 II. PSYCHOLOGICAL RISK FACTORS FOR CORONARY ARTERY DISEASE 928 III. CONCLUSIONS AND FUTURE DIRECTIONS 936

be followed by a summary of research on psychological CAD risk factors and potential neuroimmunological pathways by which these psychological factors may promote CAD progression (see “Psychological Risk Factors for Coronary Artery Disease”). This chapter concludes with possible clinical implications and directions for future research.

Inflammatory processes play an important role in the progression of coronary atherosclerosis (Danesh et al., 1997; Fahdi et al., 2003; Hansson, 2005; Libby and Theroux, 2005; Ross, 1999). Evidence also suggests that both chronic and acute psychological factors increase the risk of coronary artery disease (CAD) (Kop, 1999; Krantz et al., 1996; Rozanski et al., 1999; Suls and Bunde, 2005). These psychological risk factors can be categorized as chronic personality traits (e.g., hostility), episodic factors (e.g., depression and exhaustion), and acute psychological triggers (e.g., anger). Of direct relevance to psychoneuroimmunology are the results of an increasing number of studies suggesting that the immune system plays an important role in the relationship between these psychological risk factors and future coronary syndromes (Fricchione et al., 1996; Kop, 1994; Kop, 2003; Kop and Cohen, 2001). This review describes potential interactions of psychological factors and immune system mediators in the pathophysiology of CAD progression. We will distinguish between CAD and its clinical manifestations as acute coronary syndromes, namely myocardial infarction and sudden cardiac death. A brief overview of immune system involvement in CAD (see “Immune System Involvement in Coronary Disease Progression”) will PSYCHONEUROIMMUNOLOGY, 4E VOLUME II

I. IMMUNE SYSTEM INVOLVEMENT IN CORONARY DISEASE PROGRESSION Risk for acute coronary syndromes is based on plaque, blood, and myocardial vulnerability markers (Naghavi et al., 2003a). These vulnerability markers are part of the atherosclerotic disease process and are influenced by the immune system. Immune system parameters also affect gradual CAD progression indirectly by their association with known CAD risk factors (e.g., hypertension, smoking, dyslipidemia, obesity). Clinical manifestations of CAD may be symptomatic without damage to the myocardium (i.e., angina pectoris and its equivalents). In addition, clinical manifestations of CAD may present as acute coronary syndromes such as myocardial infarction (MI) and sudden cardiac death (Alonzo et al., 1975; Braunwald, 1988; Naghavi et al., 2003a; Naghavi et al., 2003b). In most instances, acute coronary syndromes occur in the presence of underlying coronary atherosclerotic disease. In this section, we will first describe the characteristics of vascular injury in stages of CAD progression and then discuss the association between the

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severity of atherosclerotic disease and the onset of acute coronary syndromes (see “Stages of Coronary Atherosclerosis and the Onset of Acute Coronary Syndromes”). The atherosclerotic consequences of inflammation and infections will then be reviewed (see “Immunological Processes in Coronary Disease” and “Bacterial and Viral Infection”).

A. Stages of Coronary Atherosclerosis and the Onset of Acute Coronary Syndromes Given that atherosclerosis causes damage to the coronary vessel wall, the gradual progression of atherosclerotic plaques can be viewed as a “response to injury” (Ross, 1986; Ross, 1999). Several pathophysiological classifications of coronary vascular injury have been formulated based on the extent of damage to the arterial wall (Falk et al., 1995; Fuster et al., 1992a; Fuster et al., 1992b). The severity of coronary disease will be used here to clarify effects of immunological and psychosocial factors on CAD progression and its clinical manifestations as acute coronary syndromes. The initial stages of coronary atherosclerosis, characterized by fatty streaks, are associated with functional alterations of the endothelium (the lining cells of the vessel wall) without substantial morphological changes in the vessel. Mild endothelial damage is promoted by several factors including hypercholesterolemia, circulating vasoactive amines, chemical irritants such as tobacco smoke, and inflammatory processes (Fuster et al., 1992a; Fuster et al., 1992b). These early lesions promote the accumulation of lipids, macrophages, and T lymphocytes in the vascular wall (Hansson, 2005; Ross, 1999). Fatty streaks are never associated with cardiac symptoms and may either disappear or develop into atheromata (i.e., atherosclerotic lesions) (Hansson, 2005). Platelets, endothelial cells, and macrophages may secrete several growth factors that initiate migration of smooth muscle cells to other layers of the vessel wall and proliferation (growth) of these cells. Endothelial dysfunction and smooth muscle cell proliferation may result in lesions of more inward layers of the coronary vascular wall. When atherosclerosis progresses to established atheromata, vessel damage involves the endothelium and the intima. These coronary lesions are characterized by fibrin deposits in the intima and/or the development of lesions composed of lipid-laden phagocytes (i.e., foam cells). LDL cholesterol modification by oxidation results in release of phospholipids that can activate endothelial cells. Platelets are the first cells that respond to activated endothelium and may further increase endothelial activation by their glycoproteins (Ib and

IIb/IIIa). The lipid-laden lesion is then coated by a capsule-like fibrous layer of smooth muscle cells and a matrix primarily consisting of collagen. The atherosclerotic lesion is also infiltrated by T-cells, macrophages, and mast cells, particularly at sites where the atheroma grows. These activated immune cells produce inflammatory cytokines. As a result of compensatory vessel dilatation (remodeling) and an inward growth of the lesion, the arterial luminal narrowing is generally minimal at this stage. However, because of its thin layer, lipid-laden lesions can easily rupture, causing blood-clot formation and development of severe vascular lesions. Atherosclerotic plaques at the advanced stage of CAD (formerly referred to as Type III lesions) are characterized by severe damage to all three layers of the vessel wall, including the elastic lamina. Platelets may activate several growth factors that cause the smooth muscle cells to proliferate and migrate from the medial vascular layer to the intima. Following the formation of the initial platelet clot, an extracellular matrix may develop which includes fibrotic organization of the clot. Platelet adhesion and aggregation, as well as coagulation and fibrinolytic processes, are involved in the formation and stabilization of the blood clot (thrombus). The nature and speed of the processes causing progression from moderate to severe lesions are mediated by immunological processes and are associated with major clinical consequences, including unstable anginal complaints and acute coronary syndromes. Anginal symptoms such as chest pain and shortness of breath are indicative of CAD. Such symptoms occur when coronary artery disease has progressed to become flow-limiting (i.e., >50% stenosis of luminal diameter). The ischemia caused by increased cardiac demand in the setting of reduced coronary supply accounts for the symptoms of chest pain and other angina equivalents. Acute coronary syndromes are often the first clinical manifestations of CAD and occur in vulnerable individuals with advanced lesions (Fuster et al., 1992a; Muller et al., 1994). Acute coronary syndromes commonly occur in the setting of relatively stable but nonobstructive (

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