Allergy and Allergic Diseases, 2 Volume Set

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Allergy and Allergic Diseases

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

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To our wives: Rosemary, Lee, Hélène and Barbara

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

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Allergy and Allergic Diseases Editor-in-Chief

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

Editors

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

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

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

SECOND EDITION

FOREWORD BY K. FRANK AUSTEN, MD

In two volumes Volume 1

A John Wiley and Sons, Ltd., Publication

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

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Contents

List of Contributors, ix

Part 2 Inflammatory Cells and Mediators

Foreword, xvii

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

Preface, xviii

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

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

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

41 Airway Smooth Muscle, 874 Stuart J. Hirst

Part 3 Pharmacology

Part 5 Allergens

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

42 Biochemistry of Allergens and Recombinant Allergens, 895 Rudolf Valenta

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

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

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

Part 6 Animal Models of Asthma

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

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

40 Biology of Vascular Permeability, 857 Peter Clark

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

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

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

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

Index

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

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

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

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

Part 10 Asthma and its Treatment

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

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

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

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

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

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

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

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

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Contents

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

Part 12 Allergy and the Skin

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

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

Part 14 Prevention of Allergic Disease

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

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

90 Urticaria and Angioedema, 1853 Allen P. Kaplan

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

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

Index

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

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

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List of Contributors

Cezmi A. Akdis

Tony R. Bai

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

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

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

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

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

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

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

Andrew J. Beavil

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

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

Pedro C. Avila

Rebecca L. Beavil

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

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

Mark V. Avdalovic

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

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

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

Bengt Björkstén The National Institute of Environmental Medicine/IMM

Division of Physiology Karolinska Institutet Stockholm, Sweden

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

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

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

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

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

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

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

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List of Contributors

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

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

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

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

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

Glenn Cruse

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

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

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

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

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

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

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

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

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

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

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

Anuk M. Das Immunobiology Centocor Radnor, Pennsylvania, USA

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

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

Alexander C. Drew Andrew S. Cowburn

Edwin R. Chilvers

Peter Clark

Pascal Demoly

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

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

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

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

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

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

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

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

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

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

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List of Contributors

David J. Fear

Arturo Genovese

Trevor T. Hansel

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

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

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

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

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

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

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

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

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

Howard Fox Novartis Horsham Research Centre Horsham West Sussex, UK

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

Cathryn C. Hassett

William G. Glass Immunobiology Centocor Radnor, Pennsylvania, USA

Catherine Hawrylowicz

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

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

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

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

Nicholas J. Gross

C. Garren Hester

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

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

Avinash Gurbaxani

Stuart J. Hirst

Moorfields Eye Hospital London, UK

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

Belinda J. Hales Michael M. Frank

Institute of Environment and Health Cranfield University Cranfield, UK

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

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

Paul Harrison

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

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

Arthur N. Freed

Qutayba Hamid

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

Meakins-Christie Laboratories McGill University Montreal, Quebec, Canada

Anthony J. Frew

Hamida Hammad

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

Postdoctoral Fellow University of Ghent Laboratory of Immunoregulation Ghent, Belgium

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

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

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List of Contributors

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

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

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

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

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

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

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

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

Royal Brompton Hospital London, UK

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

John Lacey Deceased

Paige Lacy

Department of Respiratory Medicine Ghent University Hospital Ghent, Belgium

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

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

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

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

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

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

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

Onn Min Kon

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

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

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

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

Guy F. Joos

Debbie L. Jarvis

Peter K. Jeffery

Kevin Kwong

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

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

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

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

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

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

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

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

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List of Contributors

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

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

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

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

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

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

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

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

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

University of California Davis, California, USA

St George’s Hospital London, UK

Miriam F. Moffatt

Robyn E. O’Hehir

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

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

Rafael I. Monsalve

Thomas G. O’Riordan

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

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

Redwan Moqbel

David I. Orton

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

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

Ulrich R. Müller

Reinhard Pabst

Allergy Division Medical Department Spital Bern Ziegler Bern, Switzerland

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

Michelle J. Muessel

Giovanni Passalacqua

Research Associate Glenfield Hospital Department of Respiratory Medicine Leicester, UK

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

Lynne A. Murray

Ian D. Pavord

Manager of Pharmacology Promedior Inc Malvern, Pennsylvania, USA

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

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

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

Dean D. Metcalfe

Wolfgang A. Nockher

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

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

Lisa A. Miller

Yee-Ean Ong

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

Consultant Physician and Honorary Senior Lecturer

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

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

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

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List of Contributors

Emily M. Pieczarka

Jamie A. Robyn

Edward S. Schelegle

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

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

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

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

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

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

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

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

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

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

Anna Pomés INDOOR Biotechnologies Inc. Charlottesville, Virginia, USA

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Rebecca Slack

Wayne R. Thomas

Laura S. Van Winkle

Institute of Environment and Health Cranfield University Cranfield, UK

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

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

Catherine Thornton

Ulrich Wahn

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

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

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

Suzette M. Smiley-Jewel

Stephen J. Till

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

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

Sanchaita Sonar

Isabelle Tillie-Leblond

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

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

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

Taniawati Supali Department of Parasitology University of Indonesia Jakarta, Indonesia

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

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

Department of Pneumology and Immunoallergology University Hospital Lille, France

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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List of Contributors

Reen Wu

Maria Yazdanbakhsh

Michael C. Zacharisen

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

Department of Parasitology Leiden University Medical Center Leiden, The Netherlands

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

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Foreword

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

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

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Preface

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

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

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

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

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

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Immunology of the Allergic Response

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

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Allergy and Hypersensitivity: History and Concepts A. Barry Kay

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Allergy and Hypersensitivity: History and Concepts

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

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

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

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

From reagin to IgE

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

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

6

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

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

(a)

Allergy and Hypersensitivity: History and Concepts

(b)

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

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

Mediator cells and mediators

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

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

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

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

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

8

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

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

(a)

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

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

Allergy and Hypersensitivity: History and Concepts

(b)

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

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

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(b)

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

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

10

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

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

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

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

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

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

Allergy and Hypersensitivity: History and Concepts

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

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

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

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

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

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

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

(a)

12

(b)

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

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

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

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

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

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

(a) (b)

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

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

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

14

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

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

Shen Nung

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

2698 BC 2641 BC

Huang Ti Menes

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

460–365 BC

Hippocrates

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

25 BC–AD 40

Aulus Celsus

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

AD

c. 40

Marcus Terrentius Varro

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

AD

b.41

Britannicus

Reported to be afflicted by acute allergic reactions to horses

AD

c. 60

Pedanius

Remedies for asthma in classical pharmacology treatise

Aretaeus of Cappadocia

First detailed description and coining of the word asthma

120–AD 180 AD

b.131

Galen

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

AD

b.865

Rhazes

Description of seasonal catarrh due to roses in Persia

1135–1204

Moses Maimonides

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

b.1306

John of Arderne

Prescription of a “syrup” for asthma

1530

Thomas Moore

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

1552

Girolamo Cardano

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

1565

Leonardhus Botallus

Description of “rose cold” in Pavia

1570

Pietro Mattioli

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

c. 1584

Johann Schenk

Coins the term “idiosyncrasy”

b.1603

Kenelm Digby

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

1603

Felix Platter

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

c. 1630

Sanctorius

Description of asthma to cat hair

1656

Pierre Borel

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

1662

K.V. Schneider

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

1665

Philipp Jacob Sachs

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

1673

Johannes Binneringus

Description of seasonal rose coryza in Basle

1675

Theophile Bonet

Idiosyncrasies to bread, strawberries and wine

1680

Thomas Willis

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

c. 1680

Nehemiah Green

First microscopic studies of pollen grains

1682

Joan van Helmont

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

1691

Jacob de Rebecque

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

1698

John Floyer

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

1713

Bernardino Ramazzini

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

1765

Debrest

Description of sudden death by bee sting in Montpellier

1775

William Cullen

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

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Table 1.1 (Cont’d ) 1776

Johann Murray

Ipecac root (emetine), causes asthma attacks in pharmacists

1778

Stolpertus

Description of acute angioneurotic oedema following ingestion of eggs

1783

Friedrich Schademantal

Allergic urticaria due to ingestion of fresh pork meat

1783

Philipp Phoebus

Comprehensive monograph on hayfever. First epidemiological enquiry

1802

William Heberden

Description of summer catarrh and asthma; differentiation from common cold

1816

Henri Laennec

Invents stethoscope. Identifies bronchospasm as important component of asthma

1819

John Bostock

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

1839

François Magendie

Description of sudden death of dogs repeatedly injected with egg albumin

1853 1886

Jean Martin Charcot and Ernst van Leyden

Description of Charcot–Leyden crystals in sputum of asthmatics

1868

Henry Hide Salter um

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

1872

Heinrich Quincke

First description of angioneurotic oedema

1872

Morrill Wyman

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

1873–80

Charles Blackley

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

1877

Paul Ehrlich

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

1894

Samuel Flexner

Experimental “toxic death” whilst injecting dog serum into rabbits

1895

Josef Jadassohn

Establishment of patch tests in contact dermatitis

1895

Josef Jadassohn

Description of various types of drug reactions in the skin

1900

Solomon Solis-Cohen

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

1902

Charles Richet Paul Portier

Discovery of experimental anaphylaxis in dogs

1903

Theobald Smith

Observations of anaphylactic reactions of guinea pigs to horse serum

1903

Maurice Arthus

Experimental localized acute necrotizing vasculitis, first described as local anaphylaxis

1905

Bela Schick

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

1905

Clemens von Pirquet

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

1906

A. Wolff-Eisner

Relationship of human hayfever and urticaria to experimental anaphylaxis

1909

William Schultz

Detection of anaphylaxis by contraction of isolated smooth muscle in vitro

1909

William B. Osler

Asthma associated with neurotic disease

1910

S. Meltzer

Bronchial asthma as a phenomenon of anaphylaxis

1910

William Dunbar

Methodology for pollen extraction; first approaches to pollen immunotherapy

1911

Leonard Noon John Freeman

Wide use of immunotherapy with pollen extracts in hayfever patients

1911

Henry Dale

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

1911

Tomaso Casoni

Skin test in patient infected with Echinococcus

1912

Oscar M. Schloss

Use of scratch test in allergy to foods

1913

William Dunbar

Methodology for pollen extraction, identification of allergenic protein

1914

A.T. Waterhouse

Anaphylactic reactions of beekeepers to stings

16

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Table 1.1 (Cont’d ) 1915

Warfield T. Longcope

Experimental lesions in organs from animals repeatedly injected with foreign proteins

1916

Robert A. Cooke

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

1918

Francis Rackemann

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

1919

M.A. Ramirez

Horse dander asthma following blood transfusion

1921

Carl Prausnitz Heinz Küstner

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

1921

Arent de Besche

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

1922

Fernand Widal Pierre Abrami J. Lermoyez

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

1923

Arthur Coca

Reagent for allergen extraction

1923

Arthur Coca

Coined the term “atopy”

1924

F.T. Codham

First description of mould allergy

1924

Ko Kuei Shen Carl F. Schmidt

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

1927

Thomas Lewis

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

1927

Bret Ratner

Experiments on allergic sensitization in utero

1928

Storm van Leeuwen

Inhalation allergy to house dust

1928

Edward Dienes

Induction of cellular delayed hypersensitivity by protein antigens

1934

Mataso Masugi

Experimental glomerulonephritis with anti-kidney antiserum

1935

Ulf von Euler

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

1937

Daniel Bovet

First synthesis of antihistaminic drugs

1939

Harry H. Donally

Transmission of food allergens in breast milk

1940

Charles H. Kellaway Everton T. Trethewie

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

1940

Mary Loveless

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

1941

Louis B. Jaques

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

1941

Joseph Harkavy

Bronchial asthma with recurrent pulmonary eosinophilic infiltration and polyserositis

1942

Merrill W. Chase

Transfer of tuberculin sensitivity by cells from immunized animals

1945

Merrill W. Chase

Transfer of contact dermatitis to simple chemicals by sensitized leukocytes

1945

Robin A. Coombs

Described the antiglobulin (“Coombs”) test

1949

Philip S. Hench Edward C. Kendall

Isolation of cortisone from adrenals for therapy of rheumatoid arthritis

1952

Zoltan Ovary

Development of passive cutaneous anaphylaxis (PCA) for quantification

1953

James F. Riley Geoffrey B. West

Mast cell granules as major source of histamine in tissues

1954

William Frankland Rosa Augustin

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

Continued p. 18

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Table 1.1 (Cont’d ) 1957

Ernest Witebsky

Experimental autoallergic thyroiditis: pathology and criteria of immune diseases

1958–60

Zoltan Ovary Herman N. Eisen

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

1960

Bernard. B. Levine Alain L. de Weck

Identification of major and minor antigenic determinants in penicillin allergy

1962

Alain L. de Weck Charles W. Parker

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

1963

Robin A. Coombs Philip Gell

Classification of the hypersensitivity reactions

1963

K. Frank Austen

Biochemistry of mast cell mediator release

1963

Jack Pepys

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

1964

Lawrence M. Lichtenstein Abraham G. Osler

Development of allergen-specific histamine release test

1964

T.E. King

Identification of major allergen in ragweed

1966

Barry R. Bloom John R. David

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

1966

David Marsh

Identification of major grass allergens

1966

J.J. Curry

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

1967

Kimishige Ishizaka Teruko Ishizaka

Characterization of reagins as IgE immunoglobulins

1967

Hans Bennich Gunnar Johansson

Identification of myeloma ND as IgE immunoglobulin

1967

L. Wide Gunnar Johannsson

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

1967

Reindert Voorhorst Frederick Spieksma

Identification of Dermatophagoides mites as major allergen source in house dust

1967

Bernard Halpern

Lymphocyte stimulation test in drug allergy

1967

Roger Altounyan

Discovery of sodium cromoglycate as anti-asthmatic drug

1981

K. Frank Austen

Biological properties of leukotrienes

1983

Bengt I. Samuelson

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

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

18

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

Soluble

Antigen

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

b

Antagonist

Neutrophil-rich inflammatory response

APC

MHC Class II

TCR

Macrophage-rich inflammatory response

Type 1 cytokines

AF

CD4+ type 1

T Lymphocyte

Soluble

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

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

Agonist

IgG

Microvasculature

Soluble

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

• • • •

MHC Class II

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

Eosinophil- and basophil-rich inflammatory response

Type 2 cytokines

APC

CD4+ type 2

T Lymphocyte

Soluble

MHC Class I

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

Cytotoxicity (apoptosis)

Target cell

CD8+ cytotoxic

T Lymphocyte

Cell-associated

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

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

Tissue injury by cytotoxic T lymphocytes

Th2

Th1

Type IV cytotoxic

Type IV

Type IV

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

Type III

Receptor Antigen-antibody complexes + ligand complement

Target cell Receptor

Cell-associated

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

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

Type II

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

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

Complement lysis or removal by the RE system

+/– complement

IgG

Cell surface antigen

Target cell

Cell-associated

IgG antibody interacting with cell surface antigen

Cytolytic, or cytotoxic, reactions

a

CHAPTER 1

Examples in humans

Allergen

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

IgE

Mast cell/basophil

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

Initiating event

Simplified scheme of the proposed mechanism

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

Descriptive term

Type I

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

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

20

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

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

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

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

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

Allergy and Hypersensitivity: History and Concepts

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

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

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

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

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

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

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

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

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

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

ia ter

ve

iti pos

Gram negative bacteria (including endotoxin) RSV F protein

?

bac

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

Flagellated bacteria

m Gra gi u F n

TLR10

TLR4

TLR6

TLR5

TLR2

Plasma membrane

TLR1 Viral ssRNA Viral/bacterial DNA Viral dsRNA

TLR7 TLR8 TLR3

24

TLR9

Intracellular vesicle membrane

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

OLD MODEL Th1

NEW MODEL Th2

Th1

Th2

Treg

Dendritic cell

Dendritic cell

Microbial stimulation

Microbial stimulation

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

ENDOTOXIN

Dendritic cell

Th1

Dendritic cell

Allergen

Th2

Th1

Allergen

Th2 Allergic inflammation Tissue remodeling

No allergic disease

Epithelium

ENDOTOXIN Allergic disease exacerbation

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

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

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

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

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

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

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

Development of Allergy and Atopy

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

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

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

mCD14+ cells

Membrane CD14-negative cells

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

Signal

LPS

LBP

CD14

TLR4

MD2

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

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ALLERGIC DISEASE IgE sensitization

Environment

Tissue-specific factors

Development

Genetics

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

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

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

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T Cells and Cytokines in Asthma and Allergic Inflammation Chris Corrigan

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

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

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

Immature T cell

Thymic cortical epithelium Primary positive selection

CD4+

CD4+

Intermediate anti-self affinity

High anti-self affinity

HC

TSLPactivated DC

TSLP

Secondary positive selection

AIRE

Non-self reactive T cells eliminated

CD4+

Immature DC

Thymic medullary epithelium CD4+

CD4+ CD25+ FoxP3+

Low-affinity anti-self T cells

Negative selection

Naive T cells in the periphery

Death

Treg cell in the periphery

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

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

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

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Th2

Th1

Signature cytokines

IFN-g

IL-4, IL-5, IL-13

IL-12 receptor b2-chain

+++

+/−

Chemokine receptors

CXCR3 CCR5

CCR3 CCR4 CXCR4 CCR8

Chandra protein

+

−

Costimulatory molecule ICOS

−

+

CRTH2

−

+

TIM (mice)

TIM-3

TIM-2

Signaling

STAT4 T-bet

STAT6 GATA3 c-Maf

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

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

Table 3.1 The IL-17 cytokine family. Name

Alternative names

Cellular sources

Receptor

IL-17A

IL-17, CTLA-8

IL-17RA (IL-17R)

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

CXI, NERF CX2 IL-27 IL-25

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

IL-17F

ML-1

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

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

52

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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Table 3.2 T-cell costimulatory molecules and their receptors.

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

Naive T cells

Activated Th2 cells

Memory T cells

Other cells*

+++ ++ +++ +++ − − − − + − −

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

+++ +++ +++ ++ + − − − − − −

Yes Yes Yes Yes No No Yes Yes No No No

Counterreceptor(s)

Action

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

+ + +? − + + + + + − −?

CD80, CD86 PD-L1, PD-L2

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

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

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

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

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

T Cells and Cytokines in Asthma and Allergic Inflammation

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

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

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

56

age-old question of why only some subjects sensitized to allergens develop allergic diseases such as asthma and rhinitis in the target organs. Using CD1d tetramers loaded with α-GalCer and PCR analysis of the expression of the invariant TCR of NKT cells, it has recently been suggested that, in human asthma, approximately 60% of airway CD4+ cells are not conventional MHC class II restricted T cells, as has hitherto been assumed, but NKT cells expressing the invariant Vα24/Jα18 receptor (Akbari et al. 2006). These cells exhibited a cytokine expression profile similar to that of conventional Th2 cells. This finding was striking considering that NKT cells comprise <1% of the CD4+ population in the peripheral blood of humans. These studies suggest that CD4+ NKT cells are selectively recruited to the asthmatic airways, possibly through CCR9 expression (Sen et al. 2005). At present these studies are contentious (Mutalithas et al. 2007; Vijayanand et al. 2007) but if verified by scientific consensus may force a shift in our current understanding of the relative contributions of conventional CD4+ Th2 cells and NKT cells to the production of IL-4 and IL-13 in asthma and of the contribution of these cells to asthma pathogenesis in general. It is unclear what could be activating NKT cells in this situation, but glycolipids indigenous to the mucosal environment are obvious theoretical candidates. In addition, human NKT cells have been shown to be activated by exogenous lipids such as those found in certain tree pollens (Agea et al. 2005). Finally, it is of interest that NKT cells are relatively resistant to glucocorticoid inhibition (Tamada et al. 1998), which could, at least in theory, form one explanation for clinical glucocorticoid refractoriness in asthma (assuming that NKT cells are involved).

T-cell memory The adaptive immune system is characterized by the presence of immunologic memory, which augments or at least maintains subsequent immune responses to encounters with particular antigens, including allergens, following initial exposure. Expression of the CD45RO isotype on memory T cells distinguishes them from their naive counterparts that express CD45RA. Memory CD4+ T cells have been further classified into two distinct types based on the presence or absence of expression of the chemokine receptor CCR7. Central memory T cells (TCM) expressing CCR7 preferentially migrate to secondary lymphoid tissues, and proliferate on antigen encounter, giving rise to a progeny of effector T cells. In contrast, effector memory T cells (TEM), which lack CCR7, traffic directly to inflamed tissue and respond rapidly on activation, thereby exerting their effector functions in the tissues. Thus, memory cells have been defined as CD45RO+CCR7+CD62L+ (TCM) and CD45RO+CCR7−CD62L− (TEM) (Fig. 3.3) (Sallusto et al. 2004). TCM cells are maintained long term in the immune system by the cytokines IL-7 and IL-15, which induce “homeostatic proliferation” of these cells to prevent them from disappearing (Geginat et al. 2001). On

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Pre-Th1 TCM

CXCR3

Pre-Th2 TCM

CCR4

Th2 TCM

CRTH2

Th1 TEM

CXCR3

Th2 TEM

CCR4

Th2 TEM

CRTH2

CCR7

TCM CD62L +

Fig. 3.3 Phenotypes of human CD4 memory T-cell subsets. Th2 TEM cells have not been formally demonstrated (hence the question mark) but such cells have been identified at sites of ongoing Th2 inflammation and would be predicted to exhibit a TEM phenotype. The question mark by the large arrow indicates that it is controversial whether effector memory cells develop from central memory cells or de novo from naive T cells. TM, memory T cell; TCM, central memory T cell; TEM, effector memory T cell. See Rivino et al. (2004), Sallusto et al. (2004) and Wang et al. (2006).

?

Antigen reexposure

TM CD45RO

reencounter with antigen, they can differentiate into effectors (TEM). Distinct patterns of chemokine receptor expression are also said to distinguish pre-Th1 and pre-Th2 TCM cells. CXCR3-bearing cells differentiate into Th1 effectors, whereas CCR4-expressing cells differentiate into Th2 effectors (Rivino et al. 2004), although this distinction is not absolute since reports suggest that CCR4 can be expressed by both pre-Th1 and pre-Th2 TCM cells, as well as T regulatory cells (Andrew et al. 2001; Iellem et al. 2001). In addition, selective expression of a novel seven-transmembrane G protein-coupled receptor called CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) has been described on a subpopulation of memory cells committed to the production of IL-4, IL-5 and IL-13 but not IFN-γ following TCR triggering (Nagata et al. 1999; Hirai et al. 2001), suggesting that these cells are committed memory Th2 cells. Very recent studies (Wang et al. 2006) show that CRTH2+CD4+ cells from the blood of normal human donors exhibit a central memory phenotype and respond to allergens in vitro. In this particular study, priming of dendritic cells with the IL-7-like cytokine TSLP appeared to be necessary for generating allergen-specific recall responses in CRTH2+CD4+ T cells, although it is not clear whether this is an obligatory activation pathway for all Th2 committed memory T cells since, in vitro, T-cell recall responses are regularly generated in response to allergen in the absence of exogenous TSLP. TSLP-activated dendritic cells can maintain CRTH2+CD4+ T cells in vitro, without altering their central memory phenotype or Th2 commitment (Watanabe et al. 2004). This is one possible mechanism whereby allergenspecific Th2 memory cells may persist in vivo for many years. TSLP is emerging as an important mediator of T-cell

TEM

?

responses to allergens, since it not only maintains Th2 committed memory T cells but also strongly activates dendritic cells which then prime naive T cells to differentiate into Th2 cells (Fig. 3.4). Furthermore, TSLP expression has been described, as mentioned above, in lesional skin keratinocytes of patients with atopic dermatitis as well as the bronchial mucosa of asthmatics (Soumelis et al. 2002; Ying et al. 2005). The presence of TSLP at the critical interface between the mucosal surface and the environment raises important questions for the future concerning how TSLP expression is regulated by environmental influences such as allergen exposure and infections. Despite this augmentation of knowledge about the physiology of T-cell memory in recent years, many fundamental questions regarding the maintenance of T-cell memory remain. Whether memory T cells arise from effector T cells, or naive cells, or both has long been debated and is currently uncertain. How long it takes allergen-specific memory Th2 cells to develop is also unclear. Furthermore, it is not clear whether maintenance of the memory T-cell repertoire against a particular antigen depends on, or is shaped by, chronic exposure. It is clear that antigen-specific T cells persist for at least many years in the peripheral blood of humans (Bohle et al. 1998). The mechanisms discussed above for the maintenance of TCM cells are likely important in maintaining immunologic memory, and it may well be the case that continued exposure to antigen is not necessary to maintain it. It is conceivable, for example, that environmental influences maintaining TSLP expression at mucosal surfaces may preserve antigen-specific TCM cells without the necessity for the persistence of the antigen. The studies described above

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Immunology of the Allergic Response Allergen Mucosal surface DC

TSLP

Other sources?

TSLP DC

MHC/peptide/TCR DC

OX40/OX40L

Effector Th2

Lymph node

Th2 CRTH2

Th2 TEM CRTH2

Effector memory Th2

Naïve T cell

Trafficking to target organ

Th

Central memory Th2

Th2 TCM CRTH2

Inflammatory Th2 Th2 CRTH2

suggest that interaction between the costimulatory molecule OX40 expressed on memory T cells and its ligand OX40L expressed on dendritic cells is important not only for the persistence of TCM cells but also recall responses to allergens (Soumelis et al. 2002; Wang et al. 2006). Therapeutic strategies directed against the formation or persistence of TCM cells may constitute an important (and largely neglected) strategy in atopic diseases involving long-term sensitization to allergens (Epstein 2005). Equally important is the prospect of directing memory T-cell responses in early life, which is a corollary of the hygiene hypothesis, but much more work must be done to clarify the cellular targets of environmental factors such as endotoxin, whether the timing of exposure to such factors is critical, and whether redirection of allergen-specific Th2 type immunologic memory by agents such as endotoxin requires continued allergen exposure. The effects of T regulatory cells on immunologic memory have not yet begun to be explored.

T regulatory cells Although Th1 and Th2 cells are mutually inhibitory, it has long been apparent that simple, mutually antagonistic interactions between these cells cannot explain the regulation of Th2 activity in humans. For example, in animal models of asthma, allergen-specific Th1 cells have in some cases been reported to augment allergic inflammation (Hansen et al. 1999). Helminthic infections, which promote Th2 responses, do not appear to be associated with increased risk of allergy and asthma and on the contrary in some studies appear to be protective (Yazdanbakhsh et al. 2002). Finally, epidemiologic data in the last 40 years have shown a parallel rise in the prevalence of “Th1-type” diseases such as diabetes and “Th2type” diseases such as asthma (Sheikh et al. 2003), an observation that, while not necessarily reflecting direct mutual Th1/Th2 antagonism, certainly does not support it. It is in this setting that T regulatory cells have come to the fore as being capable of suppressing Th1 and Th2 cells independently. Environmental influences altering the function of T

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Fig. 3.4 Dendritic cells (DC) activated by thymic stromal lymphopoietin (TSLP) produced at mucosal surfaces migrate to lymph nodes and induce Th2 inflammatory cells while maintaining Th2 central memory cells. At the mucosal surface, DC activate allergen-specific effector/memory T cells in situ. OX40/OX40L interaction is necessary for both these processes. TCR, T-cell receptor.

regulatory cells seem a more likely candidate to explain the aforementioned data, rather than mutual antagonism of Th1 and Th2 responses within individuals. T regulatory cells may be “natural” (arising in the course of normal development of the immune system) or “adaptive” (arising because of immune encounters). As described previously, natural T regulatory cells arise largely to promote tolerance to self antigens. They constitute 5–10% of circulating CD4+ T cells (Fehervari & Sakaguchi 2004), and there is some evidence that they recognize self antigens but with broad specificity (Hsieh et al. 2004). They are characterized by high expression of CD25 (which confounds their identification since this is also an activation marker of effector T cells). They proliferate poorly in vitro in response to conventional T-cell activation stimuli and appear to inhibit adaptive T-cell responses through contact with T cells when they synapse with dendritic cells during activation. The precise mechanisms are unclear, but signaling through surface molecules such as CTLA-4, Notch 3, and LAG-3 has been implicated. Ligation of CTLA-4 on dendritic cells induces the enzyme indoleamine 2,3-dioxygenase (IDO), which starves T cells of tryptophan (Mellor & Munn 2004). In addition to CD25, natural T regulatory cells express CTLA-4, CD45RO, CD38, CD62L, the glucocorticoid-induced TNF receptor gene GITR, and neuropilin-1, although none of these markers is specific. As mentioned above, the transcriptional regulator FoxP3 is overexpressed in natural T regulatory cells as compared with resting and conventionally activated T cells, although again this is troublesome as an identification marker for these cells since FoxP3 is not a surface marker, and again FoxP3 expression is not entirely limited to T regulatory cells. The precise actions of FoxP3 are not clear, but retroviral transfer of FoxP3 is sufficient to confer regulatory T-cell function (Yagi et al. 2004). Humans carrying loss-of-function mutations in FoxP3 develop the IDEX syndrome (see above). They also have an increased incidence of eczema and food allergy, suggesting that underlying defects in T regulatory cells cause these symptoms, and more importantly that efficient natural

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T regulatory cell function is important for maintaining normal immune homeostasis in health. Adaptive T regulatory cells arise generally in the periphery from naive T cells on encounter with antigens presented by tolerogenic dendritic cells. Tolerogenic dendritic cells are generally semimature cells with increased expression of MHC class II molecules and CD86, but low expression of CD40 and secretion of proinflammatory cytokines such as IL-6 and TNF-α (Lutz & Schuler 2002). Repetitive stimulation of T cells with such dendritic cells generates IL-10-producing Tr1 adaptive T regulatory cells (Jonuleit et al. 2001). Similarly, inhibition of dendritic cell maturation by inhibiting NF-κB activation with 1,25-dihydroxyvitamin D3, glucocorticoids, or mycophenolate mofetil induces development of Tr1 T regulatory cells; this may also occur directly in the presence of such drugs by activating naive CD4+ T cells in the absence of dendritic cells (Barrat et al. 2002). Again the mechanism is not completely understood, but appears to depend on secretion of IL-10 (Tr1) or TGF-β (Tr3) by immature dendritic cells. Tolerance induced to mucosally applied antigens also appears to induce tolerogenic dendritic cells producing IL-10 or TGF-β (Weiner 2001). As has been discussed, the role of T regulatory cells in controlling the development and expression of allergic diseases in asthma is only beginning to be explored (reviewed by Akdis et al. 2005). Whether these cells play an important role in sensitization to allergens in intrauterine or early life is not yet clear. In patients with established allergy, evidence of deficiency of allergen-specific natural T regulatory cells has been reported in some (Ling et al. 2004) but not all (Bellinghausen et al. 2003) studies. The former study suggested that allergen-specific T regulatory cell activity was reduced compared with nondiseased controls in subjects with allergic rhinitis during the pollen season when they have symptoms, rather an outside it, suggesting that deficient T regulatory cell activity may somehow be involved with the clinical expression of allergic disease, although these experiments do not make it clear if clinical disease is caused by reduced natural T regulatory cell activity or is a consequence of it. What these studies do show for certain is that external allergens seem to be included in the antigenic repertoire of natural T regulatory cells. A further study (Karlsson et al. 2004) showed increased numbers of CD4+CD25+ βlactoglobulin-specific T cells in the blood of children who had outgrown cows’ milk allergy compared with others who had not. The inhibitory effect of these cells appeared to depend on cell contact, implying a role for natural T regulatory cells. There are similar data suggesting deficiency of allergen-specific IL-10-producing Tr1 adaptive T regulatory cells in patients who develop sensitization and symptoms in response to aeroallergens compared with those who do not (Akdis et al. 2004). Therapeutic intervention also appears to be able to regulate T regulatory cell activity. For example, inhaled or systemic

T Cells and Cytokines in Asthma and Allergic Inflammation

glucocorticoid therapy of asthmatics was associated with elevated expression of both FoxP3 and IL-10 mRNA in peripheral blood CD4+ T cells (Karagiannidis et al. 2004), suggesting the induction of T regulatory cells, although the precise phenotype of these cells (natural or Tr1) could not be concluded from this study since it is still controversial whether the development of adaptive as opposed to natural T regulatory cells always requires expression of FoxP3 (Fehervari & Sakaguchi 2004; Yagi et al. 2004). Allergen immunotherapy may also induce adaptive, allergen-specific T regulatory cells. Studies have shown increases in allergenspecific IL-10-producing Tr1 and TGF-β-producing Tr3 type cells in the blood or airways following immunotherapy (Jutel et al. 2003; Nouri-Aria et al. 2004), although what role these cells play, if any, in mediating the clinical effects of immunotherapy (i.e., reduced responsiveness of mucosal surfaces to allergen exposure) is not clear (this is secondary to the fact that the immunologic processes underlying the clinical effects of allergen immunotherapy in general are poorly characterized). Furthermore, increased IL-10 production following allergen immunotherapy is not limited to T cells, but may also be observed in monocytes/macrophages and B cells (Akdis et al. 1998; Jutel et al. 2003). These are all potential antigen-presenting cells and could conceivably directly suppress T effector cells or contribute to the induction of adaptive T regulatory cells. Despite all these uncertainties, it would seem well worth pursuing therapeutic induction of adaptive T regulatory cells as a means of long-term, directed, and allergen-specific therapy for allergic diseases. Since allergen presentation by immature dendritic cells and production of cytokines such as IL-10 and TGF-β appear important in this respect, combination of allergen immunotherapy with administration of drugs that inhibit dendritic cell maturation and induce IL-10 production, such as 1,25-dihydroxyvitamin D3 or glucocorticoids, may be a fruitful strategy. Finally, a return to the hygiene hypothesis, which in essence suggests that lack of exposure to environmental infectious agents is one factor responsible for the increasing prevalence of allergy in the past 30 or 40 years. As originally formulated, it cannot now apply to the concept of directing a battle between Th1 and Th2 cell supremacy. Environmental influences on the development and persistence of allergic disease, if they truly exist, must now be examined in the light of their possible influences on T regulatory cells. Interestingly, pathogen-specific Tr1 adaptive T regulatory cells are induced in the course of infections with many viruses, bacteria, and parasites (reviewed by Mills 2004). In some cases this results in incomplete elimination of the pathogen, which could theoretically result in “bystander” suppression of allergic responses by both natural and adaptive T regulatory cells. Such findings may necessitate investigation of the hygiene hypothesis in the context of a much broader range of infectious agents, not only pathogens but also commensal bacteria such as the intestinal flora (Kalliomaki & Isolauri 2003).

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TIMs and SOCS The TIM family of proteins was first recognized in mice during a random search for markers that would differentiate between Th1 and Th2 cells. The protein now called TIM-3 is one of very few found to be uniquely expressed on Th1 differentiated T cells (Monney et al. 2002). Subsequent positional cloning techniques uncovered eight related TIM genes in the mouse, although only four of them, TIM1–4, are known to be expressed. Corresponding genes were identified in humans. Human TIM-1 shows homology with mouse TIM-1 and TIM-2 and may have evolved to serve the purposes of both, although this is not certain. Human TIM-3 and TIM-4 show reasonable homology with their mouse counterparts. These genes encode type 1 transmembrane proteins with an immunoglobulin variable domain, and an intracellular signaling sequence with tyrosine phosphorylation motifs (TIM-4 exceptionally lacks such sequences, supporting the suggestion, referred to below, that it may serve as a ligand for the other TIM molecules). In humans, the genes encoding the TIM proteins are on chromosome 5q33.2, not far from the IL-4/IL-5/IL-13 cluster: indeed linkage studies in mice suggest that it is this locus and not the IL-4/IL-5/IL-13 cluster that is linked most closely to susceptibility to experimental asthma (McIntire et al. 2001). Experiments with TIM-3–immunoglobulin fusion protein in mice suggest that the TIM-3 ligand is found predominantly on naive CD4+ T cells. Blockade of TIM-3 stimulation in mice with this fusion protein results in highly augmented Th1 responses (Sabatos et al. 2003). The function of its putative human homolog is as yet poorly characterized. In mice, TIM-2 is preferentially expressed on Th2 cells, whereas TIM-3 is present to an extent on all activated T cells. The use of a TIM-2–immunoglobulin fusion protein has demonstrated a ligand for TIM-2 on activated antigen-presenting cells. Administration of this fusion protein in various models of inflammation tends to augment Th2 responses. The putative human analog of TIM-1 and TIM-2 is the hepatitis A virus receptor (Feigelstock et al. 1998). Epidemiologic studies suggest that patients previously infected with hepatitis A are less likely to have asthma and allergy (Matricardi et al. 2002). In addition, TIM-1 is preferentially expressed on human Th2 cells, whereas TIM-3 is preferentially expressed on Th1 cells (Khademi et al. 2004). A polymorphism of TIM-1 resulting in a six-amino-acid insertion has been linked to the risk of hepatitis A infection and atopy in a North American population (McIntire et al. 2003). Recently, it has been reported that another member of the TIM family, TIM-4, serves as a natural ligand for TIM-1 in mice. Accordingly, TIM-4 is not expressed on T cells but instead on antigen-presenting cells, particularly mature dendritic cells (Meyers et al. 2005). In general, engagement of TIM-1 with a soluble TIM-4–immunoglobulin fusion protein produces a general increase in T-cell proliferation and cytokine production, although the effect can be inhibitory in vitro

60

at lower concentrations (Meyers et al. 2005). The mechanism of this effect has not been elucidated, although it suggests there may be more than one ligand for TIM-4 on T cells. Understanding the mechanisms by which the TIM gene family contributes to immune regulation might uncover new therapeutic opportunities. Because all the TIM molecules studied so far seem to be involved with the balance of expression of Th1- and Th2-type cytokines, they could act as useful drug targets in any system where one would hope to shift this balance, as in allergic diseases. Finally, the TIM genes are highly polymorphic in humans, and one or more of these polymorphisms may be particularly important in directing the strength of T-cell polarization in diseases including allergy and asthma. Cytokines generally signal through the Janus family kinases Jak1–3 and Tyk2, and the STAT (signal transducer and activator of transcription) proteins 1–6. Interaction of a cytokine with its receptor results in reciprocal tyrosine phosphorylation of receptor-associated Jaks and Jak-mediated tyrosine phosphorylation of a cytoplasmic domain of the receptor. The STAT molecules then dock with the latter and are themselves tyrosine phosphorylated, which allows them to dimerize and translocate to the nucleus where they act as transcriptional regulators. The SOCS (suppressor of cytokine signaling) proteins appear to act as negative regulators of this mechanism. Eight members of the SOCS family have been identified: cytokine inducible SH2-containing protein (CIS) and SOCS1–7. They are induced by activation of STATs and limit, by negative feedback, docking of STATs to activated cytokine receptors, thus turning off the initial activation signal. SOCS proteins are induced by a variety of cytokines, growth factors, and PAMPs such as lipopolysaccharide and bacterial DNA sequences acting on TLRs. SOCS1 appears to be particularly important for regulation of IFN-γ-mediated signaling. SOCS1-deficient mice die early with widespread activation of peripheral T cells, an effect that can be delayed by removal of IFN-γ-producing T and NKT cells (Alexander et al. 1999). However, SOCS1 is also capable of inhibiting Jak/STAT signaling initiated by a wide variety of cytokines other than IFN-γ, including IL-2, IL-4, IL-6, IL-7, IL-9, and IL-13 as well as the α and β interferons. SOCS1deficient T cells exhibit sustained phosphorylation of STAT6 or STAT4 when stimulated with IL-4 or IL-12 respectively, accompanied by extreme polarization toward a Th2 or Th1 phenotype (Fujimoto et al. 2002), but these cells spontaneously revert to a high IFN-γ-producing Th1 phenotype. This net effect may represent a complex balance of untamed Th1- and Th2-inducing signals from cytokines such as IFN-γ, IL-12, IL-6, and IL-4. SOCS5 is more clearly implicated in enhancing Th1 T-cell polarization, since it may limit Jak1/Jak3/STAT6 signaling induced by engagement of the IL-4 receptor α chain, which is selectively impaired in Th1 cells (Huang & Paul 1998) although the expression of Jak1, Jak3, and STAT6 is similar

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may come from in the first place before Th2 differentiation has taken place (it certainly does not come from dendritic cells). Work from Yong-Jun Liu’s group (Liu et al. 2006) suggests TSLP as a candidate for the counterpart of IL-12 in promoting Th2 responses. If ratified by scientific consensus, this finding may move TSLP to center stage as a prime molecular target in allergic diseases. The critical difference between TSLP and other dendritic cell stimuli such as CD40L and TLR ligands is that TSLP does not induce the Th1-polarizing cytokines IL-12, or members of the IL-12 family, or other proinflammatory cytokines such as IFN-8, IL-1β, and IL-6, but does induce chemokines such as TARC and MDC which may attract Th2 differentiated T cells. Further to this, when TSLP-activated dendritic cells stimulate naive CD4+ T cells in vitro, they induce a unique type of Th2 cell that produces the “signature” Th2 cytokines IL-4, IL-5 and IL-13, and large amounts of TNF-α, but not IL-10 (Soumelis et al. 2002). That TNF-α also plays a role in asthmatic inflammation has been confirmed by the therapeutic success of TNF-α blockade in this disease (see below). Conversely, IL-10, although conventionally classified as a Th2type cytokine, exerts a number of antiinflammatory actions in asthma as described below. Liu has proposed that Th2 cells producing large amounts of TNF-α, in addition to the “signature” cytokines IL-4, IL-5 and IL-13, may be regarded as inflammatory Th2 cells. Conversely, those Th2 cells producing large amounts of IL-10 but relatively little TNF-α in addition to these signature cytokines could exert a regulatory function. A similar principle may apply with Th1 cells (Fig. 3.5). As described above, interaction of OX40L expressed on TSLP-activated dendritic cells with OX40 on T cells may be a critical trigger for the differentiation of inflammatory Th2 cells. This effect is independent of IL-4 but may be overridden by IL-12, which provides a mechanism whereby environmental stimuli enhancing IL-12 production could turn off development of inflammatory Th2 cells, which is the basis of the hygiene hypothesis. In this scenario IL-4, while acting as an autocrine promoter of Th2 cell differentiation once initiated, is not a critical signal for this initiation. TSLP is expressed in “stromal” cells, such as skin keratinocytes, bronchial epithelial cells, and lung smooth muscle cells

in both Th1 and Th2 cells. Furthermore, SOCS5 is expressed in fully committed Th1 but not Th2 cells, in which its expression appears to be driven by IL-12 (Seki et al. 2002). Only Th1 cells characteristically express a functional IL-12 receptor. Thus, selective induction of SOCS5 during initial T-cell activation might play a role in regulating the direction of Th1 cell differentiation. A caveat is that T cells from SOCS5 gene-deleted mice did not show a tendency toward Th2 differentiation (Brender et al. 2004). Conversely, SOCS3 is implicated in inhibiting Th1 cell differentiation. It is selectively expressed in a Th2-type cytokine environment and in Th2 cells (Seki et al. 2003). SOCS3 can block STAT4 activation by the IL-12 receptor β chain in the presence of IL-12 (Seki et al. 2003). Furthermore, its expression in circulating T cells in patients with asthma correlates with disease severity, while SOCS3 overexpression in transgenic mice enhances Th2-type cytokine expression and airway eosinophilia in experimentally induced asthma (Seki et al. 2003). These observations suggest that SOCS3 blockade may be a worthwhile goal for pharmacologic and other approaches to limiting the Th2 responses in diseases such as asthma, although the consequences may be wide ranging, nonspecific, and possibly ultimately deleterious.

Finale: beyond the Th1/Th2 paradigm Th2 cytokines have been implicated in IgE switching, eosinophil infiltration, and mucous hyperplasia in the target mucosa in allergic diseases and asthma. Dendritic cells play a critical role in directing these responses, which in turn depend on developmental signals acting on these dendritic cells as they mature. As discussed earlier, a link between dendritic cells and induction of Th1 responses is relatively well defined. Microorganism-derived PAMPs activate dendritic cells through TLRs inducing IL-12 production, which primes naive CD4+ T cells to undergo Th1 differentiation. The situation has currently been less clear with induction of Th2 responses. It is not clear whether environmental PAMPs acting through TLRs are involved in initiation of Th2 responses. Furthermore, a clear cytokine counterpart of IL-12 in promoting Th2 responses is not clear. The cytokine IL-4 has been presumed to serve this role, but the vexing question is where this IL-4

Th2

Th2 IL-10

Fig. 3.5 The new Th1/Th2 paradigm. Whereas Th2 cells produce the “signature” cytokines IL4, IL-5, and IL-13, and Th1 cells produce the “signature” cytokine IFN-g, subsets of these cells may serve overall proinflammatory or inhibitory/regulatory functions according to their additional production of TNF-a or IL-10 respectively.

Regulatory IL-4, IL-5, IL-13

TNF-a

Inflammatory IL-4, IL-5, IL-13

Th1

Th1

Regulatory IFN-g

Inflammatory IFN-g

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and fibroblasts, although it may also be expressed by at least some blood leukocytes. The stimuli leading to its expression are as yet uncharacterized, but induction by environmental agents such as allergens, pollutants, and viral infections could provide an important link between environmental influences and induction and exacerbation of allergic diseases such as atopic dermatitis, atopic rhinitis, and asthma. A recent study from our own group shows that TSLP mRNA expression is increased in asthmatic airways and correlates with the expression of both Th2-attracting chemokines and disease severity (Ying et al. 2005). Finally mast cells activated by surface IgE cross-linkage also produce TSLP, which could provide a link between mast cell activation and induction of inflammatory Th2 cells following acute allergen exposure of atopic individuals.

Role of T cells and cytokines in asthma pathogenesis As recently as the late 1980s, asthma was seen as a disease driven by smooth muscle contraction as a result of release of mast cell mediators such as histamine, and the proposition that T cells might be involved was treated with some derision. In the past 25 years, overwhelming evidence, to which the author of this chapter has made a substantial contribution, has accumulated for a critical role for T cells and their cytokine products in asthma, and the proposition is now generally accepted. Indeed, this discovery was hailed as one of the significant milestones in asthma research in the 200th centenary publication of the American Thoracic Society. Asthma is now regarded as a chronic inflammatory disease of the bronchial mucosa characterized by eosinophil infiltration and airway remodeling. Despite these findings, the evidence for a role for T cells has remained largely circumstantial in the sense that there remains a great gulf between descriptive studies of the nature of the inflammatory changes in the bronchial mucosa in asthma and how precisely these are related to the clinical features of the disease, namely variable airways obstruction (causing wheeze and shortness of breath) and nonspecific airway hyperresponsiveness (AHR) (causing cough and inappropriate bronchial constriction in response to environmental stimuli such as cold air, smoke, and exercise). Another major question which remains to be answered is what activates T cells in asthma. While allergen exposure clearly exacerbates asthma in atopic subjects, not all asthmatics are atopic and, even in atopic asthmatics, it cannot be assumed, as it largely has been, that T-cell activation is exclusively initiated by allergens. It has been suggested, for example, that self antigens might also drive T-cell responses in asthma (Lassalle et al. 1993; Nahm et al. 2002). It is possible that T-cell activation in asthma may occur initially in response to allergens, but that other (hypothetical) circumstances intervene in those patients

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in whom it persists, rather than regresses, to maintain chronic T-cell activation and thereby chronic disease.

T cells and Th2 cytokines in asthma The concept of T-cell involvement in asthma arose from the known involvement of these cells in allergen-specific IgE synthesis by B cells in atopic subjects, and the belief that T-cell products might also have direct effects on the airways through recruitment of inflammatory cells, particularly eosinophils. My early studies demonstrated activation of CD4+ T cells in the peripheral blood of patients with acute severe asthma, as shown by expression of the activation marker CD25 (now a source of some uncertainty since this is now also recognized as a marker of T regulatory cells) and correlations of the percentages of activated T cells with disease severity (Corrigan et al. 1988; Corrigan & Kay 1990). Since glucocorticoids are powerful inhibitors of T-cell activation, it was reassuring that T-cell activation was inhibited by systemic glucocorticoid therapy in asthmatics to a degree which correlated with clinical improvement, as measured by changes in spirometry (Corrigan et al. 1991a). Indeed, a subgroup of patients relatively clinically resistant to glucocorticoid therapy failed to show inhibition of T-cell activation, although this could be effected by other immunosuppressive drugs such as cyclosporin A (Corrigan et al. 1991a,b). Activated T cells could similarly be demonstrated in other compartments of asthmatic airways such as the bronchial mucosa and bronchoalveolar lavage fluid, and again the degree of activation could be correlated with eosinophil infiltration and impairment of lung function (Azzawi et al. 1990; Walker et al. 1991; Robinson et al. 1992, 1993a). After the description of the Th1/Th2 dichotomy, and the development of the technique of in situ hybridization to investigate expression of mRNA encoding cytokines in T cells, it became apparent that the overall profile of expression of cytokine mRNA in CD4+ T cells in asthmatics was Th2-type, that is IL-4, IL-5 and IL-13 predominant, in the peripheral blood (Doi et al. 1994; Corrigan et al. 1995), in bronchoalveolar lavage fluid (Robinson et al. 1992, 1993a,b; Till et al. 1995; Ying et al. 1995), and in the bronchial mucosa (Ying et al. 1995, 1997; Humbert et al. 1997a) of asthmatics as compared with controls. The prominent features of these studies were that expression of the cytokine IL-5 most consistently correlated with the degree of airways obstruction in asthma and, where studied, cytokine expression was reduced concomitantly with glucocorticoid therapy and clinical improvement. Some of these Th2-type T cells were shown to be allergen-specific (Till et al. 1998), although interestingly at least some of them retained the ability to respond to IL-12 (Varga et al. 2000). Again, cytokine production was inhibited by antiasthma glucocorticoids (Powell et al. 2001a). As mentioned, however, these experiments do not necessarily indicate that allergens are the sole or even the most important stimulus to CD4+ T-cell activation in asthma.

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Histamine Bronchospasm

Prostaglandin D2 Leukotrienes Mast cell

IL-4, IL-13, TGF-b

IgE

ACUTE CHRONIC

Allergen Other antigens?

B

Epi Fib ASM

B cell

TSLP IL-4, IL-13

TSLP IL-4

IL-13

AHR Remodeling Narrowing TGF-b

IL-5 Major basic proteins Leukotrienes

CD4+ DC Th2 T cell

Eosinophil

Fig. 3.6 Working hypothesis of asthma pathogenesis. Mast cells sensitized by surface allergen-specific IgE release acute mediators of bronchoconstriction on allergen exposure, which may serve as a mechanism for acute exacerbation of asthma in atopic patients. Th2 T-cell differentiation in the airways is driven by dendritic cells (DC) which present allergens and possibly other antigens to T cells in an MHC class II restricted fashion, possibly under the influence of thymic stromal lymphopoietin (TSLP) produced by mast cells, epithelial cells, and other cells. Th2 cytokines control IgE synthesis by B cells (IL-4, IL-13) and encourage recruitment and

activation of eosinophils (IL-5), which may damage the mucosa through release of major basic protein, and are also a source of remodeling cytokines such as TGF-b. Th2 cytokines such as IL-4 and IL-13 also induce release of growth factors by structural cells in the airways such as epithelial cells (Epi), myofibroblasts (Fib), and airway smooth muscle cells (ASM) causing remodeling changes (fibrous protein deposition, mucous hyperplasia, smooth muscle hypertrophy/hyperplasia). Mast cells are also a source of remodeling cytokines. See text for definition of other abbreviations.

These findings resulted in the concept that asthma is driven by Th2-type T cells, and that IL-5 expression by these cells drives eosinophil recruitment to, and selective accumulation in, the airways (Fig. 3.6). They also linked IL-4 and IL-13 production by these cells as promoters of IgE synthesis by inducing B-cell switching to IgE. Despite this explosion of knowledge in a period of 10 years, important questions remain. Because all the aforementioned studies represent a “snapshot” of a single period in time, they have provided no information as to how asthma severity is regulated on a day-to-day basis, and even today there is very little information on how environmental stimuli (with the possible exception of allergen exposure in those asthmatics who are also atopic) interact with this T-cell/Th2-type cytokine/ eosinophil axis to alter disease severity in the short term (such stimuli would include viral infections, exercise, cold air, smoke, and others). Furthermore, as mentioned, although eosinophil products such as basic proteins have been hypothesized to “damage” the bronchial mucosa in asthma, there is little or no understanding of how this “damage” results in the clinical features of the disease. It has also become apparent that CD4+ T cells are not the only possible sources of Th2-type cytokines in asthma. Eosinophils, mast cells, and basophils are also potential sources (Moqbel et al. 1995; Kon et al. 1998a; Lacy & Moqbel 2001; Pawankar 2001), while airway smooth muscle cells may also produce Th2-type cytokines and chemokines (see below and other chapters in this volume).

Is all asthma the same? Several clinical phenotypes of asthma have been described (atopic, nonatopic, occupational), although the fundamental clinical features of asthma (variable airways obstruction and AHR) in all these phenotypes appears to be similar. The question arises as to whether the T-cell/cytokine/eosinophil paradigm of inflammation plays a similar role in all these phenotypes. In cross-sectional studies of bronchial biopsies from patients with these phenotypes of asthma, the cellular immunopathology of the disease, at least in terms of numbers of infiltrating cells, appeared to be virtually identical (Bentley et al. 1992a,b; Humbert et al. 1996), and in every case characterized by evidence of T-cell activation and eosinophil infiltration. We have performed exhaustive comparisons of the cellular and molecular immunopathology of the bronchial mucosal infiltrate in atopic and nonatopic asthma (Humbert et al. 1996, 1997a,b,c; Ying et al. 1997) and have found that the profiles of cytokine expression, with predominant expression of IL-4, IL-5 and IL-13, as well as the cellular provenance of the cytokines (predominantly CD4+ T cells) are remarkably similar in asthma regardless of the atopic status of the patient. At first sight, this might suggest that IgEmediated mechanisms such as mast cell degranulation are neither necessary nor sufficient for the development of asthma, and that when asthma and atopy coexist, as they often do, mast cell degranulation serves merely to exacerbate the disease acutely by release of bronchoconstricting mediators such as histamine and leukotrienes into airways already

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rendered hyperresponsive by chronic T cell-mediated inflammation. The situation has been complicated, however, by our recent observation (Takhar et al. 2007) that IgE class-switch recombination in B cells may occur in the bronchial mucosa of both atopic and nonatopic asthmatics, suggesting that the latter patients may manufacture IgE, allergen-specific or otherwise, in the bronchial mucosa even though this is not detectable by conventional tests (skin-prick tests or in vitro tests) in the periphery. Thus, while the precise role of IgE in asthma pathogenesis remains to be elucidated, these experiments nevertheless stress the similarity of the immunopathologic mechanisms across the spectrum of clinical phenotypes. Studies on child asthma seem to support the T-cell hypothesis. We observed activated peripheral blood CD4+ T cells in child asthmatics, correlations with blood eosinophils and disease severity, and increased Th2-type cytokine mRNA expression that were reduced concomitantly with glucocorticoid therapy and disease improvement (Gemou-Engesaeth et al. 1994, 1997, 2002). Other phenotypes of asthma which have been much discussed are the “severe” and “difficult to treat” phenotypes (these are not synonymous since the clinical responsiveness of asthma to therapy does not necessarily parallel its severity). One problem with studying patients with severe asthma is that it is difficult to discriminate a priori abnormalities from the effects of the high dosages of glucocorticoid that these patients inevitably receive. Making a case for “severe” asthma forming a distinct immunopathologic phenotype, Wenzel et al. (1997, 1999) have shown that it is possible to delineate groups of severe, glucocorticoid-dependent asthmatics with numbers of bronchial mucosal eosinophils within the range observed in normal controls. One of these studies suggested that these patients also had increased numbers of airway neutrophils, whereas the other did not. Pursuing this theme, it has recently been suggested that milder asthmatics with “noneosinophilic” asthma respond less well, at least in the short term, to inhaled glucocorticoid therapy (Berry et al. 2007). There is intense interest in linking (if possible) particular immunopathologic changes with particular clinical phenotypes of asthma, since this has obvious implications for distinct approaches to therapy, and doubtless these studies will multiply. Nevertheless, aside from these isolated (though intriguing) observations, there is as yet no extensive evidence base supporting the hypothesis that asthma can be subdivided on molecular or cellular pathologic grounds, or that clinical phenotypes of asthma have distinct immunopathologic features.

Airway remodeling There has been much discussion about the possible role of changes in the airways, collectively termed “remodeling,” that may be observed in association with asthmatic inflammation. These changes include deposition of new fibrous

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proteins, hyperplasia of mucous glands, neovascularization, and airway smooth muscle hypertrophy and hyperplasia. In theory such changes, once established, might be resistant to therapy and could cause irreversible blockage of the airways and AHR, although none of these possibilities has yet been convincingly demonstrated. Experiments in animals (see below) suggest that all the changes characteristic of remodeling, as well as AHR and inflammatory cell infiltration, can be recapitulated in the airways by the transgenic expression of single cytokines such as IL-5, IL-11, or IL-13 in the airways. In the case of transgenic IL-13 expression (Kibe et al. 2003), glucocorticoid administration abolished the associated cellular infiltrate but not the remodeling changes. In human studies, anti-IL-5 therapy was shown to reduce deposition of extracellular proteins in the bronchial mucosa while partly but not completely abolishing the infiltration of eosinophils (FloodPage et al. 2003). Together these experiments strongly suggest that Th2-type and other cytokines may participate in remodeling, but leave open the question of whether leukocytes such as eosinophils are necessarily involved in the process. In humans, Th2-type cytokines such as IL-5 and IL-13 have been shown to have the ability to increase airway smooth muscle contractility to cholinergic agonists in vitro (Hakonarson et al. 1999; Laporte et al. 2001), and it has been suggested that such changes alone might be sufficient to sustain AHR (Maclem 1996). Bronchial biopsies from asthmatics compared with those from patients with eosinophilic bronchitis (characterized by cough without reversible airways obstruction or AHR) showed no differences in bronchial mucosal T cell or eosinophil infiltration, but mast-cell invasion of smooth muscle cells only in the asthmatics, suggesting AHR may be more related to mast cell/smooth muscle interactions than the T-cell/cytokine/eosinophil axis (Brightling et al. 2002). This mast-cell myositis was also observed in a second study (Berry et al. 2007). If reinforced by scientific consensus, and if specific for asthma, these findings suggest that mechanisms other than those mediated by T cells may contribute to AHR (although cytokines may still be involved), and even that attention might be better focused on inflammatory changes in airway smooth muscle rather than the mucosa. Having said all this, there is no firm evidence, as with postulated “damage” to the bronchial mucosa by eosinophil products, that remodeling changes contribute to the clinical features of asthma. If they do, one might expect that remodeling changes, which might be less susceptible to reversal once established, would be more prominent in those asthmatics who show a poor response to T cell-directed therapy, such as glucocorticoid, compared with those who show a good response. This possibility remains to be investigated. Two sets of experiments provide more direct evidence for T-cell involvement in ongoing regulation of asthma severity. Treatment of severe, glucocorticoid-dependent asthmatics with a single infusion of a chimeric anti-CD4 monoclonal antibody, which temporarily ablated circulating CD4+ T cells,

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resulted in measurable improvement in lung function compared with a parallel group treated with placebo (Kon et al. 1998b). Such an approach is unlikely to be used in routine clinical practice, but it does provide “relatively” clear proof of principle that direct inhibition of CD4+ T cells can ameliorate asthma. Conversely, it was shown that intradermal injection of short peptides derived from the cat allergen Fel d 1 could induce an isolated late bronchoconstrictor response in asthmatics with cat allergy (Haselden et al. 1999, 2001). These peptides bound to MHC class II molecules, but did not activate basophils by IgE cross-linking in a histamine release assay. The phenomenon was MHC class II-restricted in the sense that it was observed only in individuals whose MHC class II molecules were able to bind to the injected peptides. These interesting data show not only that a presumably “pure” CD4 + T-cell stimulus can cause bronchoconstriction in asthmatics, but also that this stimulus is capable of being transmitted from the site of injection, presumably to the lungs, within hours. The mechanism of this phenomenon remains unclear since it was not accompanied by any detectable T-cell activation within the airways, cellular infiltration, or changes in the production of IL-5, IL-13, histamine or leukotrienes, although inhaled challenge with the same peptides was accompanied by increased eosinophils in induced sputum (Ali et al. 2002).

New cytokines in asthma In addition to the “classical” Th2-type cytokines, many other cytokines have been implicated in the mechanism of asthma (Table 3.3). Many of these demonstrations have relied on mouse models of asthma, which usually involve relatively short-term sensitization of the animals to a protein (typically ovalbumin) by intraperitoneal injection of the protein in adjuvant, followed by repeated inhalational challenge with the same protein. Although animals challenged in this way develop “AHR,” usually demonstrated by increased airways resistance and/or reduced compliance in response to aerosolized bronchoconstricting drugs such as methacholine, the data obtained are very dependent on the precise protocol employed, not to mention the strain of mouse. Rather than being truly representative of the mechanism of chronic human asthma, these models have provided insight into how individual cytokines may effect airway changes when either overexpressed (as in transgenic animals) or ablated (by gene deletion or blocking antibodies). One of the most striking realizations from these models is that many cytokines may cause airway changes associated with AHR and remodeling apparently through direct effects on the airways, pushing effector granulocytes somewhat out of the limelight, at least in this context although, as has been discussed, direct interactions of granulocytes such as mast cells with airway smooth muscle cells may yet have an important role to play in the generation of AHR. In addition to these animal models, studies of cytokine expression have been performed

T Cells and Cytokines in Asthma and Allergic Inflammation

in human asthma, although many of these have been simply observational. In mouse models, adoptive transfer of differentiated allergen-specific Th2, but not Th1, cells to naive mice can induce airway eosinophil infiltration and AHR on allergen rechallenge (reviewed by Lloyd et al. 2001). The idiosyncratic nature of these models was well demonstrated by two studies published in the Journal of Experimental Medicine in 1996, one of which (Foster et al. 1996) suggested that IL-5 blockade abolished airway eosinophil infiltration, AHR, and remodeling changes, while a second (Corry et al. 1996) suggested that IL-4 but not IL-5 blockade had the same range of effects. IL-5 appears to have an important eosinophil-regulating function in asthma, as do the cytokines IL-3 and GM-CSF. The heterodimeric receptors for all three of these cytokines share a common β chain, although the IL-5 receptor is expressed among the granulocytes only on eosinophils and basophils, unlike IL-3 and GM-CSF receptors which are expressed on a wider range of granulocytes, as well as monocyte/macrophages and dendritic cells. All three cytokines activate eosinophils by signaling through p38 MAPK, causing increased expression of integrins such as ICAM-1, α6β2 integrin (CD18), and CD44 (Ip et al. 2005). Once activated, eosinophils in asthmatics release IL-5 in an autocrine fashion, which prolongs their life through upregulation of the antiapoptotic factor Bcl-2 (Huang et al. 2005). IL-3 upregulates CD48 expression on eosinophils, cross-linking of which causes release of granule proteins (Munitz et al. 2006). This is an important observation since although eosinophil granule proteins have been postulated to damage the bronchial epithelium in human asthma, an effect that is in turn postulated to increase AHR, the precise physiologic stimulus to eosinophil degranulation in vivo in human asthma remains ill-defined. IL-5, IL-3, and GM-CSF also have an important regulatory role in the formation of eosinophil progenitors in the bone marrow (Dorman et al. 2004). There has been much interest in a possible role for IL-9 in asthma. In addition to Th2 cells, eosinophils, neutrophils and mast cells are potential sources. IL-9 is produced by allergenspecific T cells (Devos et al. 2006) and is implicated in inducing tissue infiltration with mast cells (Nouri-Aria et al. 2005). It is overexpressed in the bronchial mucosa of asthmatics (Ying et al. 2002). Interestingly, IL-9 was reported to be overexpressed in infants with respiratory syncytial virus bronchiolitis, which causes wheeze, where neutrophils appear to be a major source (McNamara et al. 2004). Bronchial epithelial cells express IL-9 receptors (Tsicopoulos et al. 2004), which control epithelial cell differentiation and repair by signaling through the transmembrane receptor tyrosine kinase family of receptors called the ErbBs. MUC-4, a large membranebound glycoprotein, is a potential ligand for the ErbB-2 receptor in epithelial cells. Thus IL-9 also causes increased MUC-4 expression, leading to increased production of mucous glycoproteins (Damera et al. 2006). Cultured human airway

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66

Th1

Th1/Th2

Th2

Th2

Th1/Th2

IL-2

IL-3

IL-4

IL-5

IL-6

Fibroblasts, macrophages, endothelial cells, epithelial cells

AHR, eosinophilia, mucus hypersecretion, airway remodeling

IL-16

Chemoattractant for CD4 cells, monocytes, eosinophils; reduces Th2-type cytokines but increases IL-10

Increased

Increased

Increased

CD8+, MC, eosinophils

T-cell growth factor; expands Treg

Th1

IL-15

Increased: soluble IL-13 receptor blocked AHR but not eosinophils or IgE. Transgene developed airways inflammation, remodeling, AHR

Decreased: exogenous IL-12 decreased eosinophils but not AHR

Increased

Deficient IL-10 response in therapy-resistant patients, restored by vitamin D3

Increased further by viral infection

Increased, especially by systemic glucocorticoid (along with IP-10)

Increased (in severe asthma)

Increased: antibody decreased eosinophils but not AHR

Increased: soluble IL-4 receptor had some steroid-sparing effect

Increased

Increased

Human asthma

Decreased

Th2 cells, eosinophils, MC, basophils

MC development, B-cell switch to IgE production, eosinophilia, AHR, mucus hypersecretion, reduced epithelial integrity, remodeling, reduced PGE2 production by epithelial cells

Th2

IL-13

Exogenous IL-12 blocked eosinophils and AHR

Transgene had AHR and changes of remodeling

Adenoviral transfer decreased inflammation

Transgene had eosinophils, AHR and mucus; knockout no effect

IL-8 receptor knockout decreased AHR and neutrophils

Transgene showed increased AHR and inflammation, knockout less; trans-signaling blockade reduces Th2 response; conventional signaling blockade favors Treg

Increased: knockout or blocking decreases eosinophils and AHR

Increased: blocking decreases AHR (but some residual)

Increased: blocking decreases eosinophilia

Decreased

Animal models of airway inflammation

Many non-T cells

DC, macrophages, B cells

Favors Th1 differentiation, inhibits IgE synthesis

Th1

IL-12

IL-11

T cells, macrophage

Endothelial cells, epithelial cells (induced by viral infection), macrophages, fibroblasts, T cells, ASM (increased by IL-17A)

Neutrophil activation and chemotaxis; weak eosinophil chemotaxis

Suppresses Th1/Th2 function, eosinophil/MC/basophil activation, favors Treg production, B-cell switch to IgG4

Th1/Th2 cells, macrophages, endothelial cells, DC, ASM, fibroblasts

T- and B-cell growth factor, cofactor for IgE synthesis, Th2 differentiation (trans-signaling), diversion of Treg to Th17 (conventional signaling)

“Regulatory” Th1/Th2, Treg (induced by glucocorticoid)

Th2 cells, eosinophils, MC, basophils

Eosinophil and basophil differentiation, maturation, and activation

IL-10

Th2 cells, eosinophils, MC, basophils

B-cell switch to IgE synthesis, MC development, eosinophil and basophil activation, mucus secretion, favors Th2 production, increased endothelial cell VCAM expression

Th2 cells, eosinophils, neutrophils, MC, basophils

Th1/Th2 cells, eosinophils, MC, basophils, macrophage/ monocytes, fibroblasts

Differentiation and activation of eosinophil, neutrophil, basophil, MC; eosinophil degranulation (CD48)

MC and eosinophil development, AHR, mucus secretion, chemokine expression by ASM

Th1 cells, eosinophils

T-cell growth factor

Th2

Producer cell in human patients

Actions

IL-9

IL-8

Th1/Th2/Th17/ Treg associated

PART 1

Cytokine

Table 3.3 Cytokines implicated in asthma.

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Suppresses Th1/Th2 function, favors Treg induction, cofactor for IgA secretion, increases mucin production and myofibroblast differentiation

Inhibits IgE synthesis, inhibits Th2 induction, activates eosinophils and macrophages

“Regulatory” Th1/Th2, Treg

Th1

TGF-b

IFN-g

Epithelial cells, endothelial cells, fibroblasts, ASM, IgE-activated MC, other leukocytes

Th1 cells, NK cells

Eosinophils, MC, basophils, T cells, monocytes, macrophages, ASM

Macrophages, NK cells, T cells

Transgene develops atopic dermatitislike phenotype and airways inflammation

Decreased

Increased

Increased

Increased: transgene has airway inflammation

Not studied

Not studied

Increased in mouse model of asthma; transgene (electroporation) had enhanced Th2-type cytokine expression

Knockout had increased eosinophils and AHR; exogenous IL-18 (with IL-12) decreased AHR and eosinophils; exogenous IL-18 alone increased Th2-type cytokines

Transgene showed augmented allergen-induced airways eosinophilia, Th2-type cytokines, mucus secretion

Wide variety of targets increasing cytokines, chemokines, and metalloproteinases

Increased in the bronchial mucosa; correlates with CC chemokine expression

Increased in viral infection and exacerbations

TGF-b2 increased (particularly in eosinophils), TGF-b1 and b2 after allergen challenge

Increased: blockade by antibody or soluble receptor improves asthma symptoms and AHR

Increased

Not studied

Not studied

Elevated in serum

Reduced

Not yet measured

Increased

ASM, airway smooth muscle cell; DC, dendritic cell; IFN, interferon; MC, mast cell; NK, natural killer; TGF, transforming growth factor; TNF, tumor necrosis factor; Treg, T regulatory cell; TSLP, thymic stromal lymphopoietin; VCAM, vascular cell adhesion molecule.

Activates DC to induce “inflammatory” Th2 cells and CC chemokines; induces OX40L on DC; may be primary trigger for Th2 differentiation

Activation of endothelial cells and epithelial cells

“Inflammatory” Th1/Th2

TNF-a

Th1/Th2 cells, macrophages, eosinophils, MC, basophils, fibroblasts, epithelial cells, endothelial cells

Macrophages, DC

Macrophages, DC

T cells

Macrophages, epithelial cells, DC

Th2 central memory cells

Th17 cells, CD8 T cells, gd cells, NK cells, neutrophils, epithelial cells, endothelial cells, fibroblasts

CHAPTER 3

TSLP

Differentiation and activation of eosinophils, neutrophils, basophils, MC

Th1/Th2

Acts conversely to IL-23 in limiting Th17 development of naive T cells

IL-27

GM-CSF

Amplifies Th17 development once initiated by TGF-b and IL-6

In presence of IL-12 induces IFN-g by T cells, NK cells, favors Th1 differentiation; with IL-2 alone increases Th2type cytokines in T and NK cells; with IL-3 increases Th2type cytokines in MC and basophils

IL-23

Th1

IL-18

Maintains Th2-type T-cell activation and central memory

T-cell growth factor

Th2

IL-17E (IL-25)

Induces inflammatory cytokine production by fibroblasts, macrophages, epithelial cells, endothelial cells; CXC chemokine production by ASM

IL-19

Th17

IL-17A/F

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smooth muscle cells also express IL-9 receptors, stimulation of which triggers release of Th2-type chemoattracting chemokines by these cells (Gounni et al. 2004). Overexpression of IL-9 in the airways of transgenic mice causes airway inflammation, mast cell hyperplasia, and AHR (Temann et al. 1998). As with IL-9, transgenic expression of IL-11 and IL-13 in the airways of mice was sufficient to cause eosinophil infiltration, mucus hypersecretion and remodeling changes (Tang et al. 1996; Zhu et al. 1999). IL-11 is produced by normal human lung fibroblasts in response to mediators of neovascularization such as endothelin A (Gallelli et al. 2005). It is also overexpressed in the asthmatic bronchial mucosa (Minshall et al. 2000). In addition to Th2-type cells, IL-13 may originate from mast cells, basophils, and eosinophils. It is induced in mast cells by TNF-α through an NF-κB-dependent pathway (Lee et al. 2004). IL-13 is highly implicated in remodeling (Wills-Karp 2004), increasing the production of remodeling mediators such as platelet-derived growth factor and periostin by lung fibroblasts (Ingram et al. 2006; Takayama et al. 2006). It may compromise epithelial cell basement membrane attachment by inhibiting production of proteins such as paxillin (Ramirez-Icaza et al. 2004). It also stimulates the release of chemokines such as eotaxin from human airway smooth muscle cells (Peng et al. 2004). In a mouse model of asthma, soluble IL-13 receptor abrogated AHR without affecting serum IgE concentrations or infiltration of the airways with eosinophils (Grunig et al. 1998; Wills-Karp et al. 1998), reinforcing the concept that cytokines such as IL-13 may act directly to cause AHR perhaps independently of granulocytic infiltration, at least in the airways mucosa. Most interestingly, IL-13 has been implicated in the aspirinsensitive phenotype of asthma. It reduces the production of prostaglandin (PG)E2 in airway epithelial cells by reducing the activities of the enzymes cyclooxygenase-2 and PGE synthase 1, while increasing the activity of the PGE2-catabolizing enzyme 15-prostaglandin dehydrogenase, as well as reducing expression of the PGE2 receptor EP2 (Trudeau et al. 2006). Finally, it has been shown to increase expression of the cysteinyl leukotriene receptors CysLT1 and CysLT2 in mouse lungs (Chavez et al. 2006). IL-16 is a CD4+ T-cell chemoattractant in vitro (Center et al. 1994) and is overexpressed in the asthmatic bronchial mucosa. IL-16 reduces production of IL-5 and IL-13 by allergen-stimulated T cells from atopic donors, while increasing production of IL-10 (El Bassam et al. 2006). Thus it may play a role in suppression of Th2 T-cell activation and, conceivably, induction of allergen-specific T regulatory cells. The role of the IL-17 family of cytokines has been discussed above. IL-25 (IL-17E), the most distant relative of the IL-17 cytokine family, may play a pivotal role in activating Th2 cells and, more importantly, maintaining Th2-type TCM cells. Overexpression of IL-25 in mice increased antigen-induced but not spontaneous eosinophil and T-cell recruitment to the

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airways, and also caused goblet cell hyperplasia (Tamachi et al. 2006). IL-17A has a number of effects on human airway smooth muscle cells, including induction of CC chemokines such as eotaxin (Rahman et al. 2006) and augmentation of TNF-α-induced production of CXC chemokines such as IL-8 (Henness et al. 2006). IL-6 is a member of a family of cytokines that includes IL-11 and ciliary neurotrophic factor. Although most soluble cytokine receptors act as antagonists and compete with their membrane-associated counterparts for binding to cytokine ligands, soluble receptors of the IL-6 family of cytokines can, when coupled with their cytokines, act as agonists by ligating the universal signal-transducing receptor for all IL-6 family cytokines called gp130 (Rose-John et al. 2006), which is expressed on a range of target cells that do not necessarily express the membrane-bound form of the receptors. This process has been termed “trans-signaling.” In some cells, transsignaling by soluble IL-6/IL-6R complexes ligating gp130, and “conventional” signaling, whereby IL-6 acts on its membranebound receptor, may exert very disparate effects. Thus IL-6 increases proliferation of Th2 cells by trans-signaling, but by conventional signaling through the membrane-bound receptor diverts the development of CD4+CD25+ T regulatory cells toward a more proinflammatory Th17 phenotype (see above). In mouse models, blockade of the soluble IL-6 receptor using a gp130–immunoglobulin heavy chain fusion protein reduced Th2 T-cell development in the airways, whereas direct blockade of the membrane-bound IL-6 receptor increased the development of T regulatory cells (Doganci et al. 2005a,b). The possible origins of IL-6 are manifold and include dendritic cells, airway smooth muscle cells (Shan et al. 2006), and fibroblasts (in which it is induced by Th2-type and other cytokines as well as TGF-β). Activated eosinophils have been reported to increase IL-6 release from human fibroblasts (Gomes et al. 2005), probably through release of cytokines including IL-1β and TGF-β. Endothelin-1, a profibrotic mediator, also increases production of IL-6, as well as IL-11 by human lung fibroblasts (Gallelli et al. 2005). Such observations link airway remodeling in asthma with cytokine products of airway smooth muscle cells and eosinophils. IL-8 is a CXC chemokine that attracts and activates neutrophils. Epithelial cell expression of IL-8 is increased in viral infections where it may contribute to viral-induced airways inflammation (Funkhouser et al. 2004; Newcomb et al. 2005). The cytokine IL-17A enhances the stability of TNF-α-induced IL-8 mRNA in human airway smooth muscle cells (Henness et al. 2006). Expression of IL-8 and its sister CXC chemokine IP-10 is increased in the human bronchial mucosa in association with systemic glucocorticoid therapy, in contradistinction to that of eosinophil-attracting CC chemokines such as eotaxin which is reduced (Fukakusa et al. 2005). This may contribute to the neutrophil infiltration of the airways observed in severe asthmatics treated with systemic glucocorticoid.

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IL-19 belongs to the IL-10 family of cytokines, which includes IL-10, IL-19, IL-20, IL-22, melanoma differentiationassociated gene 7 (IL-24), and AK155 (IL-26). Apart from IL-10 (see below), there have been few studies of a possible role of these cytokines in human asthma. Human asthmatics were found to have elevated serum concentrations of IL-19 compared with controls, and IL-19 expression was also elevated in a mouse model of asthma induced by house-dust mite allergen (Liao et al. 2004). Overexpression of the IL-19 gene by electroporation in this mouse model increased IL-4, IL-5, and IL-13 expression in a T cell-dependent fashion. Thus IL-19 must be added to the ever-expanding list of cytokines that may regulate Th2-type cytokine expression in human asthma. IL-18 is produced by epithelial cells and activated monocytes/ macrophages. It is traditionally regarded as an inducer of Th1 T-cell differentiation and increases IFN-γ production by Th1type T cells, CD8+ T cells, and NK cells. It synergizes with, but acts independently of, IL-12 (El-Mezayen & Matsumoto 2004). In mouse models of asthma, IL-12 administration inhibited AHR, airway cellular infiltration, and Th2-type cytokine expression (Gavett et al. 1995); coadministration of IL-12 and IL-18 had a similar effect (Hofstra et al. 1998). Expression of IL-12 and IL-18 has been shown to be deficient in human asthmatics compared with controls (Ho et al. 2002; McKay et al. 2004). However, there is a twist in the tale of the IL-18 story, since it has been reported that IL-18 in combination with IL-2 promotes Th2-type cytokine production by T cells and NK cells, while in combination with IL-3 it promotes the production of these cytokines by mast cells and basophils. Intranasal administration of IL-18 with IL-2 caused eosinophilic infiltration, AHR, and mucous hyperplasia in the airways of naive mice in a T cell-, STAT6-, and IL-13-dependent fashion (Ishikawa et al. 2006). Thus, in conditions where IL-18 may be produced independently of IL-12, as for example following direct activation of epithelial cells, IL-18 may actually enhance asthmatic inflammation. With the realization that IL-10 is a key mediator of suppression of both Th1 and Th2 T-cell proliferation and cytokine production by certain subsets of adaptive (Tr1) T regulatory cells, its importance in the regulation of disease severity and its potential for the long-term regulation of allergen-specific T-cell responses in human asthma has moved to center stage. IL-10 is one of the few cytokines whose expression is increased, rather than inhibited, by glucocorticoid. It has a wide range of inhibitory actions on cells of the immune system, including T cells and antigen-presenting cells and, when expressed by T regulatory cells, it can inhibit the function of both Th1 and Th2 cells (reviewed by Hawrylowicz & O’Garra 2005). Deficiency of IL-10 production, for example by dendritic cells, has been associated with the development of atopy (Gentile et al. 2004). We identified a defect in glucocorticoid-induced IL-10 production in clinically glucocorticoid-refractory asthmatics compared with responsive asthmatics, implying that

T Cells and Cytokines in Asthma and Allergic Inflammation

glucocorticoid ameliorates human asthma at least partly through the actions of IL-10 (Hawrylowicz et al. 2002). Some of the IL-10 produced by glucocorticoid therapy of asthmatics originates from FoxP3-expressing Tr1 T regulatory cells (Karagiannidis et al. 2004). We have also shown that IL-10-secreting regulatory T cells can be induced in vitro by glucocorticoid and long-acting β2 agonist (Peek et al. 2005), providing one possible mechanistic explanation for the now well-recognized effects of concomitant long-acting β2-agonist therapy in sparing glucocorticoid therapy while improving symptoms and reducing disease exacerbations in moderate/ severe asthmatics. Finally, we have shown that the defect in glucocorticoid-induced IL-10 production by T cells from clinically glucocorticoid-resistant asthmatics is subject to reversal by therapeutic manipulation, for example by 1,25dyhidroxyvitamin D3 (Xystrakis et al. 2006). IL-10 has also been shown to be a key mediator of allergen-specific adaptive T regulatory cells induced in the course of allergen immunotherapy (Durham & Till 1998). Manipulation of the immune response by induction of IL-10 production, for example with drugs like vitamin D3 or by administration of glucocorticoid in the course of allergen immunotherapy, promises the possibility of long-term allergen-specific suppression of T-cell responses in asthma and allergic disease. Another cytokine implicated in mediating the functions of adaptive T regulatory cells is TGF-β, which exists in three isoforms: TGF-β1, TGF-β2, and TGF-β3 (reviewed by SchmidtWeber & Blaser 2004). TGF-β is produced by a wide variety of cells, including T cells, monocyte/macrophages, mast cells, and basophils. Eosinophils are also an important potential source in human asthma (Balzar et al. 2005). TGF-β plays a dichotomous role in inflammation, since in addition to being a mediator of T regulatory cell suppression it also exerts widespread effects on structural cells in the airways that may be relevant to remodeling (Howell & McAnulty 2006). In a group of severe asthmatics, expression of TGF-β2 but not the other isoforms was elevated in the airways and expressed prominently in eosinophils (Balzar et al. 2005). TGF-β2 but not TGF-β1 was also elevated in the asthmatic bronchial epithelium and increased mucin expression in cultured human bronchial epithelial cells (Chu et al. 2004). In a study in milder atopic asthmatics employing segmental allergen bronchial challenge, only TGF-β2 was elevated in lavage fluid at baseline, but both TGF-β1 and TGF-β2 were increased following allergen challenge. Both isoforms induced myofibroblast differentiation of fibroblasts, as measured by de novo expression of smooth muscle α-actin (Batra et al. 2004). It is not possible from these studies to determine whether any of this TGF-β expression reflects the activity of T regulatory cells. TGF-β1 has been reported to synergize with other profibrotic growth factors such as fibroblast growth factor (FGF)-2 to increase proliferation of human airway smooth muscle cells and increase production of connective tissue growth factor, which induces fibronectin and collagen I

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synthesis by fibroblasts (Burgess 2005; Bosse et al. 2006; Johnson et al. 2006). It is also itself produced by airway smooth muscle, for example in response to neutrophil elastase (Lee et al. 2006). The antiasthma glucocorticoid flunisolide has been reported to reduce TGF-β synthesis and fibronectin release by cultured induced sputum cells from asthmatics (Profita et al. 2004), suggesting that glucocorticoid may be able to inhibit at least some ongoing remodeling changes in asthma, even if it may not reverse established changes. Finally, leave cannot be taken of this section without reminding the reader of the increasing interest in the IL-7like cytokine TSLP as a “master switch” of Th2-type T-cell development through its priming effects on dendritic cells, and in maintaining Th2-type T-cell memory. Mice expressing a TSLP transgene have been shown to develop an atopic dermatitis-like phenotype (Yoo et al. 2005) and airways inflammation resembling that of asthma (Al Shami et al. 2005; Zhou et al. 2005). TSLP expression is increased in lesional skin of human patients with atopic dermatitis (Soumelis et al. 2002) and the bronchial mucosa of asthmatics (Ying et al. 2005). TSLP may prove to be a key therapeutic target in allergic disease since its elimination might not only block the development of allergen-specific Th2-type T-cell activation but also the maintenance of allergen-specific TCM cells. Furthermore, these effects would be expected to be considerably greater in extent than those of blocking single cytokines released by activated “inflammatory” Th2-type end effector cells. New data regarding the role of TNF-α in asthma are discussed below. An overview of the putative roles of the cytokines produced by inflammatory Th2 cells is presented in Fig. 3.7.

Microbial fragments

Table 3.4 Responsiveness of subsets of T cells to chemokines. (Modified after Sebastiani et al. 2002) Chemokine

Th1-type

Th2-type

IP-10 Mig I-TAC RANTES MIP-1b MCP-1 Eotaxin TARC MDC I-309

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

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

T regulatory cells are more sensitive to I-309, MCP-1, MIPs and TARC.

Do T cells home to the airways in asthma? Homing receptors that attract activated T cells to particular organs are obviously of great interest when considering new therapeutic approaches to putative T cell-mediated diseases such as asthma. Although homing receptors that preferentially attract T cells to the lymph nodes, skin, and gut mucosa have been identified, a search for homing receptors directing T cells to the airway mucosa has so far been fruitless. In the absence of a specific set of homing receptors, attention has switched to the possibility that particular functional subsets of T cells may express receptors for particular chemoattractant molecules. Chemokines, which pay a particular role in diapedesis of leukocytes into and out of tissues, have been particularly studied in this regard (Table 3.4). Dogma has

IL-9, IL-13 IL-4, IL-9, IL-13

TLR Blockade

OX40L NF-AT TSLP

DC

AHR

IL-12

DC

IL-4, IL-13

IL-4

Mucus hypersecretion IgE switching

OX40 IL-4, IL-5, IL-9, IL-13 Eosinophil and basophil activation

GATA-3 STAT6 C-maf

TSLPR

Mast cell activation Smooth muscle activation, remodeling

IL-4, IL-9, IL-13 IL-4R IL-25

IL-25R

CD4+ inflammatory Th2 T cell (IL-4, IL-5, IL-13, TNF-a high, IL-10 low)

Fig. 3.7 Activation and functions of inflammatory Th2 cells in asthma. Thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DC) promote the induction of inflammatory Th2 cells through OX40/OX40L costimulatory molecule interaction, which induces nuclear factor of activated T cells (NF-AT), which triggers IL-4 production and then IL-4dependent GATA-3 transcription. This process is reinforced by IL-25

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TNF-a

but overridden by IL-12 produced, for example, by DC exposed to microbial fragments and activated through Toll-like receptors (TLR). Cytokine products of inflammatory Th2 cells exhibit considerable pleiotropism and have been implicated in all the pathophysiologic changes that characterize asthma. TSLPR, TSLP receptor. See text for definition of other abbreviations.

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it that human Th2-type T cells preferentially express the chemokine ligands CCR3, CCR4, and CCR8 (Bonecchi et al. 1998). CCR4 in particular is said to be a marker of Th2-type central memory and effector cells (Rivino et al. 2004), and indeed we have identified preferential expression of CCR4 and CCR3 on IL-4-expressing bronchoalveolar lavage T cells from both asthmatics and controls (Morgan et al. 2005). Furthermore, in another study (Panina-Bordignon et al. 2001) it was observed that nearly all T cells in bronchial mucosal biopsies from atopic asthmatics obtained 24 hours after allergen challenge expressed IL-4 and CCR4, while CCR8 was coexpressed only on a minority of these cells (28%), and CCR3 was not expressed at all on T cells but was prominent on eosinophils. Thus, while CCR3 may play an important role in the recruitment of granulocytes such as eosinophils and basophils to sites of inflammation (see other chapters of this volume), its role in attracting T cells in human asthma is less certain. Despite these observations, other studies do not paint a clear picture of preferential bronchial mucosal expression of chemokines that attract Th2-type cells in human asthma. We (Ying et al. 2005) and others (Miotto et al. 2001; Bochner et al. 2003) have described elevated expression not only of chemokines whose ligands are said to be preferentially expressed on Th2-type T cells, such as TARC/CCL17 and MDC/CCL22 (both CCR4 ligands), but also of chemokines whose ligands are said to be preferentially expressed on Th1type T cells, in particular IP-10/CXCL10 (a CXCR3 ligand), both in “steady-state” asthma and following bronchial allergen challenge. At the same time, expression of CCR4 as a marker of Th2-type T cells has been questioned. For example, CCR4 expression has been detected on both Th1 and Th2 differentiated T cells, as well as T regulatory cells (Andrew et al. 2001; Iellem et al. 2001). If T cells are attracted to sites of inflammation according to their expression of chemokine receptors and the types of chemokines expressed in that particular type of inflammation, one would expect to be able to correlate expression of chemokines with infiltration of T cells bearing their receptors. In addressing this, however, we showed (Ying et al. 2005) that while expression of the chemokine CCR4 ligands TARC/CCL17 and MDC/CCL22 was elevated in the bronchial mucosa of asthmatics compared with controls, the total number of cells expressing CCR4 was no different in the two groups of subjects, although slightly elevated percentages of CD4+ T cells expressed CCR4 in the asthmatics as compared with the controls. Overall, these data support a scenario, at least in the case of T cells, in which cells are attracted to the bronchial mucosa in health and disease according to a particular pattern of chemokine expression, rather than one in which selective expression of chemokines results in selective influx of cells bearing their particular ligands. Examination of the effects of deletion of the genes encoding CCR3, CCR4, and CCR8 in mouse models of asthma has provided similarly

T Cells and Cytokines in Asthma and Allergic Inflammation

disappointing equivocal results with regard to Th2 T-cell infiltration (Chensue et al. 2001; Humbles et al. 2002; Schuh et al. 2002). As mentioned above, the CRTH2 receptor, which is a receptor for PGD2, may be a better marker for Th2 central and effector memory cells (Cosmi et al. 2000; Wang et al. 2006) but whether this molecule is involved in T-cell trafficking is unknown. In summary, then, there is at present little justification for the hope that specific blockade of chemokine receptors will provide a practical approach to blocking infiltration of the asthmatic bronchial mucosa by Th2-type cells, although it still holds some promise for the prevention of infiltration of other leukocytes such as eosinophils and basophils.

CD8, gd, and NKT cells in asthma This account has dealt principally with CD4+ T cells. Although traditionally regarded as cytotoxic cells, CD8+ T cells might play a proinflammatory role in the airways of asthmatics. For example, we showed that, in both atopic and nonatopic asthmatics, mRNA encoding IL-4 and IL-5, while localizing predominantly to CD4+ T cells, was also detectable in CD8+ T cells (Ying et al. 1997). Furthermore, we also found that CD8+ as well as CD4+ T-cell lines from the airways of asthmatic patients were capable of elaborating IL-5 protein (Till et al. 1995). All these observations are compatible with the concept of the population of CD8+ T cytotoxic type 2 (Tc2) lymphocytes. In a mouse model of asthma, viral infection was shown to induce virus-specific CD8+ T cells that were also capable of secreting IL-5 (Schwarze et al. 1999). Thus, CD8+ T cells may also contribute to the production of Th2type cytokines, perhaps under particular circumstances such as viral exacerbation of asthma (Dahl et al. 2004). As mentioned earlier, little is known about how cytokine production by T cells of any phenotype may contribute to the day-to-day regulation of severity of asthma, as for example when it is exacerbated by a rhinoviral infection. Studies in mouse models of asthma have implicated γδ T cells as having both regulatory and stimulatory roles in the genesis of airways inflammation (Zuany-Amorim et al. 1998; Lahn et al. 1999; Jin et al. 2005). There are currently only three observational reports of these cells in human asthma suggesting increased numbers of γδ T cells in bronchoalveolar lavage fluid of atopic asthmatics both at baseline and following segmental bronchial allergen challenge (Spinozzi et al. 1996; Krug et al. 2001), with corresponding reduced numbers in the blood (Chen et al. 1996). Although it is not inconceivable that these cells were allergen-specific as claimed in one of these reports (Spinozzi et al. 1996), very little is known about the antigen repertoire of γδ T cells, and those studies that do exist (see above) suggest that this repertoire is likely to be very limited. Consequently it is not possible to make any definitive statement about the role, if any, of γδ T cells in human asthma. With regard to NKT cells, the current hot topic is the controversial possibility that many of the CD4+ cells observed in

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the bronchial mucosa of moderate to severe asthmatics are not conventional T cells but NKT cells expressing the T-cell invariant receptor chain (Akbari et al. 2006; see above). NKT cells are certainly potentially abundant sources of Th2-type cytokines, and if these preliminary observations are verified by scientific consensus, they could force a change in our current thinking about how such cytokines are generated in the asthmatic airways, as well as the nature of the stimuli which cause their production.

What have new approaches to treatment taught us about the T-cell hypothesis of asthma? Glucocorticoids remain the cornerstone of asthma therapy, and we and many others have consistently demonstrated evidence of T-cell inhibition in asthmatics in association with clinical improvement (see above). There are similarly many studies showing reduction of Th2-type cytokine expression and eosinophil infiltration in the airways of asthmatics following glucocorticoid therapy (Robinson et al. 1993b; Bentley et al. 1996). Conversely, patients who are refractory to glucocorticoid therapy have T cells that are refractory to the inhibitory effects of glucocorticoid both in vitro and in vivo (Corrigan et al. 1991a; Leung et al. 1995). The mechanisms of this refractoriness are likely multifactorial and depend on interaction of positive T-cell regulatory signals induced by the inflammatory milieu and the environment, and negative regulatory signals influenced by glucocorticoid. For example, we have recently shown (Loke et al. 2006) that systemic glucocorticoid reduces bronchial mucosal activation of the components of the transcriptional regulator activator protein (AP)-1 in glucocorticoid-sensitive but not glucocorticoidresistant asthmatic patients, which may be one mechanism accounting for glucocorticoid refractoriness in some patients and which is likely centered on T cells. One stimulus for the activation of AP-1 components is excessive oxidative stress, which could conceivably arise from dietary deficiency or other environmental influences such as cigarette smoking, which has been reported to impair glucocorticoid responsiveness in asthma (Chaudhuri et al. 2003). Other investigators have reported signaling through p38 MAPK (Irusen et al. 2002) or following exposure to bacterial superantigens (Hauk et al. 2000) as stimuli that induce glucocorticoid refractoriness of T cells. Such interactions are likely complex and dynamic, but will likely repay further dissection since they may clarify how glucocorticoid exerts its inhibitory actions on T cells, how this may be confounded by environmental influences, and how these in turn might be overcome by new therapeutic approaches. Notwithstanding this paucity of knowledge, all these experiments reinforce the concept that, within individual asthmatics, glucocorticoid inhibition of T cells can be correlated with clinical responsiveness. They do not, however, indicate what the critical aspects of this responsiveness are or, as mentioned above, whether all the

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changes effected by T cells in asthma can be fully reversed by glucocorticoid. Other immunomodulatory drugs have also been assessed for a possible therapeutic role in asthma. Since many of these agents have potentially serious unwanted effects, attention has been focused on severe asthmatics requiring systemic glucocorticoid therapy (i.e., those in whom the risk–benefit ratio of therapy is most likely to be acceptable). Methotrexate is a folic acid analog used at low dosage for its immunomodulatory activity in a variety of chronic diseases. It exerts a delayed but sustained therapeutic effect even with intermittent weekly dosing, probably because it accumulates in cells as polyglutamate complexes, resulting in accumulation of S-adenosyl methionine and adenosine, both of which are inhibitory to T-cell function. Indeed, we have shown that methotrexate therapy of oral glucocorticoid-dependent asthmatics increases the sensitivity of their blood T cells to glucocorticoid inhibition (Corrigan et al. 2003). A metaanalysis (Marin 1997) of the effects of concomitant methotrexate therapy of oral glucocorticoid-dependent asthmatics suggested an oral glucocorticoid sparing effect (overall about 20%, but only in about 60% of responsive patients) if used for a minimum of 3–6 months, but with no significant improvement in lung function. Cyclosporin A is a lipophilic cyclic undecapeptide. In a complex with the cytoplasmic binding protein cyclophilin, it inhibits calcineurin-mediated dephosphorylation and nuclear translocation of the cytoplasmic subunit of the T-cell transcriptional activator NF-AT, thus inhibiting T-cell proliferation and cytokine production relatively specifically. Two blinded placebo-controlled trials in severe, oral glucocorticoid-dependent asthmatics (Alexander et al. 1992; Lock et al. 1996) both showed that concomitant cyclosporin A therapy improved lung function (slightly) while reducing oral glucocorticoid requirements. It was further shown that pretreatment of mild atopic asthmatics with cyclosporin A inhibited the late-phase bronchoconstrictor response of these patients following bronchial allergen challenge (Sihra et al. 1997). These approaches to therapy have not been widely embraced because the overall effects of these drugs have proven small. Not all patients respond, and response cannot be predicted a priori (Alexander et al. 1996). Furthermore, they have a high incidence of unwanted effects and chronic immunosuppression raises the specter of possible increased malignancy and opportunistic infection. Because they affect T cells they provide broad support for the T-cell hypothesis of asthma, although it is somewhat disappointing that they have not proven more effective, suggesting that, as with glucocorticoid, external unidentified influences may confound their activity in certain individuals or that there is more to the antiasthma activity of glucocorticoid than turning off cytokine production. The precise effects of these drugs in vivo in the airways of asthmatics have not been systematically studied, although one study showed that attenuation by

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cyclosporin A of allergen-induced late-phase bronchoconstriction in atopic asthmatics was accompanied by reduced infiltration of eosinophils as well as asthma-relevant cytokines and chemokines (Khan et al. 2000). We have shown (Haczku et al. 1994; Powell et al. 2001b) that other immunomodulatory drugs such as sirolimus (rapamycin) and mycophenolate mofetil exert inhibitory effects on T cells from glucocorticoidresistant asthmatics, but the data from existing trials have so far dampened enthusiasm for clinical trials of these newer drugs in severe asthma. As discussed above, in the new concept of “inflammatory” Th2 cells, IL-5, IL-4, IL-13, and TNF-α may be regarded as key effector cytokine products. It was disappointing, therefore, when a single infusion of a humanized monoclonal IL-5blocking antibody (mepolizumab), while markedly reducing circulating blood eosinophils and recruitment of eosinophils to the airway lumen following allergen bronchial challenge of patients with mild asthma, had no effect on the ensuing bronchoconstrictor response or on basal AHR (Leckie et al. 2000). More prolonged studies of anti-IL-5 antibody in patients with moderate/severe asthma were similarly disappointing in terms of improvements in asthma symptoms or lung function. A biopsy study (Flood-Page et al. 2003) showed that while anti-IL-5 antibody profoundly reduced circulating eosinophils, it was less effective in removing eosinophils from the bronchial mucosa, but did appear to reduce deposition of extracellular structural remodeling proteins (see also discussion above). These data have provided asthma researchers with much food for thought. They do not entirely discount a role for eosinophils in regulating asthma severity or AHR, since in none of the studies has eosinophils been completely removed from the bronchial mucosa (although in view of the many factors regulating eosinophil recruitment to the airways, IL-5 blockade alone may never accomplish this). They also suggest, however, the possibility that direct effects of cytokines on the structure of the airways are more important that effects mediated through granulocytes such as eosinophils, which might be acting in a subservient role or even as innocent bystanders. Soluble humanized IL-4 receptor (altrakincept), administered by nebulization, has been tested in clinical trials and shown to prevent deterioration in lung function induced by withdrawal of glucocorticoid in patients with moderately severe asthma (Borish et al. 1999, 2001) but these relatively promising data have not so far been pursued. Nebulized therapy is cumbersome and wasteful, and may be followed in the future by trials of blocking monoclonal antibodies or soluble IL-4 receptor (which would also incidentally be expected to block the actions of IL-13). The pivotal role of IL-13 in airway remodeling has been discussed above, and trials of IL-13 blockade in asthma might be of interest. In addition, apart from the IL-4 receptor, IL-13 has two other specific receptors named IL-13Rα1 and IL-13Rα2. The latter

T Cells and Cytokines in Asthma and Allergic Inflammation

receptor exists naturally in a soluble form, and may act as a “decoy” receptor, scavenging active IL-13. Human soluble IL-13Rα2 is currently in clinical development. As will be apparent from the discussion above, IL-9 and IL-25 are also relatively promising theoretical targets for cytokine blockade in the treatment of asthma. A blocking humanized anti-TNF-α antibody (infliximab) and soluble TNF-α “decoy” receptor (etanercept) have been shown in preliminary studies to have remarkable (and somewhat unprecedented) clinical efficacy in human asthma (Berry et al. 2006; Erin et al. 2006; Howarth et al. 2006). In one study (Howarth et al. 2006), treatment of moderate/severe asthmatics with etanercept 25 mg subcutaneously twice weekly for 12 weeks was associated with improvements in symptoms and reductions in AHR comparable to that seen with systemic glucocorticoid therapy. This therapy seems therefore to hold considerable promise, although experience with the use of TNF-α blockade in other diseases has shown that this approach is not without its dangers (such as reactivation of tuberculosis, opportunistic infection, and possible increased malignancy). Because this therapy has to be given by injection, a search is on for small-molecule TNF-α inhibitors. TNF-α-converting enzyme (TACE) is a matrix metalloproteinase-related enzyme required for the release of TNF-α from cell surfaces where it is stored as an extracellular matrix “reservoir.” Small-molecule TACE inhibitors are currently in development. The importance of IL-10 as an inhibitory cytokine in asthma has already been emphasized. Recombinant IL-10 has proven effective in controlling other inflammatory diseases such as inflammatory bowel disease and psoriasis, where it is given by weekly injection. It is reasonably well tolerated, although hematologic unwanted effects have been noted. The possibility of manipulation of IL-10 production through the induction of T regulatory cells, for example by modified allergens (Klinman 2004; Simons et al. 2004) or by the use of relatively innocuous agents such as vitamin D, as distinct from therapy with IL-10 itself, offers considerable promise for future asthma therapy. As mentioned above, T-cell IL-10 production is increased rather than inhibited by glucocorticoid, and it could possibly prove that this is a critical effect of glucocorticoid not shared by other immunosuppressive drugs, which might explain the disappointing antiasthma efficacy of the latter. The reverse strategy to blocking asthma-relevant cytokines is to attempt to reduce their production by cytokines that inhibit Th2-type T-cell development. IL-12 is a “master regulator” of Th1 T-cell development, as emphasized above. IL-12 and Th1-type cytokines such as IFN-γ strongly inhibit Th1 responses. Recombinant human IL-12 has been administered to humans but it has a range of toxic effects that are somewhat minimized by slow acceleration of the dosage. In a single study on atopic asthmatics (Bryan et al. 2000), weekly

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infusion of IL-12 over 4 weeks, at escalating dosage, caused a progressive fall in circulating eosinophils and also reduced ingress of eosinophils into induced sputum following allergen bronchial challenge. Unfortunately, as with anti-IL-5, there was no evidence of reduction of the allergen-induced bronchoconstrictor response or of the associated increase in AHR. Most of the patients suffered with malaise and one with cardiac dysrhythmia. Consequently, IL-12 alone is unlikely to be developed as a therapeutic agent for asthma. Cytokines such as IL-18 and IL-23, which share some of the effects of IL-12 but have a rather ambiguous role in the disease at present (see above), are other possible therapeutic agents, although problems with unwanted effects similar to those seen with IL-12 seem likely. In summary, these studies have done little more than highlight the chasm of ignorance about how the cellular, immunologic, and structural changes one can observe in asthma are related, if at all, to the clinical manifestations of disease. After more than 25 years of the T-cell/cytokine hypothesis of asthma, there still remains a pressing need to delineate those precise functions of cells and cytokines that are clearly related to causation of the disease, otherwise other breakthroughs in cytokine-directed therapy will remain serendipitous. It is essential to understand what mechanisms cause irreversible airways obstruction in asthma so that therapy can be directed against these. Specific, cytokinedirected therapies offer perhaps the best chance of removing particular cytokines from the equation, but owing to the redundancy of cytokine action, as evidenced by the ventures into anticytokine therapy thus far attempted, they may not necessarily offer viable therapeutic solutions.

Back to the future In summary, there is considerable evidence that T cells and their cytokine products play a role in asthma pathogenesis, but key questions, which have not so much been generated by extant work but have always been lurking in the wings, remain. These may be summarized as follows: • Do T-cell products cause the clinical features of asthma, and if so how? • Will T cell-directed therapy cure asthma or will irreversible changes induced by T-cell products preclude this? • What drives T-cell activation in asthma? • What drives T-cell refractoriness to inhibition in asthma? • Can new forms of therapy, which target multiple cytokines but are nevertheless more specific than glucocorticoids, be developed? • Can we keep effector T cells out of asthmatic airways? • Can we abolish T-cell memory of allergenic and other antigenic, asthma-relevant triggers? • Does the development of asthma in certain individuals reflect alterations in immunology or underlying heterogeneity, as yet uncharacterized, in the structure of the bronchial mucosa?

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Addressing all these challenges provides exciting prospects for future studies.

Acknowledgment I wish to thank my excellent assistants Pamela Robertin and Christine Grace for their many hours of work on this manuscript.

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Regulatory T Cells and Other Tolerogenic Mechanisms in Allergy and Asthma Catherine Hawrylowicz and Cezmi A. Akdis

Summary The induction of immune tolerance and specific immune suppression are essential processes in the control of immune responses. Regulatory T cells (Treg) play a central role in immune control in the periphery. Two broad categories of Treg have been described: naturally occurring Treg that are present in all individuals and antigen-induced Treg that secrete inhibitory cytokines such as interleukin (IL)-10 and/or transforming growth factor (TGF)-β. In allergic disease, the balance between allergen-specific Treg and disease-promoting T helper 2 (Th2) cells appears to be decisive in the development of an allergic versus a nondisease-promoting or “healthy” immune response against allergen. Treg specific for common environmental allergens represent the dominant subset in healthy individuals, arguing for a state of natural tolerance to allergen in these individuals. In contrast, there is a high frequency of allergenspecific Th2 cells in allergic individuals. Treg function appears to be impaired in active allergic disease. Both allergen-specific immunotherapy and certain nonspecific therapies, such as glucocorticoids, enhance Treg function. For example, induction of IL-10- and TGF-β-producing Treg cells, IgG4 isotype blocking antibodies, and suppression of mast cells, basophils and eosinophils represent major components of a relatively normalized immune response after allergen-specific immunotherapy. Through the application of recent advances in our knowledge of Treg biology and related mechanisms of peripheral tolerance, the hope is that more rational and safer approaches for improved prevention and cure of allergic diseases will emerge.

Prevention of the development of allergic responses Many common biological mechanisms are likely to exist to prevent immune responsiveness to innocuous substances or allergens in our environment and to self-antigens, for which an extensive literature exists (Hogquist et al. 2005). While the Allergy and Allergic Diseases, 2nd edition. Edited by A.B. Kay, A.P. Kaplan, J. Bousquet, P.G. Holt. © 2008 Blackwell Publishing. ISBN: 978-1-405-15720-9.

majority of autoreactive T cells undergo selection and clonal deletion in the thymus (central tolerance), a minority escape and are detectable in the periphery. The importance of thymic selection in control of immune responses to allergen is unclear since allergens themselves are unlikely to be present at the time of thymic selection, although negative selection on some cross-reactive determinants may occur. Additional immunologic controls are therefore in place to eliminate or inactivate potentially hazardous effector cells that emerge from the thymus into the periphery. Allergens themselves are often not highly immunogenic compounds. Many allergens enter the body via the respiratory tract or the gut which, unlike other routes of immunization, in the steady state generally results in the induction of tolerance (Macaubas et al. 2003). In addition to this, specialized T-cell subsets, broadly termed regulatory T cells (Treg), play an essential and protective role in immune suppression in the periphery. This review focuses on the evidence that Treg cells play a central role in the prevention of disease-promoting allergen-specific Th2 responses in health, that Treg function may be impaired in allergic disease, and how therapeutic strategies aim to restore, boost and/or induce de novo Treg activity.

Regulatory T cells (Fig. 4.1) The existence of suppressor cells, which limit ongoing immune responses and prevent autoimmune disease, was postulated over 30 years ago (Gershon 1975). The recent phenotypic and functional characterization of these cells has led to a resurgence of interest in their therapeutic application in a number of immune-mediated diseases. Two broad subsets of CD3+CD4+ suppressive or Treg cells have been described: constitutive or naturally occurring versus adaptive or inducible Treg. Subsets of Treg cells and their suppressor mechanisms are shown in Table 4.1. There are other Treg populations, including CD8+ Treg cells with the reported capacity to both inhibit and exacerbate allergic airway disease (Stock et al. 2004a; Noble et al. 2006). In addition, double-negative (CD4− CD8−) T-cell receptor (TCR)αβ+ Treg cells that mediate tolerance in several experimental autoimmune diseases (Strober

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Induction of IgG4, IgA suppression of IgE

Suppression of Th2 cell homing to tissues Endothelial cells

Epithelial activation, release of chemokines IP-10, mig and iTac

B cell

IL-

4,

IL-

13

Decreased Th2 cytokines

Epithelial apoptosis

13 ion t 4, duc IL ro p E Ig IL-

Th2

13

L,I

9 IL-

IL-10 TGF-b TReg

IL-

3,

IL-

4,

IL-10 TGF-b

IL-

5,

IL-

9

Mucus production

Suppression of Th0/Th1/Th2 effector cells

Direct and indirect suppressive effects on mast cells, basophils and eosinophils

Fig. 4.1 Suppression of Th2 and Th1 cell-mediated features of allergic inflammation by Treg cells. Treg cells utilize multiple suppressor factors to regulate undesired activity of effector Th2 cells. Interleukin (IL)-10 and transforming growth factor (TGF)-b suppress IgE production and induce noninflammatory immunoglobulin isotypes IgG4 and IgA, respectively. Furthermore, these two cytokines directly suppress allergic inflammation induced by effector cells such as mast cells, basophils, and eosinophils. In addition, Th2 cells are suppressed by Treg cells and can therefore no longer provide cytokines such as IL-3, IL-4, IL-5, IL-9, and IL-13. These cytokines

T regulatory cells

Suppressor mechanism(s)

Reference

Tr1, IL-10-Treg

IL-10, TGF-b, CTLA-4, PD-1

Akdis (1998) O’Garra & Vieira (2004) Hawrylowicz & O’Garra (2005)

Th3

TGF-b, IL-10

Li et al. (2006) Faria & Weiner (2006)

CD4+CD25+ Treg

Membrane TGF-b, CTLA-4, PD-1, GITR, IL-10

Maloy & Powrie (2001) Bluestone & Abbas (2003) O’Garra & Vieira (2003, 2004) Shevach (2002) Sakaguchi (2004)

CD8+CD25+CD28− Treg

Same as CD4+CD25+

Jutel (2003) Akdis (2004) Stock et al. (2004a) Noble et al. (2006)

CD4−CD8− Treg

Induction of apoptosis

Thomson et al. (2006)

TCRgd Treg

IL-10, TGF-b

Strober et al. (1996)

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Th0 Th1 IFN-g TNF-a

Smooth muscle cell activation and apoptosis

are required for the differentiation, survival, and activity of mast cells, basophils, eosinophils, and mucus-producing cells, as well as for the tissue homing of Th2 cells. The Th1 cytokine interferon (IFN)-g, in combination with tumor necrosis factor (TNF)-a and/or FasL, induces apoptosis of smooth muscle cells, keratinocytes, and bronchial epithelial cells as essential tissue injury events in atopic dermatitis and asthma. Treg cells suppress the stimulation of Th0/Th1 cells, leading to the abrogation of tissue injury mechanisms (red lines indicate suppression, black lines stimulation). (See CD-ROM for color version.)

Table 4.1 Subsets of T regulatory cells and their suppressive mechanisms.

See text for definition of abbreviations.

IFN-g TNF-a

Basophil Eosinophil mast cell

Suppression of mucus production

FasL

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Table 4.2 General immunologic properties of IL-10* and TGF-b†. Cell type

IL-10

TGF-b

Dendritic cells

Inhibits DC maturation, leading to reduced MHC class II and costimulatory ligand expression Inhibits proinflammatory cytokine secretion Inhibits APC function for induction of T-cell proliferation and cytokine production (Th1 and Th2)

Promotes Langerhans cell development Inhibits DC maturation and antigen presentation Downregulates FceRI expression on Langerhans cells

T cells

Suppresses allergen-specific Th1 and Th2 cells Blocks B7/CD28 costimulatory pathway on T cells

Promotes T-cell survival Inhibits proliferation, differentiation, and effector function, including allergen-specific Th1 and Th2 cells Promotes the Th17 lineage

B cells and immunoglobulin

Enhances survival Promotes immunoglobulin production, including IgG4

Inhibits proliferation Induces apoptosis of immature or naive B cells Inhibits most immunoglobulin class switching Switch factor for IgA

IgE

Suppresses allergen-specific IgE

Suppresses allergen-specific IgE

CD25+

Treg

Upregulates Foxp3 Promotes generation in the periphery Potential effects on homeostasis

IL-10-Treg

Promotes IL-10-Treg induction

Can promote IL-10 synthesis

Monocytes/macrophages

Inhibits proinflammatory cytokine production and antigen presentation

Inhibits scavenger and effector functions including proinflammatory cytokine production, and antigen presentation Promotes chemotaxis

Eosinophils

Inhibits survival and cytokine production

Chemoattractant

Mast cells

Inhibits mast cell activation, including cytokine production

Promotes chemotaxis Variable effects on other functions; may inhibit expression of FceR

Neutrophils

Inhibits chemokine and proinflammatory cytokine production

Potent chemoattractant

* Extensive reviews of the immunologic properties of IL-10 can be found in Moore et al. (1993), Hawrylowicz & O’Garra (2005) and Asadullah et al. (2003). † Extensive reviews of the immunologic properties of TGF-b can be found in Li et al. (2006) and Faria & Weiner (2006). See text for definition of abbreviations.

et al. 1996) and TCRγδ+ Treg cells that can play a role in the inhibition of immune responses to tumors (Seo et al. 1999; Hayday & Tigelaar 2003; Jiang et al. 2006; Thomson et al. 2006) have been described. An immunoregulatory role for interleukin (IL)-10-secreting B cells and dendritic cells (DCs) which have regulatory/suppressor properties has been recently suggested (Steinbrink et al. 1997; Akbari et al. 2001; Mauri et al. 2003). In addition natural killer (NK) cells, epithelial cells, macrophages, and glial cells express suppressor cytokines such as IL-10 and transforming growth factor (TGF)-β (Table 4.2) (Moore et al. 1993; Li et al. 2006). Although the role of many of these cell types has not been coined as professional regulatory cells, as yet we know little regarding their capacity and importance in modulating the allergic response and it is feasible that some of these cells

contribute efficiently to the generation and maintenance of a regulatory/suppressor-type immune response.

Naturally occurring CD4+CD25+Foxp3+ Treg Naturally occurring Treg constitute less than 5% of the CD3+CD4+ population in healthy unmanipulated animals and humans (Maloy & Powrie 2001; Shevach 2002; Bluestone & Abbas 2003; O’Garra & Vieira 2003, 2004). They are characterized by the constitutive expression of high levels of the α chain of the IL-2 receptor, CD25, which is generally used to isolate and study this population. There are two major hypotheses concerning the generation of these Treg cells. One suggests that Treg cells emerge from the thymus as a distinct subset of mature T cells with defined functions (Jordan et al. 2001; Sakaguchi 2004). Conversely, several studies have

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shown that Treg cells may differentiate from naive T cells in the periphery, discussed further below (Chen et al. 2003; Hawrylowicz & O’Garra 2005). The relative contribution of the two pathways is still unclear (Sakaguchi 2003). While a number of studies suggest that thymic differentiation accounts for Treg cells that are specific for self-peptides, peripheral differentiation may be required for environmental antigenspecific T cells for which an undesired immune response results in pathology.

Markers of naturally occurring CD25 + Treg These cells constitutively express cytotoxic T-lymphocyte antigen (CTLA)-4, a negative regulator of adaptive immune responses (Boden et al. 2003), which binds the B7 (CD80 and CD86) costimulatory molecules on antigen-presenting cells (APC). They also express PD-1, an immunoreceptor tyrosinebased inhibitory motif-containing receptor expressed upon T-cell activation. PD-1/PD-ligand interactions inhibit IL-2 production and may play a role in the suppressive function of Treg cells (Carter et al. 2002). Glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) is also constitutively expressed by unactivated Treg and abrogates Treg suppression probably by mediating resistance to suppression in effector T cells (McHugh et al. 2002; Shimizu et al. 2002; Stephens et al. 2004). CD103 (αEβ7 integrin) and CD122 (β chain of IL-2 receptor) are highly expressed on Treg cells, which correlates with their suppressive activity (Lehmann et al. 2002; Wing et al. 2002). Other proposed markers include certain chemokine receptors, Toll-like receptors (TLRs), membrane-bound TGF-β, neuropilin-1 (Bruder et al. 2004), lymphocyte activation gene (LAG)-3, and granzymes. However, none of these markers are specific and many are also expressed by other regulatory and effector T-cell populations (Banham 2006; Banham et al. 2006). Gene profiling studies have identified additional markers, including G-proteincoupled receptor 83 (Gpr83), Ecm1, and Helios (Pfoertner et al. 2006; Sugimoto et al. 2006). Further validation of their profile of expression on a range of effector and regulatory T-cell populations will determine their value as unique markers of Treg. There remains an urgent need for lineagespecific cell-surface markers to facilitate the study of the naturally occurring Treg population in patients.

The forkhead winged transcription factor, Foxp3 The intracellular forkhead winged transcription factor Foxp3 (forkhead box P3) appears to be specifically expressed by naturally occurring Treg cells (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003), particularly in mice, although in humans there is evidence of upregulation of Foxp3 in all T cells on activation. Foxp3 is required for the development and function of naturally occurring Treg (Fontenot et al. 2005) and expression is sufficient to convert nonregulatory CD4+CD25− T cells into cells with regulatory activity. Conversion of peripheral CD4+CD25− naive T cells to

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Foxp3+CD4+CD25+ Treg cells can be induced by TGF-β (Chen et al. 2003). In a murine asthma model, these TGF-β-induced Treg prevented house-dust mite-induced allergic pathogenesis in lungs (Chen et al. 2003). A single independent report has suggested that IL-4 and IL-13 also induce Foxp3+CD25+ Treg from CD4+CD25− precursors (Skapenko et al. 2005). Studies in both humans and mice of mutations affecting the function of the gene encoding Foxp3 show loss of the naturally occurring Treg compartment. Human subjects affected by the X-linked autoimmune and allergic dysregulation syndrome (XLAAD) or immune dysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) suffer from autoimmunity and severe atopy from an early age, including eczema, food allergy, and eosinophilic inflammation (Chatila et al. 2000; Bennett et al. 2001; Wildin et al. 2001). Analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. In mice, original studies in the scurfy mouse and more recently using a targeted loss-of-function mutation in the murine Foxp3 gene have described an intense multiorgan inflammatory response associated with allergic airway inflammation, a striking hyperimmunoglobulinemia E, eosinophilia, and dysregulated Th1 and Th2 cytokine production in the absence of overt Th2 skewing (Lin et al. 2005). These types of data, reflecting the absence of Foxp3+ Treg control at the time of initial allergen exposure support a role for the naturally occurring Treg compartment in prevention of sensitization to allergen.

Mechanisms of suppression Several modes of action of Treg have been proposed, including cell-contact dependent mechanisms, observed in most in vitro studies, as well as cytokine-dependent ones (Maloy & Powrie 2001; Shevach 2002; Bluestone & Abbas 2003; O’Garra & Vieira 2003, 2004). Suppression may be targeted at effector T cells and/or APC to reduce the ability of APC to prime T cells via modulation of costimulation and cytokine production, or the increase of tryptophan metabolism. Cell-contact inhibitory mechanisms can involve delivery of negative costimulatory signals via CTLA-4, although this mechanism is not exclusive, since Treg cells isolated from mice with deletion of the CTLA-4 gene were still suppressive in vitro (Paust et al. 2004; Tang et al. 2004). A role for cellsurface TGF-β has also been proposed. CD4+CD25+ Treg have been reported to directly kill T-cell effectors in a perforin and granzyme-dependent cytolysis (Grossman et al. 2004). In vitro studies have suggested that human thymus-derived CD25+ Treg inhibit Th2 responses less efficiently than Th1 responses (Cosmi et al. 2004). CD25+ Treg may efficiently inhibit Th2 differentiation, but are less effective for inhibition of cytokine production and proliferation of established Th2 cells, requiring preactivation in vitro for strong inhibition of Th2 responses (Stassen et al. 2004). In studies using peripheral blood, T cells from healthy nonatopic donors show

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poor responses to allergen in culture for proliferative and Th2 cytokine responses in comparison with atopic patients. If, however, peripheral blood mononuclear cells (PBMCs) from nonatopic donors are depleted of the CD4+CD25+ Treg compartment prior to stimulation with allergen, increased proliferative and Th2 cytokine responses are observed (Ling et al. 2004). These studies imply that active control of the allergic response occurs rather than the lack of T cells with allergic potential in these individuals. The mechanism of suppression in vivo appears to be highly dependent on the experimental system being studied and may vary according to the tissue, type of inflammation, and animal model under study. Several early studies demonstrated that naturally occurring CD25+ Treg inhibit allergic airway disease in mice (reviewed and fully referenced in Hawrylowicz 2005; Hawrylowicz & O’Garra 2005). In a recent mechanistic study, CD4+CD25+ T cells suppressed the Th2 celldriven response to allergen in vivo by an IL-10-dependent mechanism whereby CD25+ Treg induced expression of IL-10 by resident lung CD4+ T cells (Kearley et al. 2005), while a second study suggested that naturally occurring lung CD25+ T-cell regulation of airway allergic responses was dependent on IL-10 induction of TGF-β (Joetham et al. 2007). A further study concluded that inhibition was mediated by CD4+CD25+ Treg cell suppression of DC activation and that the absence of this regulatory pathway contributed to disease susceptibility (Lewkowich et al. 2005). The maintenance of protective Treg activity depends on continuing allergen stimulation (Strickland et al. 2006). While most studies to date have indicated at least some capacity to inhibit allergic airway inflammation, recent studies have also highlighted that some inhibition of airway hyperresponsiveness occurred. Reports on the capacity to inhibit structural remodeling in the airways are awaited with interest.

Loss of suppression during acute inflammatory responses An emerging concept is that proinflammatory signals lead to loss of Treg function. Pasare and Medzhitov (2003) demonstrated that activation of DCs through TLRs led to the production of signals, including IL-6, which blocked the suppressive effect of CD4+CD25+ Treg. Subsequent studies support these observations. For example in a mouse model of allergic airway disease, IL-6 is proposed to act via two mechanisms to promote disease: direct enhancement of Th2 responses and by overcoming the suppressive function of CD4+CD25+ Treg (Doganci et al. 2005). Tumor necrosis factor (TNF) (Valencia et al. 2006; Nadkarni et al. 2007) as well as IL-7 and IL-15 (Ruprecht et al. 2005) have also been proposed to overcome regulatory activity in other human immunologic diseases. In humans with strong responses to allergen or in patients tested during hay-fever season, regulation by peripheral blood-derived CD4+CD25+ Treg is impaired (Bellinghausen

et al. 2003; Grindebacke et al. 2004; Ling et al. 2004). The mechanism for this is unclear and could variously imply dilution of Treg cells by activated effector T cells in the periphery, direct loss of regulatory activity, refractoriness of T cells or APCs to suppression, or other mechanisms. One study suggests that only a proportion of allergic patients exhibit defective CD4+CD25+ Treg activity because the function of Treg depends on the concentration and type of the respective allergen, with different thresholds for individual allergens and patients (Bellinghausen et al. 2005). In conclusion, CD4+CD25+ Treg clearly have the capacity to control diseasepromoting Th2 responses to allergen in both animal models and in human cultures. While it would clearly be desirable to fully restore the activity of this Treg compartment in allergic patients, it is as yet unclear whether this would be sufficient for long-term disease resolution. If proinflammatory signals overcome CD25+ Treg activity, then dampening down of the proinflammatory environment might be predicted to restore CD25+ Treg function. Glucocorticoids represent the cornerstone of asthma therapy, and exhibit well-established antiinflammatory actions. Several studies have assessed the effects of glucocorticoids on Treg activity in vitro and in vivo, with all implying some enhancement of regulatory function (Dao Nguyen & Robinson 2004; Karagiannidis et al. 2004; Chen et al. 2006). The immunosuppressive agent rapamycin reportedly expands human CD25+ Treg in vitro as well as in type 1 diabetic patients in whom a defect in these cells has been reported (Battaglia et al. 2006).

Therapeutic application of naturally occurring Treg The capacity to either expand existing CD25+ Treg populations or convert CD4+CD25− T cells to a regulatory phenotype offers the potential for adoptive transfer therapy for various immune-mediated pathologies (Levings et al. 2001; Bluestone 2005; Kretschmer et al. 2006). A major limitation may be the capacity to generate sufficient numbers of antigen-specific T cells. New technologies for the rapid in vitro generation of antigen-specific T cells by transduction of appropriate receptors (TCR) in a manner which promotes preferential transduced α- and β-chain pairing, increased total surface expression of the introduced TCR, and reduced mismatching with endogenous TCR have been pioneered for the treatment of malignancy (Kuball et al. 2006), and such techniques clearly have the potential to be applied to other immunemediated diseases, including allergy. However it is debatable whether such therapy will be appropriate for the treatment of allergic disease, based on both safety considerations and cost.

Antigen-specific Treg Naive peripheral T cells are able to acquire regulatory characteristics as a consequence of peripheral induction after

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exposure to antigen (induced or adaptive Treg) (O’Garra & Vieira 2004; Hawrylowicz & O’Garra 2005). These cells are characterized by expression of the antiinflammatory cytokines IL-10 (Tr1 or IL-10-Treg) and/or TGF-β (Th3), and in many cases also express CD25. Factors influencing development of adaptive Treg include soluble antigen or peptide in the absence of adjuvant, immature or cytokine-activated DC populations, and certain soluble mediators in the microenvironment in which antigen is encountered.

β and Th3 TGF-β TGF-β is a potent regulatory cytokine produced by a wide range of cell types, including T lymphocytes (reviewed in Li et al. 2006; see Table 4.2). TGF-β plays a pivotal role in the maintenance of tolerance within the immune system, particularly oral tolerance (reviewed and fully referenced in Faria & Weiner 2006; Li et al. 2006). TGF-β inhibits both B- and T-lymphocyte proliferation, differentiation and survival. It inhibits most immunoglobulin isotype switching, but notably promotes the differentiation of IgA-secreting plasma cells, which is associated with defense to microbial infection in the mucosa as well as successful allergen immunotherapy (see below). TGF-β promotes the differentiation of Langerhans cells and DCs with an immature phenotype. It acts on monocytes and macrophages, promoting a proinflammatory phenotype in the former, but a largely inhibitory phenotype in macrophages. In mast cells TGF-β promotes mast cell chemotaxis, but may inhibit FcεRI expression. Eosinophils can produce significant quantities of TGF-β, which has complex effects on their survival and activation. TGF-β is also associated with the resolution of immune responses and the induction of regulatory T-cell populations, as discussed above. Given these broad-ranging functional properties, the effects of TGF-β in allergic disease are complex, with evidence of both disease-inhibitory and -promoting effects. TGF-β has a demonstrated capacity to inhibit human Th2 responses in vitro (Kunzmann et al. 2003). In a murine model of ovalbumin (OVA)-induced allergic airway asthma, overexpression of TGF-β1 in OVA-specific CD4+ T cells abolished airway hyperresponsiveness and airway inflammation induced by OVA-specific Th2 cells (Hansen et al. 2000). A recent study of TGF-β1 heterozygous mice, which produce smaller quantities of TGF-β1 compared with wild-type mice, exhibited exacerbated allergic airway immunopathology (Scherf et al. 2005), suggesting that endogenous TGF-β1 can suppress airway disease in mice. T cells appear to be an important target of TGF-β1 action, since antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice, which show a block in TGF-β signaling. Both OVA-induced airway hypersensitivity and airway inflammation were enhanced in the transgenic mice and were associated with high production of Th2 cytokines (Nakao et al. 2000). Involvement of TGF-β in the regulation of allergic

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airway disease by naturally occurring Treg has also been reported. In a study by Ostroukhova et al. (2004), tolerance induced by repeated exposure to low doses of inhaled allergen involved CD4+ T cells expressing both membrane-bound TGF-β and Foxp3, while Joetham et al. (2007) suggested that lung CD4+CD25+ Treg controlled airway disease through local induction of TGF-β production. These studies suggest that endogenous TGF-β acts to minimize airway sensitization and inflammation, and all use mouse models of acute airway inflammation. They do not address the potential of TGF-β for inducing structural remodeling in the lung, where clear associations of TGF-β with chronic human lung disease have been documented (Gauldie et al. 2006). In a mouse model using prolonged exposure to allergen that exhibited many properties of chronic asthma, blockade of TGF-β even after the onset of established eosinophilic airway inflammation significantly reduced peribronchiolar extracellular matrix deposition, airway smooth muscle cell proliferation, and mucus production in the lung without affecting established airway inflammation and Th2 cytokine production (McMillan et al. 2005), leading the authors to suggest it may be possible to uncouple airway inflammation and remodeling during prolonged allergen exposure. The contribution of TGF-β in the regulation of human asthma remains to be established. Basal levels of TGF-β1 are reported to be higher in the airways of atopic asthmatics compared with normal controls (Redington et al. 1997) and these levels increase further in response to segmental bronchoprovocation with allergen. These authors conclude that their findings are consistent with the hypothesis that TGF-β1 is implicated in airway wall remodeling in asthma. However from all the published data it is possible to conclude that TGF-β1 in patients may represent a combination of the following: 1 a negative feedback mechanism to control airway inflammation; 2 since TGF-β1 is important in healing, increased TGFβ1 production may be involved in the repair of asthmatic airways; 3 TGF-β1 may also induce fibrosis to exaggerate disease development (Branton & Kopp 1999).

IL-10 and IL-10-secreting Treg IL-10 is of interest in the control of allergy and asthma, since it inhibits many effector cells and functions associated with disease and is inversely correlated with disease incidence and severity (reviewed and fully referenced in Moore et al. 1993; Asadullah et al. 2003; Hawrylowicz & O’Garra 2005; Urry et al. 2006). IL-10 is synthesized by a wide range of cell types, including APCs and T cells. It inhibits proinflammatory cytokine production, as well as Th1 and Th2 cell activation, which is likely to be largely attributable to effects on APCs, although direct effects on T-cell function have also been noted. IL-10 impairs mast cell and eosinophil activation, effector

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cells associated with early- and late-phase allergic responses. It promotes IgG4 synthesis and the induction of IL-10 following allergen immunotherapy has been suggested to account for the favorable change in ratio of IgG4 to IgE associated with successful allergen immunotherapy (discussed at length below). Studies in both murine models and in humans have proposed that IL-10 plays a role in maintaining immune homeostasis in the lung in health (Hawrylowicz & O’Garra 2005; Urry et al. 2006). An inverse correlation exists between the presence of IL-10 and the incidence and severity of asthmatic disease in the lung (Borish et al. 1996; Lim et al. 1998). For example, the bronchoalveolar lavage of asthmatic patients contains lower levels of IL-10 than healthy individuals and T cells from asthmatic children produce less IL-10 than those from healthy ones (Borish et al. 1996; Hawrylowicz & O’Garra 2005). Additionally, an inverse correlation also exists with the levels of IL-10 and skin-prick test reactivity to allergen (Heaton et al. 2005). IL-10-positive T cells consistently represent the dominant subset specific for common environmental allergens in the peripheral blood of healthy individuals; in contrast, there is a high frequency of allergen-specific IL-4-secreting T cells in allergic individuals (Akdis, M. et al. 2004). In addition bee-keepers who are naturally tolerant to bee venom allergen, due to multiple bee stings, demonstrate a similar high IL-10 response (Akdis et al. 1998). IL-10 production by T-cell clones derived from the blood of children with cows’ milk allergy produced IL-4 and IL-13, whereas those from control nonallergic children were characterized by the production of IL-10 and, to a lesser extent, interferon (IFN)-γ (Tiemessen et al. 2004). All these studies suggest that increased levels of IL-10 synthesis, including allergenspecific production, are associated with the lack of allergic symptoms. A large body of published data describes the capacity of IL-10 to control allergic airway disease in mice. For example, studies where recombinant murine IL-10 was instilled intranasally at the same time as allergen challenge (ZuanyAmorim et al. 1995) or studies of IL-10 gene delivery to the airways demonstrated suppression of cellular recruitment and airway inflammation (Stampfli et al. 1999; Nakagome et al. 2005). Similarly, CD4 T-helper cells engineered to produce IL-10 prevented allergen-induced airway hyperreactivity and inflammation (Oh et al. 2002), while adoptive transfer of IL-10-secreting cells (Tr1) inhibited a Th2-specific response in vivo (Cottrez et al. 2000). Studies describing the induction of tolerance in the airways highlight the association of IL-10 with protection (Akbari et al. 2001, 2002; Stock et al. 2004b). Suppression of airway eosinophilia by a killed Mycobacterium vaccae preparation induced allergen-specific Treg and protection was associated with the synthesis of both IL-10 and TGF-β (Zuany-Amorim et al. 2002). Finally as discussed above, protection by adoptive transfer of naturally occurring CD4+CD25high Treg was dependent on the induc-

tion of IL-10 in some studies (Kearley et al. 2005; Joetham et al. 2007).

Generation and therapeutic application of inducible Treg cells A wide variety of protocols have been developed to generate induced Treg cells from naive cells either in vitro, using antigen, cytokines, and DCs, or in vivo by antigen administration (protocols of tolerance induction) (extensively reviewed and referenced in Bluestone & Abbas 2003; O’Garra & Vieira 2004; Hawrylowicz & O’Garra 2005). Other inducible Treg cells have been described as deriving from naive T-cell populations after, for example, in vitro induction in the presence of IL-10 alone, IL-10 together with other cytokines such as IL-4 or IFN-α, or after repetitive stimulation in the presence of immature DCs. Similarly, a wide range of antigen administration protocols have been used to induce Treg cells not only in vitro but in vivo as well (O’Garra & Vieira 2004). A common feature of many of these induced Treg populations is the production of not only IL-10 and/or TGF-β, but also Th1- and Th2-associated cytokines, and therefore they have the potential to exacerbate inflammatory reactions. Additionally, since many exhibit a limited potential to expand, they may have a restricted clinical application. A strategy designed to generate Treg secreting IL-10, in the absence of Th1- and Th2-associated cytokines, employed in vitro stimulation of CD4+ cells in the presence of glucocorticoids, either alone or, more effectively, plus the active form of vitamin D, 1α,25-dihydroxyvitamin D3. These cells were able to regulate autoimmunity in vivo (experimental allergic encephalomyelitis or EAE) in an IL-10-dependent manner (Richards et al. 2000; Barrat et al. 2002). Interestingly in mice, these drug-induced IL-10-secreting Treg cells were shown to have similar functional suppressive properties to the naturally occurring Treg cells in vitro and were able to also inhibit naive T cells via cell contact-dependent pathways, even though they did not express Foxp3 (Vieira et al. 2004). In humans both these drug-induced IL-10-Treg inhibited the proliferation and cytokine responses of naive as well as established Th1 and Th2 cells, including allergen-specific Th2 cell lines (Xystrakis et al. 2006). Additional studies suggest that it may be possible to use other drug combinations to effectively generate IL-10-Treg populations through effects on either the T-cell or APC compartment or both (Adorini 2002; Hawrylowicz & O’Garra 2005; Peek et al. 2005).

Therapies associated with the induction of Treg β and the production of IL-10 and/or TGF-β A number of examples of both antigen-specific and nonantigen-specific treatments for allergic diseases, including asthma, are associated with the induction of IL-10, as well as TGF-β in some cases. In the case of nonantigenspecific therapies, glucocorticoids highlight this concept. Glucocorticoids induce the synthesis of IL-10 both in patients

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(Wan et al. 1997; John et al. 1998) and in vitro (Richards et al. 2000). The relevance to clinical efficacy of steroid action is illustrated by the significantly reduced capacity of T cells from asthma patients who are clinically insensitive to steroid treatment, to respond to dexamethasone in culture by the synthesis of IL-10. This is in marked contrast to T cells from either healthy donors or steroid-sensitive asthma patients of comparable age and disease severity (Hawrylowicz et al. 2002). These steroid-insensitive patients represent a challenge to the respiratory physician, utilize a disproportionate amount of asthma healthcare budgets, and represent those most at risk of hospitalization or death from their asthma. The capacity of the active form of vitamin D, 1α,25dihydroxyvitamin D3 to enhance steroid-induced IL-10 and induce an IL-10-Treg phenotype was discussed above (Barrat et al. 2002). Notably, combining dexamethasone and 1α,25-dihydroxyvitamin D3 in cultures of T cells from steroidinsensitive asthma patients greatly enhanced IL-10 synthesis, with levels comparable to those observed in steroid-sensitive patients stimulated in the presence of steroid alone (Xystrakis et al. 2006). In a proof-of-concept study, ingestion of 1α,25dihydroxyvitamin D3 (calcitriol/rolcatrol) by these patients for just 1 week, was sufficient to restore their responsiveness to steroids for the induction of IL-10 synthesis (Xystrakis et al. 2006). These studies open up the exciting possibility of a potential clinical steroid-sparing effect of 1α,25dihydroxyvitamin D3 in asthma. This could be applicable not only to those patients who are completely refractory to steroid treatment, but also to those who demonstrate high-dose long-term dependency for steroids. Studies to date suggest that 1α,25-dihydroxyvitamin D3 may act to rescue the glucocorticoid receptor from ligand-dependent downregulation (Xystrakis et al. 2006).

Mechanisms of allergen-specific immunotherapy and the involvement of Treg cells Allergen-specific immunotherapy is highly effective in the treatment of IgE-mediated diseases such as rhinitis, conjunctivitis, asthma, and venom hypersensitivity. It is the only treatment that leads to lifelong tolerance against previously disease-causing allergens due to restoration of the normal immunity. However, it is not an alternative but an important part of the complex treatment that includes antihistamines, antileukotrienes, β2-adrenergic receptor antagonists, and corticosteroids, aiming at suppression of mediators and immune cells. Allergen-specific immunotherapy is most efficiently used in allergy to insect venoms and allergic rhinitis, particularly seasonal pollinosis (Bousquet et al. 1998; Durham et al. 1999; Kussebi et al. 2003; Bonifazi et al. 2005). Immunotherapy also improves asthma and inhibits seasonal increases in bronchial hyperresponsiveness (Walker et al. 2001). It

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has also been shown to prevent onset of new sensitizations (Pajno et al. 2001) and reduce development of asthma in patients with rhinitis caused by inhalant allergens (Eng et al. 2002; Moller et al. 2002). The allergen specificity of immunotherapy is crucial in the understanding of its benefits and the underlying mechanisms, which are slowly being elucidated. In 1911, the original report of Noon suggested that grass pollen extracts, used for immunotherapy of hay fever, induced a toxin that caused allergic symptoms. It was suggested that in response to injection of pollen extract, antitoxins develop and prevent the development of disease. Indeed, generation of neutralizing antibodies was demonstrated during specific immunotherapy (Cooke et al. 1935; Lichtenstein et al. 1966). It has now been acknowledged that activated T cells and their products play a major role in the pathogenesis of allergic diseases and allergen-specific T cells were considered the major target for specific immunotherapy (Table 4.3) (Jutel et al. 1995, 2003; Akdis et al. 1996, 1998; Bellinghausen et al. 1997; Marcotte et al. 1998; Müller et al. 1998; Akdis & Blaser 1999; Durham et al. 1999). Specific immunotherapy was found to be associated with a decrease in IL-4 and IL-5 production by CD4+ Th2 cells, and a shift toward increased IFN-γ production by Th1 cells. New light was shed when a further subtype of T cells, with immunosuppressive function and cytokine profiles distinct from both Th1 and Th2 cells and termed “regulatory/ suppressor” or Treg cells, was described (Chen et al. 1994; Powrie et al. 1994; Groux et al. 1997; Jordan et al. 2001; Shevach 2002; Chen et al. 2003; Sakaguchi 2004; Akdis et al. 2005; Hawrylowicz & O’Garra 2005). The evidence for their existence in humans has been demonstrated (Akdis et al. 1998, 2005; Jonuleit et al. 2000; Taams et al. 2001; Hawrylowicz & O’Garra 2005). Skewing of allergen-specific effector T cells to Treg cells appears as a crucial event in the control of healthy immune response to allergens and successful allergen-specific immunotherapy (Akdis, M. et al. 2004; Akdis, C.A. et al. 2004a). Thus, subsequent variants of the theory of “yin–yang” balance (i.e., T suppressor/Th, Th1/ Th2, or Treg/Th2) have been proposed. In addition, mediators of allergic inflammation that trigger cAMP-associated G protein-coupled receptors, such as histamine H2 receptor, may contribute to peripheral tolerance mechanisms (Jutel et al. 2001; Akdis & Blaser 2003). Most studies available so far have examined the mechanisms of subcutaneous immunotherapy. There is much less clear evidence on the immunologic effects of immunotherapy by alternative, especially sublingual, routes (Bousquet 2005).

Treg cells in allergen-specific immunotherapy T cells constitute a large population of cellular infiltrate in atopic/allergic inflammation and a dysregulated immune response appears to be an important pathogenetic factor. Cardinal events during allergic inflammation can be classified as activation, organ-selective homing, survival and

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Table 4.3 Effects of allergen-specific immunotherapy on immunologic parameters related to immune tolerance. Mechanism T cells

Decreased allergen-induced proliferation Induction of Treg cells Increased secretion of IL-10 and TGF-b Suppression of Th2 cells and cytokines Decreased T-cell numbers in late-phase response

B cells

Decreased specific IgE production Increased specific IgG4 production Increased specific IgA production Suppressed IgE-facilitated antigen presentation

Dendritic cells

Suppressed IgE-facilitated antigen presentation

Eosinophils

Reduction of tissue numbers Decrease in mediator release

Mast cells

Reduction of tissue numbers Decrease in mediator release Decrease in proinflammatory cytokine production

Basophils

Decrease in mediator release Decrease in proinflammatory cytokine production

reactivation, and effector functions of immune system cells (Akdis et al. 2000; Akdis, C.A. et al. 2004b). T cells are activated by aeroallergens, food antigens, autoantigens, and bacterial superantigens in allergic inflammation. They are under the influence of the skin, lung, or nose-related chemokine network and show organ-selective homing. Prolonged survival of the inflammatory cells in the tissues and consequent reactivation is observed in the subepithelial tissues (Simon & Blaser 1995; Akdis et al. 1999, 2003). Finally, T cells display effector functions, resulting in the induction of hyper-IgE, eosinophil survival, and mucus hyperproduction (Simon & Blaser 1995; Akdis et al. 1999; Whittaker et al. 2002), and interact with bronchial epithelial cells and keratinocytes causing their activation and apoptosis (Akdis, C.A. et al. 2004b). Peripheral T-cell tolerance to allergens can overcome all the above pathologic events in allergic inflammation, because they all require T-cell activation (see Table 4.3). The initial event responsible for the development of allergic diseases is the generation of allergen-specific CD4+ T helper cells. Under the influence of IL-4, naive T cells activated by APCs differentiate into Th2 cells (Romagnani 1994; Mosmann & Sad 1996; Rincon et al. 1997; Corry 1999). Once generated, effector Th2 cells produce IL-4, IL-5, and IL-13 and mediate several regulatory and effector functions. These cytokines induce the production of allergen-specific IgE by B cells, development and recruitment of eosinophils, production of mucus, and contraction of smooth muscles (Romagnani 1994; Mosmann & Sad 1996; Rincon et al. 1997;

Corry 1999). The degranulation of basophils and mast cells by IgE-mediated cross-linking of receptors is the key event in type I hypersensitivity, which may lead to chronic allergic inflammation. Distinct type 1 and type 2 subpopulations of T cells discriminated on the basis of cytokine secretion and function counterregulate each other and play a role in distinct diseases. Importantly, Th1 cells also contribute to chronicity and effector phase in allergic diseases, particularly by activation and apoptosis of resident tissue cells (Trautmann et al. 2000, 2001a,b, 2002; Yssel & Groux 2000; El Biaze et al. 2003). IFN-γ, TNF-α, and Fas pathways play essential roles in epithelial cell activation and apoptosis, which leads to spongiosis in atopic dermatitis and epithelial shedding in asthma (Akdis et al. 2000; Akdis, C.A. et al. 2004b). Although in early studies a switch from Th2- to Th1-type cytokines has been reported (Jutel et al. 1995; Durham & Till 1998), recent studies have demonstrated that peripheral T-cell tolerance is crucial for a healthy immune response and successful treatment of allergic disorders (Jutel et al. 1995, 2003; Francis et al. 2003; Akdis, M. et al. 2004). The tolerant state of specific cells results from increased IL-10 secretion (Jutel et al. 1995). The cellular origin of IL-10 was demonstrated as being the antigen-specific T-cell population and activated CD4+CD25+ T cells as well as monocytes and B cells (Jutel et al. 1995). Consistently, the increase in IL-10 both during specific immunotherapy and natural allergen exposure has been demonstrated (Jutel et al. 1995, 2003; Francis et al. 2003; Akdis, M. et al. 2004). A recent study has been performed using IFN-γ-, IL-4- and IL-10-secreting allergenspecific CD4+ T cells that resemble Th1, Th2, and Tr1-like cells, respectively. Healthy and allergic individuals exhibit all three subsets, but in different proportions. In healthy individuals IL-10-secreting Tr1 or IL-10-Treg cells represent the dominant subset for common environmental allergens, whereas a high frequency of allergen-specific IL-4-secreting T cells (Th2-like) is found in allergic individuals (Akdis, M. et al. 2004). Hence, a change in the dominant subset may lead to either the development of allergy or recovery. Peripheral tolerance to allergens involved multiple suppressive factors such as IL-10, TGF-β, cytotoxic T lymphocyte antigen (CTLA)-4, and programmed death-1 (PD-1) (Akdis, M. et al. 2004). Accordingly, allergen-specific peripheral T-cell suppression mediated by IL-10 and TGF-β and other suppressive factors, and a deviation toward a Treg cell response was observed in normal immunity as a key event for the healthy immune response to mucosal antigens. The analysis of other IL-10 family cytokines such as IL-19, IL-20, IL-22, IL-24, and IL-26 demonstrated that suppressor capacity for allergen/ antigen-stimulated T cells is only a function of IL-10 in this family (Oral et al. 2006). Successfully treated patients develop specific T-cell unresponsiveness against the entire allergen as well as T-cell epitope-containing peptides. These decreased proliferative responses do not arise from deletion as they are restored by

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the addition of IL-2 and IL-15. However, unlike in mucosal allergies no increases in TGF-β production during specific immunotherapy were observed in venom allergy. Differences in the control mechanism that regulates immune responses to venoms and aeroallergens might be due to different routes of natural allergen exposure as well as the induction of chronic events of allergic inflammation, leading to tissue injury and remodeling in the latter case. Apparently, T cells, which become predominant during specific immunotherapy and natural antigen exposure, represent the Tr1 or IL-10-Treg cells in humans. CD4+ Treg cells that specialize in the suppression of immune response are pivotal in maintaining peripheral tolerance (Read & Powrie 2001; Barrat et al. 2002; Shevach 2002; Wood & Sakaguchi 2003). Treg cells are enriched within the CD4+CD25+ cells (Ke & Kapp 1996; Strober et al. 1996; Weiner 1997, 2001). Increases in numbers of CD25+ cells in the skin and nasal mucosa were also observed (Varney et al. 1993; Francis et al. 2003). In humans, there is circumstantial evidence to suggest that Treg cells play a major role in the inhibition of allergic disorders. It has been reported that IL-10 levels in bronchoalveolar lavage fluid of asthmatic patients are lower than those in healthy controls, and that T cells from children suffering from asthma also produce less IL-10 mRNA than T cells from control children (Borish et al. 1996; Koning et al. 1997). Although some reports imply a role for TGF-β in the pathogenesis of asthma, particularly in remodeling of injured lung tissue in humans (Vignola et al. 1997), a recent report indicated that the increased allergic inflammation observed after blocking of CTLA-4 is clearly associated with decreased TGF-β levels in bronchoalveolar lavage fluid of mice (Hellings et al. 2002). In the vast majority of studies, cultures of PBMCs were examined. The question of whether this reflects the changes in the immune response in the mucosal tissues is of interest. T-cell responses after grass pollen immunotherapy have been examined in nasal mucosal and skin tissue. Increased IL-10 mRNA-expressing cells after specific immunotherapy with grass pollen during the pollen season was demonstrated. However, unlike the findings in the periphery, IL-10 was not increased in nonatopic subjects exposed during the pollen season. Increased Th1 activity was demonstrated in both the skin and nasal mucosa (Varney et al. 1993; Hamid et al. 1997; Varga et al. 2000). In addition, there was reduced accumulation of T cells in skin and nose after allergen challenge, but no decrease in T-cell numbers during the pollen season. Increases in IFN-γ observed after allergen challenge outside the pollen season correlated with clinical improvement (Durham et al. 1996). During the summer pollen season, increases in both IFN-γ and IL-5, with the ratio in favor of IFN-γ, were observed (Wachholz & Durham 2004). However, it seems that the demonstration of modulation of peripheral immune responses is pivotal for the effects of specific immunotherapy. Local tissue responses do not necessarily reflect peripheral tolerance and are dependent on a number of mechanisms

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such as cell apoptosis, migration, homing and survival signals, which very much depend on natural allergen exposure and environmental factors (Akdis, C.A. et al. 2004b).

Allergen-specific immunotherapy and Treg cells influence allergen-specific antibody responses Specific IgE in serum and on effector cells in tissues of allergic patients is a hallmark of atopic disease. Although peripheral T-cell tolerance is rapidly induced during specific immunotherapy, there is no evidence for B-cell tolerance in the early course (Akdis et al. 1996). Natural exposure to a relevant allergen is often associated with an increase in IgE synthesis. Similarly, specific immunotherapy frequently induces a transient increase in serum-specific IgE, followed by gradua1 decrease over months or years of treatment (Gleich et al. 1982; Bousquet et al. 1986; Van Ree et al. 1997). In pollen-sensitive patients, desensitization prevents elevation of serum-specific IgE titer during the pollen season (Lichtenstein et al. 1973; Bousquet et al. 1988). However, the changes in IgE levels can hardly explain the diminished responsiveness to specific allergen due to specific immunotherapy, since the decrease in serum IgE is late, relatively small, and poorly correlated with clinical improvement after specific immunotherapy. The induction of blocking antibodies by specific immunotherapy was suggested as early as the 1930s by Cooke et al. (1935). Lichtenstein et al. (1966) assigned these blocking antibodies to IgG. Research focused on the subclasses of IgG antibodies, especially IgG4, believed to capture the allergen before reaching the effector cell-bound IgE, and thus prevent the activation of mast cells and basophils. In fact, a substantial number of studies demonstrated increases in specific IgG4 levels together with clinical improvement (Flicker & Valenta 2003; Wachholz & Durham 2004). In the case of venom allergy, the rise of antivenom IgG correlates, at least at the onset of desensitization, with protection achieved by the treatment (Golden et al. 1982; Müller et al. 1989). The concept of blocking antibodies has recently been reevaluated. Blocking antibodies seem not only to inhibit allergeninduced release of inflammatory mediators from basophils and mast cells, but also inhibit IgE-facilitated allergen presentation to T cells as well as preventing allergen-induced boost of memory IgE production during high allergen exposure in the pollen season. It has been demonstrated that grass pollen immunotherapy induced allergen-specific, IL10-dependent “protective” IgG4 responses (Nouri-Aria et al. 2004). The data established an absolute association between IgG4-dependent blocking of IgE binding to B cells in patients who underwent immunotherapy and a trend toward a correlation with clinical efficacy. It seems to be relevant to measure the blocking activity of allergen-specific IgG rather than the crude levels in sera. This can explain the lack of correlation between antibody concentration and degree of clinical improvement. However, IgG4 antibodies can be viewed

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as having the ability to modulate the immune response to allergen and thus the potential to influence the clinical response to allergen. In a study using well-defined recombinant allergen mixures, all treated subjects developed strong allergen-specific IgG1 and IgG4 antibody responses (Jutel et al. 2005). Some patients were not sensitized to Phl p5, but nevertheless developed strong IgG antibody responses to that allergen. It has been suggested that subjects without specific IgE against a particular allergen fail to mount a significant IgG4 response (Rossi & Monasterolo 2004), but recent studies do not support this view and are consistent with induction of a tolerant immune response (Jutel et al. 2005). IL-10 that is induced by, and increasingly secreted during, specific immunotherapy appears to counterregulate antigen-specific IgE and IgG4 antibody synthesis (see Table 4.2) (Akdis et al. 1998). IL-10 is a potent suppressor of both total and allergen-specific IgE, while it simultaneously increases IgG4 production (Punnonen et al. 1993; Akdis et al. 1998). Thus, IL-10 not only generates tolerance in T cells but also regulates specific isotype formation and skews the specific response from an IgE- to an IgG4-dominated phenotype. The healthy immune response to Der p1 demonstrates increased specific IgA and IgG4, small amounts of IgG1, and almost undetectable IgE antibodies in serum (Jutel et al. 2003). House-dust mite-specific immunotherapy did not significantly change specific IgE levels after 70 days of treatment; however, a significant increase in specific IgA, IgG1, and IgG4 was observed (Jutel et al. 2003). The increase of specific IgA and IgG4 in serum coincides with increased TGF-β and IL-10 respectively. This may account for the role of IgA and TGF-β as well as IgG4 and IL-10 in peripheral mucosal immune responses to allergens in healthy individuals (Sonoda et al. 1989; Akdis et al. 1998).

Treg cells are involved in the suppression of effector cells and inflammatory responses during specific immunotherapy Long-term specific immunotherapy is associated with significant reduction of not only the immediate response to allergen provocation but also the late-phase reaction (LPR) in the nasal and bronchial mucosa or in the skin. The mechanism of LPR is different from the mast cell-mediated immediate reaction and involves the recruitment, activation and persistence of eosinophils, and activated T cells at sites of allergen exposure. The immunopathologic changes in the mucosal tissues of subjects chronically exposed to inhalant allergens resemble those seen during the late phase. Since LPR is associated with increased bronchial and nasal hyperresponsiveness and mimics the pathologic condition of chronic allergic inflammation, it has been postulated that the effect of specific immunotherapy on the LPR is relevant to its clinical efficacy (Van Bever & Stevens 1989). Successful specific immunotherapy results not only in increase of allergen concentration necessary to induce imme-

diate or late-phase reaction in the target tissue, but also in decreased responses to nonspecific stimulation. Bronchial, nasal, and conjunctival hyperreactivity to nonspecific stimuli, which seems to reflect underlying mucosal inflammation, decreases after specific immunotherapy and correlates with clinical improvement (Rak et al. 1988; Varney et al. 1997). During birch pollen-specific immunotherapy, reduced plasma levels of eosinophil cationic protein (ECP), a marker of eosinophil activation, as well as chemotactic factors for eosinophils and neutrophils correlated with decreased bronchial hyperreactivity and clinical improvement (Rak et al. 1988, 1990). Inhibition by specific immunotherapy of the seasonal increase in eosinophil priming has also been demonstrated (Hakansson et al. 1997). In biopsies taken during grass pollen-specific immunotherapy, decreased eosinophil and mast cell infiltration in nasa1 and bronchial mucosa correlated with the antiinflammatory effect. In addition, plasma concentrations and in vitro production of endothelin-1 (a bronchoconstrictor and proinflammatory peptide) were significantly decreased in asthmatic children after 2 years of immunotherapy with mite extract (Creticos et al. 1985; Chen et al. 1995). The cardinal difference between true atopic diseases like allergic rhinitis, asthma or atopic dermatitis and venom allergy is the lack of many chronic events of allergic inflammation leading to tissue injury and remodeling in anaphylactoid monoallergies (Akdis, C.A. et al. 2004b). Despite the fact that definite decrease in IgE antibody levels and IgE-mediated skin sensitivity normally requires several years of specific immunotherapy, most patients are protected against bee stings at an early stage of bee venom immunotherapy. An important observation, detected from the first injection, is an early decrease in mast cell and basophil activity for degranulation and systemic anaphylaxis. The mechanism of this desensitization effect is unknown. It has been shown that mediators of anaphylaxis (histamine and leukotrienes) are released during specific immunotherapy without inducing a systemic anaphylactic response. Particularly, ultrarush protocols induce significantly increased release of these mediators to circulation (Müller 2001). Their piecemeal release may affect the threshold of activation of mast cells and basophils. Although there are fluctuations and a risk of developing systemic anaphylaxis during the course of allergen-specific immunotherapy, the suppression of mast cells and basophils continues to be affected by changes in other immune parameters such as generation of allergen-specific Treg cells and decreased specific IgE. This is particularly because they require T-cell cytokines for priming, survival, and activity, which are not efficiently provided by suppressed Th2 cells and activated Treg cells (Schleimer et al. 1989; Walker et al. 1991). Peripheral T-cell tolerance to allergens, which is characterized by functional inactivation of the cell to antigen encounter, can overcome both acute and chronic events in allergic reactions. Specific immunotherapy efficiently modulates the thresholds for mast cell and basophil activation and

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decreases IgE-mediated histamine release (Treter & Luqman 2000; Shim et al. 2003). In addition, IL-10 was shown to reduce proinflammatory cytokine release from mast cells (Marshall et al. 1996). Furthermore, IL-10 downregulates eosinophil function and activity and suppresses IL-5 production by human resting Th0 and Th2 cells (Schandane et al. 1994). Moreover, IL-10 inhibits endogenous granulocyte– macrophage colony-stimulating factor (GM-CSF) production and CD40 expression by activated eosinophils and enhances eosinophil cell death (Ohkawara et al. 1996).

Mechanisms of sublingual immunotherapy The immunologic mechanisms of sublingual immunotherapy are less established. In Cochrane analysis (Wilson et al. 2003), the authors concluded that there was an increase in IgG4 but no stable effect on IgE levels in adults. In addition, the induction of allergen-specific IgA has been reported (Bahceciler et al. 2005). There are conflicting data concerning lymphoproliferative responses (Ciprandi et al. 2005; RolinckWerninghaus et al. 2005). So far the evidence on changes in Th1/Th2/Treg activity induced by sublingual immunotherapy need to be confirmed. The effects on T-cell reactivity and cytokine secretion show strong variation in a number of studies. One preliminary study showed reduced T-cell proliferation and peripheral IL-10 production in allergic patients successfully treated with house-dust mite sublingual immunotherapy (Ciprandi et al. 2005). A recently published study showed increased IL-10 mRNA and positive correlation of TGF-β mRNA with IL-10 and negative correlation with IL-5 (Savolainen et al. 2006). Decreased ECP and serum IL-13 after 6 months of sublingual immunotherapy has also been demonstrated (Marcucci et al. 2001). In addition, nasal tryptase secretion decreased after nasal allergen challenge test (Marcucci et al. 2003). During 2 years of sublingual immunotherapy with grass pollen allergens in children, no significant effects on in vitro T-cell immune responses or immunoglobulins were observed, despite a positive effect on rescue medication (Rolinck-Werninghaus et al. 2005). However, due to its well-established safety profile, with more than 600 million doses administered to humans, sublingual immunotherapy is currently considered an alternative to subcutaneous specific immunotherapy.

Histamine type 2 receptor as a major player in peripheral tolerance Histamine is a low-molecular-weight monoamine that binds to four different G-protein-coupled receptors, and has recently been demonstrated to regulate several essential events in the immune response (Jutel et al. 2002; Akdis, C.A. et al. 2004b). The histamine receptor type 2 (HR2) is coupled to adenylate cyclase and studies in different species and several human cells have demonstrated that inhibition of characteristic features of the cells by primarily cAMP formation dominates in HR2-dependent effects of histamine (Del Valle

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& Gantz 1997). Histamine released from mast cells and basophils by high allergen doses during specific immunotherapy interferes with the peripheral tolerance induced during specific immunotherapy in several pathways. Histamine enhances Th1-type responses by triggering HR1 whereas both Th1- and Th2-type responses are negatively regulated by HR2. Human CD4+ Th1 cells predominantly express HR1 and CD4+ Th2 cells HR2, which results in their differential regulation by histamine (Jutel et al. 2001). Histamine induces the production of IL-10 by DCs (Mazzoni et al. 2001). In addition, histamine induces IL-10 production by Th2 cells (Osna et al. 2001), and enhances the suppressive activity of TGF-β on T cells (Kunzmann et al. 2003). All three of these effects are mediated via HR2, which is relatively highly expressed on Th2 cells and suppresses IL-4 and IL-13 production and T-cell proliferation (Jutel et al. 2001). Apparently, these recent findings suggest that HR2 may represent an essential receptor that participates in peripheral tolerance or active suppression of inflammatory/immune responses. Histamine also regulates antibody isotypes including IgE (Jutel et al. 2001). High amount of allergen-specific IgE is induced in HR1-deleted mice. In contrast, deletion of HR2 leads to significantly less allergen-specific IgE production, probably due to a direct effect on B cells and indirect effect via T cells. Long-term protection from honeybee stings by terfenadine premedication during rush immunotherapy with honeybee venom in a double-blind, placebo-controlled trial has been analyzed (Müller et al. 2001). After an average of 3 years, 41 patients were reexposed to honeybee stings. Surprisingly, none of 20 patients who had been given HR1 antihistamine premedication, but 6 of 21 given placebo, had a systemic allergic reaction to reexposure using either a field sting or a sting challenge. This highly significant difference suggests that antihistamine premedication during the initial doseincrease phase may have enhanced the long-term efficacy of immunotherapy. Expression of HR1 on T lymphocytes is strongly reduced during ultrarush immunotherapy, which may lead to a dominant expression and function of toleranceinducing HR2 (Jutel et al. 1997). Administration of antihistamines decreases the HR1/HR2 expression ratio, which may enhance the suppressive effect of histamine on T cells. Further studies are required to substantiate these promising findings supporting the use of antihistamine pretreatment in patients undergoing venom-specific immunotherapy.

Novel allergen-specific vaccines for immunotherapy and evidence for induction of peripheral tolerance Recombinant DNA technology has enabled the cloning of many allergens, thus facilitating investigations aimed at improving efficacy and safety of immunotherapy. Novel developments in allergen-specific immunotherapy are summarized in Table 4.4. The effectiveness of a mixture of five

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Table 4.4 Novel developments for future allergen-specific immunotherapy and evidence for immunologic tolerance. Development

Evidence

Reference

Reconstitution of an extract of five recombinant allergens

Very high levels of specific IgG4 are induced Individuals not sensitized developed IgG4 but not IgE

Jutel et al. (2005)

Hybrid and chimeric reconstitutions of several major allergens in one protein as a T-cell-directed vaccine, which bypasses IgE-mediated effects

Single dose prevents mice from developing IgE against native allergen

Kussebi et al. (2005) Karamloo et al. (2005)

Trimer and fragments of one major allergen

IgG4 is induced

Gafvelin et al. (2005)

Peptide immunotherapy

Induction of IL-10, decreased Th2 cytokines, decreased skin late-phase response

Marcotte et al. (1998) Müller et al. (1998) Haselden et al. (1999) von Garnier et al. (2000) Oldfield et al. (2002)

recombinant grass pollen allergens in reducing symptoms and need for symptomatic medication in grass pollen-allergic patients has been demonstrated (Jutel et al. 2005). In addition, all treated subjects developed strong allergen-specific IgG1 and IgG4 antibody responses. Some patients, who initially were not sensitized to allergen group 5 (Phl p 5), developed strong IgG antibody responses to that allergen. Recently, two studies have demonstrated genetic engineering of several recombinant allergens in one protein, comprising the whole amino acid sequences of major allergens (Karamloo et al. 2005; Kussebi et al. 2005). The major allergens of honeybee (Apis mellifera) venom, comprising phospholipase A2 (Api m 1), hyaluronidase (Api m 2), and melittin (Api m 3) fragments with overlapping amino acids, were assembled in a different order in the Api m (1/2/3) chimeric protein. This vaccine preserved the entire T-cell epitopes, whereas B-cell epitopes of all three allergens were abrogated. In the Api m 1/2 hybrid, both Api m 1 and Api m 2 allergens were engineered end to end, which led to a B-cell epitope abrogated phenotype (Kussebi et al. 2005). Again T-cell epitopes were preserved. In both candiate vaccines, IgE cross-linking leading to mast cell and basophil mediator release was profoundly reduced. Supporting these findings, Api m 1/2 and Api m (1/2/3) induced 100 to 1000 times less type I skin test reactivity in bee venom-allergic patients. Treatment of mice with both novel vaccines led to a significant reduction of specific IgE development toward native allergen, representing a protective vaccine effect in vivo (Karamloo et al. 2005; Kussebi et al. 2005). Another interesting approach was the use of birch pollen major allergen Bet v 1

trimers in a clinical study, which demonstrated that Bet v 1specific IgG1, IgG2, and IgG4 were significantly increased and a significant reduction of Bet v 1-reactive IL-5- and IL-13producing cells was observed (Gafvelin et al. 2005). Immunotherapy using peptides is an attractive approach for investigation of peripheral T-cell tolerance in humans. Short allergen peptides, either native sequences or altered peptide ligands with amino acid substitutions, do not contain epitopes for IgE cross-linking that induce anaphylaxis. There is a considerable rationale for targeting T cells with synthetic peptides based on such T-cell epitopes. To date, clinical trials of peptide immunotherapy have been performed in two allergies and evidence for peripheral T-cell tolerance to whole allergens has been demonstrated (Marcotte et al. 1998; Müller et al. 1998; Haselden et al. 1999; von Garnier et al. 2000; Oldfield et al. 2002). Single amino acid alterations in T-cell epitopes can modify specific T-cell activation and cytokine production (Faith et al. 1999). Rodent studies suggest that, under highly controlled experimental conditions, allergic diseases can be inhibited by altered peptide ligand administration. Whether this is due to Th2 to Th1 immune deviation or the induction of Treg cells remains to be elucidated (Faith et al. 1999; Janssen et al. 2000). Although peptide immunotherapy is theoretically attractive as a means of avoiding IgE-mediated early-phase reactions, it is important to note that serum IgE in allergic individuals may sometimes bind to relatively short linear epitopes of protein allergens (Banerjee et al. 2000). A potential barrier to peptide immunotherapy of allergy is the apparent complexity of the allergen-specific T-cell response in terms of epitope usage

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and dominant epitopes in humans (Rolland & O’Hehir 1998; Grabie & Karin 1999; Woodfolk et al. 2000). However, it is not clear if bypassing the IgE-dependent responses using peptides results in neglect of some important mechanism of allergen-specific immunotherapy, such as mast cell and basophil desensitization and effects of histamine via HR2.

a well-balanced immune response. This might be of great value, but particularly challenging to develop for children at high familial risk of developing allergic disease. In conclusion, by applying the recent knowledge in peripheral tolerance mechanisms, more rational and safer approaches will soon be available for the treatment, prevention, and cure of allergic diseases.

Conclusions Acknowledgments Peripheral T-cell tolerance is the key immunologic mechanism in promoting a healthy immune response to self-antigens and noninfectious nonself antigens. This phenomenon is clinically well documented in allergy, autoimmunity, transplantation, cancer, and infectious disease. Treg cells, both naturally occurring and inducible inhibitory cytokine-secreting Treg, play a crucial role in the maintenance of peripheral tolerance. A fine balance between allergen-specific Treg and Th2 cells is likely to be crucial in determining the development of allergic diseases (Th2 dominant over Treg) versus maintenance of health (Treg dominant over Th2). Existing successful therapies for allergic disease, notably allergen immunotherapy, appear to act by favorably altering this effector to regulatory T-cell balance. Nevertheless this treatment is only successful in selected patient groups and is associated with significant risk of adverse events. A need for novel and improved therapies remains. Allergen-specific Treg cell populations have proven possible, but are difficult to grow, expand, and clone in vitro and it is debatable whether these will ever be considered a viable treatment option in allergic disease. Further preclinical and clinical studies are needed to demonstrate whether the in vitro generation and/or adoptive transfer of Treg cells and/or their related suppressive cytokines may change the course of immunologic disease. It seems probable that studies in autoimmunity and transplantation may inform future strategies for the use of this clinical approach in allergic and asthmatic disease. An attractive alternative therapeutic approach is to induce de novo, boost or restore endogenous Treg numbers and/or function in patients in vivo. While allergen immunotherapy appears to act at this level, improvements to current regimens are under investigation. These include the use of adjuvants or modified allergen preparations in order to increase safety and efficacy. Potential adjuvants being considered include small-molecular-weight compounds, pathogen-derived molecules, and nonspecific agents such as glucocorticoids that promote the generation of Treg cells or increase their suppressive properties. In addition to the treatment of established allergy, it is essential to consider prophylactic approaches before initial sensitization has taken place. Preventive vaccines that induce Treg responses could be developed, and allergen-specific Treg cells, which will become predominant, may in turn dampen both the Th1 and Th2 cells and their cytokines, ensuring

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The authors’ laboratories are supported by the European Union (EURO-Thymaide) (C.H.), the Medical Research Council of Great Britain (C.H.), Asthma UK (C.H.), Swiss National Science Foundation grants (C.A.A.), and Global Allergy and Asthma European Network (GA2LEN).

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Shim, Y.K., Kim, B.S., Cho, S.H., Min, K.U. & Hong, S.J. (2003) Allergen-specific conventional immunotherapy decreases immunoglobulin E-mediated basophil histamine releasability. Clin Exp Allergy 33, 52–7. Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y. & Sakaguchi, S. (2002) Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 3, 135–42. Simon, H.-U. & Blaser, K. (1995) Inhibition of programmed eosinophil death: a key pathogenic event for eosinophilia. Immunol Today 16, 53–5. Skapenko, A., Kalden, J.R., Lipsky, P.E. & Schulze-Koops, H. (2005) The IL-4 receptor alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25+CD4+ regulatory T cells from CD25–CD4+ precursors. J Immunol 175, 6107–16. Sonoda, E., Matsumoto, R., Hitoshi, Y. et al. (1989) Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production. J Exp Med 170, 1415–20. Stampfli, M.R., Cwiartka, M., Gajewska, B.U. et al. (1999) Interleukin10 gene transfer to the airway regulates allergic mucosal sensitization in mice. Am J Respir Cell Mol Biol 21, 586–96. Stassen, M., Jonuleit, H., Muller, C. et al. (2004) Differential regulatory capacity of CD25+ T regulatory cells and preactivated CD25+ T regulatory cells on development, functional activation and proliferation of Th2 cells. J Immunol 173, 267–74. Steinbrink, K., Wolfl, M., Jonuleit, H., Knop, J. & Enk, A. (1997) Induction of tolerance by IL-10-treated dendritic cells. J Immunol 159, 4772–80. Stephens, G.L., McHugh, R.S., Whitters, M.J. et al. (2004) Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J Immunol 173, 5008–20. Stock, P., Kallinich, T., Akbari, O. et al. (2004a) CD8(+) T cells regulate immune responses in a murine model of allergen-induced sensitization and airway inflammation. Eur J Immunol 34, 1817–27. Stock, P., Akbari, O., Berry, G., Freeman, G.J., Dekruyff, R.H. & Umetsu, D.T. (2004b) Induction of T helper type 1-like regulatory cells that express Foxp3 and protect against airway hyperreactivity. Nat Immunol 5, 1149–56. Strickland, D.H., Stumbles, P.A., Zosky, G.R. et al. (2006) Reversal of airway hyperresponsiveness by induction of airway mucosal CD4+CD25+ regulatory T cells. J Exp Med 203, 2649–60. Strober, S., Cheng, L., Zeng, D. et al. (1996) Double negative (CD4− CD8− alpha beta+) T cells which promote tolerance induction and regulate autoimmunity. Immunol Rev 149, 217–30. Sugimoto, N., Oida, T., Hirota, K. et al. (2006) Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int Immunol 18, 1197–209. Taams, L.S., Smith, J., Rustin, M.H., Salmon, M., Poulter, L.W. & Akbar, A.N. (2001) Human anergic/suppressive CD4(+)CD25(+) T cells: a highly differentiated and apoptosis-prone population. Eur J Immunol 31, 1122–31. Tang, Q., Boden, E.K., Henriksen, K.J., Bour-Jordan, H., Bi, M. & Bluestone, J.A. (2004) Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function. Eur J Immunol 34, 2996– 3005. Thomson, C.W., Lee, B.P. & Zhang, L. (2006) Double-negative regulatory T cells: non-conventional regulators. Immunol Res 35, 163–78.

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IgE and IgE Receptors Brian J. Sutton, Andrew J. Beavil, Rebecca L. Beavil and James Hunt

Summary

Introduction

It is 40 years since the fifth and final class of human antibody, immunoglobulin E (IgE), was discovered and shown to be the factor responsible for allergic sensitivity. Since that time, two class-specific receptors have been identified to which IgE binds through the Fc region of its ε chain. FcεRI, the “high-affinity” receptor, is found principally on mast cells and basophils, but also on antigen-presenting and other cell types. Cross-linking of FcεRI-bound IgE by allergen triggers mast cell and basophil degranulation, leading to an immediate hypersensitivity reaction. FcεRII or CD23, the “low-affinity” receptor, is expressed on B cells and other antigen-presenting cells, and as both membranebound and shed soluble forms it is involved in a complex mechanism of IgE regulation that involves interaction with another B-cell surface molecule, CD21. Yet more binding partners for CD23 including various integrins have since been identified and implicated in inflammatory reactions. Thus IgE stands at the center of a network of interactions that mediate both immediate and delayed inflammatory responses following an individual’s encounter with allergen. The IgE network presents many targets for therapeutic intervention in allergic disease, some of which are now beginning to be exploited as detailed structural information becomes available. In 2000, the crystal structure of the complex between a subfragment of IgE-Fc and the IgE-binding region of FcεRI was solved, and 3 years later when the structure of the whole IgE-Fc was determined, it was unexpectedly found to adopt an acutely and asymmetrically bent conformation. These and other data imply that significant structural changes must accompany IgE engagement of FcεRI, and the targeting of these “allosteric” changes represents a novel intervention strategy. Less is known about IgE’s interaction with CD23, but the structure of the IgE-binding domain of CD23 has recently been determined and the sites of IgE, CD21, and integrin binding mapped to distinct parts of the molecule. Thus a detailed description of the IgE network at the molecular level is beginning to emerge, and with it not only a better understanding of the molecular mechanisms of allergic disease but also the prospect of developing new and effective therapeutic agents.

Antibody molecules of the class known as IgE play a crucial role in allergy and allergic diseases. The existence of a blood factor that could transfer sensitivity to allergens was first demonstrated by Prausnitz and Küstner (1921), but only much later was this identified as a novel class of antibody (Ishizaka & Ishizaka 1967; Johansson & Bennich 1967) and named IgE after the erythema that results from allergenic challenge in the skin of sensitized individuals (Bennich et al. 1968). The story of the discovery of IgE, 40 years ago, has been described by Stanworth (1993). Two cell-surface receptors for IgE have since been identified. The first, found principally on mast cells and basophils but also on antigen-presenting and other cell types, binds IgE with an affinity (Ka ≈ 1010 M −1) that is higher than that of any other receptor for an antibody (Metzger 1992). It is termed FcεRI, since it binds to the Fc region of the ε chain of IgE, and is responsible for the release of histamine and other inflammatory mediators in hypersensitivity reactions when allergen cross-links receptor-bound IgE. The second receptor also binds to the Fc region of IgE, but with a lower affinity (Ka ≈ 108 M−1), and is known as FcεRII or CD23, the name that will be used henceforth in this chapter. It was first identified on B cells, but is also found on a variety of inflammatory cells (macrophages, eosinophils) as well as follicular dendritic cells, Langerhans cells, and T cells, and has been implicated in a number of functions including IgE-dependent antigen presentation by B cells to T cells, IgE-dependent phagocytosis, cell adhesion between B and T cells, and B-cell homing in germinal centers (for reviews see Delespesse et al. 1992; Gordon 1993; Conrad et al. 1994; Gould et al. 1997, 2003). A further feature of this receptor is that soluble fragments (sCD23) are released from the cell membrane and display various cytokine activities (Delespesse et al. 1992) including the ability to stimulate B-cell proliferation and specifically upregulate IgE synthesis (Aubry et al. 1992). Since it has also been demonstrated that IgE and IgE–antigen complexes can downregulate IgE levels by means of their interaction with membrane-bound CD23 (mCD23; Sherr et al. 1989), it is clear that this receptor plays an important role in the control of IgE responses (Gould et al. 1997).

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

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The IgE molecule The basic architecture of the IgE molecule, with two identical heavy chains (ε class) and two identical light chains (κ or λ type), is very similar to that of antibodies of other classes. These polypeptide chains fold into tandemly arranged domains of 110 amino-acid residues, each with an intradomain disulfide bond and the characteristic “immunoglobulin fold” structure, as seen in crystal structures of IgG molecules and their fragments and in other members of the immunoglobulin superfamily (Williams & Barclay 1988). This arrangement of polypeptide chains, their interchain and intrachain disulfide bridges, and the nomenclature of the variable (V) and constant (C) domains, are shown in Fig. 5.1. Like IgG, IgE consists of two antigen-binding Fab fragments and a constant Fc fragment, which contains the sites of antibody receptor binding. However, in contrast to IgG, which has a flexible “hinge” segment in each heavy chain between the Fab and Fc regions, IgE has an additional constant domain (Cε2) in each chain, linked by two disulfide bridges. The Fc of IgE thus

VH

VH

VL

Fab

1

Ce 1

C

L

CL

Ce2

Ce2

Ce3

Ce3

Fc

Ce4

Ce4

104

structures of many of these molecules have been determined by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, and their interactions studied by a variety of biophysical techniques. Our aim here is to rationalize the functional properties of these molecules in terms of their structures and mutual interactions, since this understanding at the molecular level may lead to new opportunities for therapeutic intervention to control allergic disease. Indeed, as a result of recent advances in our knowledge of these molecules, new targets for antibody-directed and structurebased drug design strategies have already been identified and are being pursued.

VL

This was demonstrated in vivo with antibodies to CD23 (Flores-Romo et al. 1993), and implied by the results of gene knockout experiments in mice (Fujiwara et al. 1994; Yu et al. 1994). An anti-CD23 antibody (lumiliximab) has recently been shown to reduce patients’ IgE levels in a phase I clinical trial (Rosenwasser & Meng 2005). This functional diversity of CD23, together with the fact that it belongs to the C-type lectin superfamily (see below) and yet does not recognize the carbohydrate moieties of IgE (Vercelli et al. 1989), led to the suspicion that a countermolecule for CD23 must exist in addition to IgE. This was confirmed in 1992 by Bonnefoy, who identified the glycosylated cell-surface molecule known variously as CR2 (complement receptor 2), the EBV (Epstein–Barr virus) receptor, and CD21 (the name that will be used here) as a counterstructure for CD23 (Aubry et al. 1992; Pochon et al. 1992). CD21 is expressed on B cells, follicular dendritic cells, some T cells, and also on basophils, and the pairing of CD23 and CD21 may enhance B cell–T cell adhesion and thus contribute to the upregulation of IgE (Bonnefoy et al. 1993; Gould et al. 1997, 2003). The interaction between these two proteins also constitutes a direct link between the IgE antibody and complement systems. The importance of complement in the generation of antibody responses has long been recognized (Erdei et al. 1991), and CD21 plays a role in B cell proliferation in its own right as receptor for the complement fragments (such as C3dg) generated by antibody-dependent or antibodyindependent complement activation by antigen (Matsumoto et al. 1991; Dempsey et al. 1996; Fearon & Carroll 2000). In 1995 CD23 was discovered to have a link with yet another family of proteins, namely the integrin receptors (LecoanetHenchoz et al. 1995). CD23 binds to the CD11b and CD11c components of integrin receptor complexes CD11b –CD18 (Mac-1; CR3) and CD11c–CD18 (CR4), and this interaction accounts for the ability of CD23 to modulate monocyte activation and enhance the release of proinflammatory agents. Other integrin interactions have since been identified, as discussed below. While FcεRI is primarily considered to be involved in immediate allergic responses, and CD23 in IgE regulation and IgE-dependent antigen presentation, the discovery that FcεRI was present on Langerhans cells (Wang et al. 1992) and eosinophils (Gounni et al. 1994) indicates a closer functional relationship between these two receptors. FcεRI may therefore contribute locally to antigen presentation, and to defense against parasites (Bieber 1994). To complete this apparent reversal of roles, sCD23 was found to enhance histamine release from normal blood basophils by interacting with the CD21 molecules that these cells express on their surface (Bacon et al. 1993). There may therefore be significant functional overlap between FcεRI and CD23. The principal components of this IgE network of interactions (IgE, FcεRI, CD23, and CD21) will now be considered in more detail. In the last few years, the three-dimensional

Ce

PART 1

lgE Fig. 5.1 Polypeptide and domain structure of the IgE molecule. Interchain and intradomain disulfide bridges are shown, and the filled circles indicate the locations of N-linked glycosylation sites. (See CD-ROM for color version.)

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CHAPTER 5

Ve

Vl

nm

Ce1

6 .9

Cl

Ce2

≥10 nm Ce4

Ce3

5.3 nm

consists of a disulfide-linked dimer of the Cε2, Cε3, and Cε4 domains. (A subfragment of IgE-Fc has also been generated in studies to be described below, consisting of a dimer of the Cε3 and Cε4 domains, and the structural homolog of IgG-Fc. We refer to this subfragment as Fcε3-4, to distinguish it from the complete IgE-Fc or Fcε2-4 fragment.) The IgE molecule was initially thought to adopt an extended Y shape, and models were built by homology with IgG in which the Cε2 domain pair appeared to be a “spacer” between the Fab arms and the Fcε3-4 region (Padlan & Davies 1986; Helm et al. 1991). However, spectroscopic studies in solution, in which the distance between chromophores attached to the extremities of the molecule was measured by Förster (fluorescence) resonance energy transfer (FRET; Zheng et al. 1991, 1992), revealed that in fact it has a compact, bent structure. Whereas the distance between the antigen-binding sites and the C-termini of the Cε4 domains is expected to be 17.5 nm in a planar structure, it is in fact only 6.9 nm. This bent structure was found not only for free IgE but also when bound to FcεRI (Zheng et al. 1991, 1992). Thus a more realistic depiction of the molecule is that shown in Fig. 5.2, which incorporates distances from IgE to the membrane surface that were also determined by FRET. This compact structure is consistent with early solution X-ray scattering data (Davis et al. 1990) and has been confirmed by more recent X-ray and neutron scattering studies (Beavil, A.J. et al. 1995). The first crystal structure of an IgE fragment to be determined was that of Fcε3-4, both unliganded (Wurzburg et al. 2000) and as a complex with the soluble extracellular fragment of the IgE-binding α chain of FcεRI (sFcεRIα; Garman et al. 2000). The complex is described below, but the Fcε3-4 structures revealed flexibility in the arrangement of the four domains, with the Cε3 domains adopting a “closed” arrangement relative to the invariant Cε4 domain pair when unliganded (Fig. 5.3a), and a more “open” arrangement when bound to the receptor (Fig. 5.3b). However, these approximately symmetrical fragments (with a local twofold symmetry axis relating the two ε chains) lacked the Cε2 domains, and when the crystal structure of the complete IgE-Fc (Fcε24) was solved (Wan et al. 2002), an acutely bent and asymmetrical conformation was observed. This is shown in Fig. 5.4, where it can be seen that the Cε2 domains are bent back onto the Cε3 domains, with one Cε2 domain even making contact with one of the Cε4 domains. This very compact structure, with an acute bend between the Cε2 and Cε3 domains, agrees with the X-ray and neutron scattering studies of Fcε2-4 free in solution (Beavil. A.J. et al. 1995), and so is not an artifact of the crystallization process. If the structures of the Fcε3-4 fragments (Fig. 5.3a,b) and Fcε2-4 (Fig. 5.4) are compared, it is clear that in the complete IgE-Fc, one of the Cε3 domains adopts the “closed” conformation (chain A in Fig. 5.4; darker blue Cε3 domain), while the other is in the “open” state (chain B in Fig. 5.4; lighter blue Cε3 domain). Thus the IgE-Fc is asymmetric not only with

D2

D1

a

ITAM b

g

g

FceRI Fig. 5.2 Schematic representation of the IgE molecule bound to the extracellular domains of the FceRI a chain. The bent IgE conformation is consistent with the crystal structure of IgE-Fc, and the distances are those determined by fluorescence resonance energy transfer experiments (see text). The locations of immunoreceptor tyrosine activation motifs (ITAM) in the b and g chains are indicated. (See CD-ROM for color version.)

respect to the location of the Cε2 domains on one “side” of the Fc, but also in the disposition of the Cε3 domains. The crystal structure of Fcε2-4 also revealed the conformation of the oligosaccharide component (Wan et al. 2002). The IgE-Fc is glycosylated at Asn394 in each Cε3 domain, which is homologous to Asn297 in the Cγ2 of IgG-Fc and is a conserved site across antibodies of other classes. Unlike IgG, however, which has “complex-type” carbohydrate chains, IgE has “high-mannose” structures at this location, as reported in early studies of myeloma proteins (Baenziger et al. 1974; Dorrington & Bennich 1978) and more recently in an analysis of normal human serum IgE (Arnold et al. 2004). The branched oligomannose chains (not shown in Fig. 5.4) occupy the cavity between the two Cε3 domains and make contact with the Cε4 domains. It may be that the presence of the Cε2 domains prevents access of the processing enzymes, resulting in the high-mannose structures, in contrast to the fully processed complex-type sugars of IgG (Arnold et al. 2004). Another difference between IgG and IgE with respect to Fc glycosylation is that while removal of the Asn297 oligosaccharides in IgG leads to loss of receptor-binding activity, neither FcεRI nor CD23 binding is substantially affected by the absence of Asn394 glycosylation in IgE (Helm et al. 1988; Vercelli et al. 1989; Hunt et al. 2005). In addition to this

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a(1)

a(2) CC′ peptide a(1) Ce3

Ce3

Ce3 N

N

CD23 binding site

(a)

Ce4

Ce4

Ce4

Fig. 5.3 Crystal structures of Fce3-4, sFceRIa, and their complex. (a) Structure of free Fce3-4 showing the Ce3 domains in the “closed” conformation. (b) Structure of the complex between Fce3-4 and sFceRIa, with the Ce3 domains in the “open” conformation. The CC′ loop of the FceRI a(2) domain at the binding interface, and the predicted

B

Cys 241 Ce2 Cys 328 Ce3

Ce4

conserved glycosylation site there are two ε chain-specific N-linked sites, Asn265 in Cε2 and Asn371 in Cε3, as shown in Fig. 5.1. These are not fully glycosylated (Young et al. 1995) and were removed by mutation in the IgE-Fc that was crystallized (Wan et al. 2002).

106

To membrane

Ce4 (c)

(b)

A

a(2)

Ce3

location of the CD23 binding site in the Ce3 domain of IgE, are both indicated. (c) Structure of free sFceRIa showing the a(1) and a(2) domains folded back on each other, in the same conformation (but different view) as in the complex. PDB codes: (a) 1fp5; (b) 1f6a; (c) 1f2q.

Fig. 5.4 Structure of the complete IgE-Fc. Top panels: orthogonal views of IgE-Fc. Lower panels: schematic representations of the structures above, color-coded to identify the individual domains. The lower central schematic diagram indicates the two (“crossed”) interchain disulfide bridges in the Ce2 domains (Cys241–Cys328). The top left-hand structure clearly shows the region of contact between Ce2 and Ce4 domains. PDB code: 1o0v. (See CD-ROM for color version.)

The IgE-Fc structure also revealed the structure of the Cε2 domain pair for the first time. Unlike typical immunoglobulin C domain pairings such as Cε4–Cε4, which have principally hydrophobic and very extensive interfaces, the Cε2–Cε2 domain interface is very much smaller and hydrophilic,

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involving many hydrogen bonds and trapped water molecules that mediate further bridging hydrogen bonds (Wan et al. 2002). Two cysteine residues at positions 241 and 328 form interchain disulfide bridges that link the two Cε2 domains in this interface region, and the crystal structure shows that these are unexpectedly paired in a “crossed” fashion: Cys241 of one chain bridges to Cys328 of the other (Figs 5.1 & 5.4). It is likely however that even with these two disulfide bridges, the absence of an extensive hydrophobic interface allows conformational flexibility between the two Cε2 domains. Many attempts were made to map the binding sites for FcεRI and CD23 in the IgE-Fc employing a variety of techniques, such as peptide inhibition, site-specific mutagenesis, chimeric antibodies with swapped γ- and ε-chain domains, and anti-domain antibodies (reviewed in Sutton & Gould 1993; Sutton et al. 1997). Although there was a consensus that both sites lay in Cε3, some of these results concerning the FcεRI site appeared to be contradictory at the time, locating the site at the Cε2–Cε3 interface (Henry et al. 1997), various surface regions of Cε3 (Presta et al. 1996; Sayers et al. 1998), and even in Cε4 (Stanworth et al. 1990). The location of the FcεRI binding site was finally revealed in the crystal structure of the complex between Fcε3-4 and sFcεRIα (Garman et al. 2000). As can be seen in Fig. 5.3b, the receptor binds across both Cε3 domains, and involves amino acid residues in loops that lie adjacent to the Cε2–Cε3 linker region (although the Cε2 domains were not present in this structure). It is an extensive interface, dominated by hydrophobic interactions with receptor residues on the α(2) domain and the α(1)–α(2) linker region, which accounts for the high affinity of binding. The fact that the receptor binds to both ε chains in a region where they are close together, and also lies partly across the symmetry axis of the Fcε3-4, accounts for the 1 : 1 stoichiometry of binding despite the presence of two identical ε chains in the IgE molecule. We shall refer to the contact regions on the two Cε3 domains as subsites 1 and 2 (corresponding to chains A and B in Fig. 5.4). As described above, both Cε3 domains must open up on receptor binding, but in the Fcε2-4 structure only one of the two Cε3 domains is open and accessible for interaction with receptor (chain B, Fig. 5.4; lighter blue Cε3 domain); the other is closed and inaccessible (chain A, Fig. 5.4; darker blue Cε3 domain), and is held so by the Cε2 domains. Thus if an attempt is made to dock the bent Fcε2-4 onto the receptor, contact only at subsite 2 is possible; this modeling is shown in Fig. 5.5a. For subsite 1 to become available, the Cε2 domains would have to move away from Cε3, i.e., the Fc must “unbend”, to allow the Cε3 domains to move apart as required for the binding mode seen in the complex. Furthermore, engagement at subsite 1 requires a conformational change to occur in the CC′ loop of the α(2) domain of the receptor (Fig. 5.5a, and described in the following section). Thus receptor engagement entails conformational changes involving the Cε3 domains, the Cε2 domains, and the receptor α(2)

FceRI

IgE and IgE Receptors

Residues which interact with FceRI a1 a2

CC’ loop

Ce2 Ce3 Ce2 Ce4 (a)

Fab

Fab

Conformational change

Fab Fab

(b) Membrane

Membrane

Fig. 5.5 Proposed interaction between IgE and FceRI. (a) Model of the interaction between the complete IgE-Fc and the two extracellular domains of sFceRIa. The CC′ loop of FceRI a(2) domain, which must undergo conformational change upon complex formation, is indicated together with the amino acid residues of Ce2 that are known to contact the receptor (see text). (b) Schematic representation of the proposed conformational change in the Fc region of IgE on receptor binding. (See CD-ROM for color version.)

domain. The extensive nature of these changes in quaternary structure, involving the Cε3–Cε4, Cε2–Cε3, and even Cε2– Cε4 interfaces, explains why mutations and antibody binding, and the like, at sites distant from the interaction site were found to affect receptor binding. Evidence in support of this conformational change model comes from observation of a direct involvement of Cε2 residues in receptor binding, in an NMR study with isolated Cε2 and sFcεRIα (McDonnell et al. 2001). This analysis identified a cluster of amino acid residues on the surface of Cε2 (shown in Fig. 5.5a) that makes contact with the receptor; clearly this could only occur if the Cε2 domains move away from the Cε3 domains. This conformational change in the Cε2 domains, and “unbending” of the IgE molecule as it engages the receptor, is shown schematically in Fig. 5.5b. The necessity for all these conformational changes – in Cε2, Cε3, and the receptor CC′ loop – to occur in a concerted manner for IgE to disengage from the receptor, may account for the exceptionally slow dissociation rate, which is at least three orders of magnitude slower than IgG for any of its receptors (Ravetch & Kinet 1991; McDonnell et al. 2001). The binding site for the lower-affinity receptor CD23 has yet to be defined by X-ray crystallography or NMR, but various approaches including inhibition by monoclonal antibody

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binding, peptide inhibition, and generation of chimeric antibodies have all been employed (reviewed in Sutton & Gould 1993; Sutton et al. 1997). The site clearly lies within the Cε3 domain, and a more recent site-specific mutagenesis study identified a key residue in the AB loop region adjacent to the Cε4 domains (Sayers et al. 2004; see Fig. 5.3b). This location, distant from the twofold axis of the Fcε3-4 region and unaffected by the bending of IgE-Fc, is consistent with the observed 2 : 1 (CD23 : IgE) stoichiometry of binding (Shi et al. 1997; McCloskey et al. 2007). The FcεRI and CD23 receptorbinding sites in IgE therefore appear to be distinct, and are located such as to permit different stoichiometries of binding: 1 : 1 for FcεRI and 2 : 1 for CD23. Whereas a 1 : 1 stoichiometry is essential if IgE is not to cross-link FcεRI in the absence of allergen, a 2 : 1 stoichiometry for CD23 enables extended networks to be formed in the membrane between IgE and mCD23, or indeed sCD23 and mIgE, which may be essential for signaling (see below). Another difference between the interactions of IgE with FcεRI and CD23 has recently been discovered. The Cε3 domain, when expressed alone, was surprisingly found to adopt only a partially folded or “molten globule” state, in contrast to other isolated immunoglobulin domains that fold fully. However, on interaction with sFcεRI it takes up its fully folded tertiary structure (Price et al. 2005). The “induced folding” was not seen for CD23 binding, perhaps because this interaction may only involve a local region of sequence. Nevertheless, the Cε3 domain clearly exhibits a plasticity that may well contribute to the conformational changes associated with IgE engagement of FcεRI described above. So far we have considered only the secreted, soluble form of IgE, but it is also expressed in the membrane of B cells committed to IgE synthesis, where it constitutes the B cell receptor (BCR) complex together with the accessory molecules Igα (CD79a) and Igβ (CD79b) that contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic sequences. The cytoplasmic sequence of mIgE does not contain a signaling motif, but is involved in signal transduction events accompanying B-cell differentiation through binding specific protein(s) (Oberndorfer et al. 2006). All membrane immunoglobulin molecules also contain an additional extracellular membrane-proximal domain (EMPD), but in the case of mIgE there are two isoforms, short and long, consisting of 14 and 66 amino acids respectively (Bestagno et al. 2001). The structures of these additional domains, which lie between the Cε4 domains and the membrane (as indicated later in Fig. 5.7d), are unknown, but one (in εsh) and two (in εl) disulfide bridges connect the two ε chains within this domain. The EMPD has been shown to play a role in the control of apoptotic signaling through the εBCR (Pogianella et al. 2006); it is also a potential target domain for raising anti-IgE antibodies that would specifically bind to mIgE-expressing B cells and suppress IgE synthesis (Inführ et al. 2005). The structure and topology of mIgE presentation on the membrane surface

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is not known, although the molecule is presumably bent at the Cε2/Cε3 interface as in secreted IgE. However, both isoforms of mIgE bind to FcεRI when presented on another cell surface and lead to allergen-independent mast cell activation (Vangelista et al. 2005). The sites for CD23 binding are presumably also accessible in mIgE. The implications of these potential cell–cell contacts, mediated by FcεRI or CD23, for the pathology of allergic disease have yet to be explored (Vangelista et al. 2005).

High-affinity receptor (FceRI) FcεRI is found either as a tetramer, αβγ2, on mast cells and basophils, or as a trimer, αγ2, on antigen-presenting cells, monocytes, and eosinophils; expression on platelets and neutrophils has also been reported (reviewed in Metzger 1992; Kinet 1999; Kraft & Kinet 2007), and most recently on airway smooth muscle (Gounni et al. 2005). The two immunoglobulin-like extracellular domains of the α chain contain the IgE-binding activity (see Fig. 5.2 for schematic), and the β chain and two disulfide-linked γ chains each contain an ITAM (DxxYxxLxxxxxxxYxxL). Phosphorylation of the tyrosine residues within these motifs following receptor cross-linking by multivalent allergen initiates interactions with intracellular signaling molecules leading to cell activation (reviewed in Kraft & Kinet 2007). The involvement of the membrane itself in FcεRI signal transduction, through the segregation of ordered regions or “lipid rafts,” and partitioning of the cross-linked receptor together with intracellular membraneassociated components into these rafts, facilitates receptor phosphorylation (reviewed in Holowka et al. 2005). While the γ chains are essential for both cell-surface expression and signal transduction, the β chain appears to act principally as an amplifier for both functions. However, inhibitory properties have also been reported and an alternatively spliced, truncated isoform of the β chain, βT, negatively regulates FcεRI expression (MacGlashan 2005; Abramson & Pecht 2007). Polymorphisms in FcεRIβ have also been found to be associated with atopy and allergic disease, but the way in which they affect β-chain function and that of the whole receptor is not understood (MacGlashan 2005). However, detailed structural information is available for the extracellular domains of the α chain and their interaction with IgE. The crystal structure of uncomplexed sFcεRIα (Garman et al. 1998) revealed that the two domains, α(1) and α(2), were folded back on each other (Fig. 5.3c), in essentially the same conformation as seen 2 years later in the structure of the complex (Fig. 5.3b). The α(1)/α(2) linker region and loops of the α(2) domain form an exposed hydrophobic ridge onto which the IgE molecule binds. This site is carbohydratefree, while seven N-linked oligosaccharide chains cover much of the rest of the domains’ surface. However, one region of the α chain within the IgE-binding site does undergo a structural

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change, and this is the C′ strand as described above. In a comparison of sFcεRIα structures solved in a number of different crystal forms (with 15 different crystal packing environments), the C′ strand and adjacent CC′ loop was the only part of α(2) to exhibit conformational variability (Garman et al. 2001). It is therefore a region of intrinsic flexibility within the domain, and rearrangement of this strand is part of the conformational changes accompanying IgE engagement of the receptor. The crystal structure of the Fcε3-4/sFcεRIα complex is certainly consistent with the topology of the interaction as depicted in Fig. 5.2. However, the disposition of the antigen-binding Fab arms of the antibody and their degree of flexibility relative to the bound Fc region is not known, since no crystal structure for a whole IgE molecule has been determined. Nevertheless, it is clear that there are orientational constraints on the ligands that can effectively crosslink receptor-bound IgE molecules, and these have been investigated with bivalent and trivalent ligands with different length spacers and degrees of flexibility between haptenic groups (Holowka et al. 2007). The results with rigid spacers show a clear dependence of signaling on the distance between cross-linked receptor complexes: the shorter the distance, the greater the response. While the IgE–FcεRI interaction is clearly one target for therapeutic intervention, little is known about the way in which the three or four polypeptides of the receptor interact and function as a unit. Knowledge of the overall architecture of the receptor may well open up alternative strategies. Early spectroscopic studies of IgE binding to the intact cellbound receptor suggested a change in the conformational state of the receptor upon ligand binding (Ortega et al. 1991), but detailed structural data are still unavailable. A peptide derived from complement protein C3a, which inhibited IgEmediated mast cell secretion, was found to target the β chain (Andrásfalvy et al. 2005), although its mode of action has yet to be determined. The functional consequences of IgE binding to FcεRI in the absence of allergen has recently become a matter of considerable interest and debate. Enhanced receptor expression is certainly one well-established consequence of IgE binding on a range of cell types (MacGlashan 2005; Kraft & Kinet 2007), and this has been shown to be due simply to increased half-life of the engaged receptor. However, evidence for activation of signaling pathways by monomeric IgE binding, counter to the prevailing dogma that only cross-linked IgE can initiate a response, has recently been reported. In vitro murine mast cell survival and cytokine production following monomeric IgE binding to FcεRI have been described (Asai et al. 2001; Kalesnikoff et al. 2001). It has been suggested that this may be due to aggregation in the membrane of exceptionally high levels of receptor-bound IgE in these studies, or interaction with other membrane-bound components through multispecificity of the murine antibodies involved (James

IgE and IgE Receptors

et al. 2003; Schweitzer-Stenner & Pecht 2005), but if human IgE displays similar activities, and if this phenomenon occurs in vivo, then there is clearly more to learn about this fundamentally important step in the molecular mechanism of human allergic disease (Kawakami & Kitaura 2005; Abramson & Pecht 2007).

Low-affinity receptor (CD23 or FceRII) This molecule differs from FcεRI and almost all other immunoglobulin receptors, as it does not belong to the immunoglobulin superfamily. It is a type II integral membrane protein (i.e., it has an intracellular N-terminus), with an extracellular domain that is homologous to the C-type (Ca2+-dependent) lectins, a family that includes cell-surface molecules such as the asialoglycoprotein receptor (ASGPR) and the selectins. It is expressed on B cells, various antigenpresenting cells, eosinophils, and platelets, and has been implicated in a range of functions including antigen presentation and the regulation of IgE levels (Kijimoto-Ochiai 2002; Gould et al. 2003). Another distinctive feature of this receptor is that various soluble fragments (sCD23) of different sizes are released from the cell membrane by proteolysis, with their own functional activities. All active sCD23 fragments contain the lectin domain, but differences in overall size, and in particular their oligomeric state, determine their individual functional properties. Between the lectin domain and the single transmembrane segment lies an extensive region of sequence with a repeating pattern of amino acid residues. Not only is this region coded by a tandem array of homologous exons (three in human, four in murine CD23) – presumably the result of gene duplication – but the sequence also exhibits a repeating heptad motif of hydrophobic residues, characteristic of α-helical coiled-coil proteins (Beavil et al. 1992). The motif is compatible with either a two- or three-stranded structure, but the existence of trimers, as shown schematically in Fig. 5.6a, was subsequently confirmed experimentally by chemical crosslinking studies (Dierks et al. 1993; Beavil, R.L. et al. 1995; Chen et al. 2002). The intracellular region of the sequence exists in two forms, a and b, which differ in their first six or seven amino acid residues (not shown in Fig. 5.6a). These two forms result from separate first exons spliced to a common mRNA sequence, and are differentially expressed: CD23a is found on antigen-activated B cells before differentiation into antibodysecreting plasma cells, while CD23b expression is induced by interleukin (IL)-4 on a variety of inflammatory cells as well as B cells (Gould et al. 2003). The two forms have also been shown to mediate distinct activities: endocytosis and recycling to the cell surface in CD23a-expressing cells for antigen presentation (Pirron et al. 1990; Karagiannis et al. 2001), and IgEdependent phagocytosis in CD23b-expressing cells (Yokota

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C-type lectin head D

RG

Ca2 IgE binding site

DG

R

C-terminal tail a-helical coiled-coil stalk

avb5 integrin binding site

N

CD21 binding site C

N N N (a)

CD23

(b)

et al. 1992; Karagiannis et al. 2007). Critical residues responsible for these functional differences have been identified within the N-terminal region (Yokota et al. 1992). At the other end of the molecule, in a C-terminal ‘tail’ beyond the lectin homology region, lies the sequence DGR (a reverse of the more common RGD, Arg-Gly-Asp motif), and a potential adhesion motif (Fig. 5.6a). No counterstructure, nor function, has yet been ascribed definitively to this region of the molecule, which is absent in murine CD23. Returning to the lectin domain, the structure of this region has recently been determined by both NMR (Hibbert et al. 2005) and X-ray crystallography (Wurzburg et al. 2006). Both structures display the canonical C-type-lectin fold (Fig. 5.6b), but there are notable differences between the two studies. Regarding calcium binding, structures of other C-type lectins have revealed two sites, designated Ca1 and Ca2, and although the Ca2 site is better conserved in CD23, the NMR analysis only revealed binding to the nearby Ca1 site. In contrast, the crystal structure, solved both in the presence and absence of calcium, revealed binding only to Ca2 (as shown in Fig. 5.6b). The requirement of calcium for ligand binding is also controversial: binding studies reported together with the NMR analysis showed that calcium was not required for either IgE or CD21 binding, in contrast to earlier reports (Richards & Katz 1990; Pochon et al. 1992). Other conformational differences between the NMR and crystal structures were observed in loop regions surrounding the calcium-binding sites, and even in the packing of the domain’s core, indicative of a molecule with a considerable degree of plasticity. The NMR study allowed the binding sites for both IgE-Fc (Cε3 domain) and CD21 (N-terminal domains D1 and D2) to be mapped (Hibbert et al. 2005), and they were found to lie in entirely different parts of the lectin domain (Fig. 5.6b). In fact it was demonstrated that the CD23 lectin domain could

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Fig. 5.6 Structure of CD23. (a) Schematic representation of membrane-bound CD23, showing the extracellular trimeric a-helical coiled-coil “stalk,” the C-type lectin “heads,” and the C-terminal “tail” containing the reverse RGD motif. The N-linked glycosylation sites near the base of the stalk are shown. (b) The crystal structure of the lectin head domain showing the location of one of the calcium sites (Ca2, see text), and the binding sites for IgE (Ce3 domain), CD21 (D1–2), and the avb5 integrin. PDB code: 2h2t. (See CD-ROM for color version.)

bind both molecules simultaneously. However, the CD21 (D1–2) binding site may well extend into the “tail” region, and evidence for involvement of other parts of the CD21 molecule beyond D1–2 (see next section) may further extend the CD21 interaction region. If, as these reports indicate, other CD21 domains (D5–8) interact in a carbohydrate-dependent manner (Aubry et al. 1994), and if the carbohydrate-binding activity of CD23 occurs in a region homologous to that of other C-type lectins (e.g., mannose-binding protein at the Ca2 site; Weis et al. 1992), then the IgE and full CD21 binding regions may in fact overlap. However, the trimer would still be expected to bind both IgE and CD21 simultaneously. A model for lectin domain trimerization was proposed in the NMR analysis (Hibbert et al. 2005), different from the arrangement seen in other trimeric C-type lectins such as the mannose-binding protein (Sheriff et al. 1994; Weis & Drickamer 1994). Whatever the precise mode of oligomerization, it has also been shown experimentally that binding of monomeric sCD23 fragments to IgE-Fc induces their trimerization and the formation of large complexes (Hibbert et al. 2005; McCloskey et al. 2007). Thus sCD23 could interact with mIgE to form extended networks that may be crucial for signaling, as discussed below. Membrane-bound CD23, with its 15-nm long “stalk,” belongs to a family of trimeric cell-surface receptor molecules with predicted α-helical coiled-coil regions of different lengths, including ASGPR (7.5 nm) and the Kupffer cell receptor (45 nm) (Beavil et al. 1992). A common feature of all these molecules is the enhanced effective affinity (avidity) for their ligands that results from the multipoint attachment afforded by their oligomeric structure. Indeed, there is a striking similarity between the endocytotic role of ASGPR in glycoprotein clearance and the internalization of soluble IgE–antigen complexes by CD23 for antigen presentation.

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The binding of IgE to trimeric CD23 displays biphasic kinetics with two association constants (Ka = 106 M−1 and 108 M−1), which are interpreted as binding that involves either one or two lectin heads respectively (Dierks et al. 1993; Kilmon et al. 2001). Very similar Ka values are reported for trimeric soluble fragments binding to IgE-Fc (Hibbert et al. 2005; McCloskey et al. 2007). Thus although termed the low-affinity receptor, IgE binds to CD23 with an affinity as high as IgG for any of its receptors (Ravetch & Kinet 1991). The stalk region not only acts as a spacer between the lectin domains and the membrane, but also contains the sites of cleavage that release the sCD23 fragments. Figure 5.6a shows that there is an N-glycosylation site near the base of the stalk, and it is close to this site that the first cleavage occurs to produce a 37-kDa fragment; it may be significant that this cleavage site occurs in a region where the heptad repeat pattern appears to break down. Subsequent cleavage generates a 33-kDa fragment and a relatively stable 25-kDa fragment that has no stalk region. A 16-kDa fragment has also been identified that lacks the C-terminal tail and consists only of the lectin domain; this is essentially the fragment that was studied by NMR and crystallography (Hibbert et al. 2005; Wurzburg et al. 2006). All these soluble fragments retain IgE binding activity, but differ with respect to their cytokine activities; thus, while the larger fragments were reported to promote IgE synthesis (Delespesse et al. 1992), the 16-kDa fragment inhibits this activity (Sarfati et al. 1992). It is clear from Fig. 5.6a that while the larger fragments that include regions of the stalk may remain oligomeric in solution, the smaller fragments must be monomeric, and this may account for their functional differences. This has recently been confirmed by a study of sCD23 fragments of defined oligomeric status, and using a trimeric sCD23 stabilized with a leucine zipper motif. While a monomeric sCD23 fragment inhibited IgE synthesis in human B cells, the trimeric sCD23 stimulated IgE production (McCloskey et al. 2007). The principal endogenous protease that releases sCD23 in vivo has now been identified as ADAM 10 (a disintegrin and metalloproteinase; Weskamp et al. 2006; Lemieux et al. 2007), which might therefore be a target for therapeutic intervention. It has also been suggested that the potency of many common allergens may derive in part from their protease activity, and a direct effect of the house-dust mite allergen Der p1 on membrane-bound CD23, with release of a soluble fragment, has been demonstrated (Hewitt et al. 1995; Schultz et al. 1995; Shakib et al. 1998). The precise mode of binding of CD23 to either IgE-Fc or CD21 remains unknown, for no crystal structures of complexes have yet been determined. However, the engagement and cross-linking of CD23 on B cells by IgE immune complexes, shown schematically in Fig. 5.7a, suppresses B cell proliferation (Luo et al. 1991) and IgE synthesis (Sherr et al. 1989). In addition to this downregulatory signal, the binding

IgE and IgE Receptors

Allergen IgE

IgE mCD23

mCD23

mCD23

CD21

(a)

(b)

CD19

sCD23

sCD23

EMPD CD21

CD21 mIgE

(c)

CD19

CD19

(d)

CD21

CD19

Fig. 5.7 Interactions between IgE, CD23, and CD21. (a) IgE binds to the lectin domains of mCD23 through sites in Ce3, and IgE–allergen complexes can cross-link mCD23 leading to downregulation of IgE synthesis. (b) mCD23 and CD21 (part of a signal transduction complex that includes CD19) can form an adhesion pair between different cell types, such as B cell–T cell, or antigen-presenting cell B cell. Glycosylation sites in CD21 and CD19 are indicated; CD23 binding is thought to involve domains D1–2 and D5–8 of CD21, including carbohydrate moieties in D5 and D6. (c) Trimeric sCD23 can cross-link CD21 on the B cell surface, and deliver a signal for B cell proliferation. (d) Trimeric sCD23 can cross-link mIgE and CD21, which are expressed on B cells committed to IgE synthesis, thus providing a potential mechanism for specific upregulation of IgE production. The additional extracellular membrane proximal domain (EMPD) in mIgE is shown.

of IgE and IgE complexes is known to inhibit the release of soluble fragments from CD23 (Lee et al. 1987), which will prevent the potentially upregulatory effects of these fragments on B cell proliferation and IgE synthesis; thus IgE and IgE immune complexes exert their negative effect in two ways. It is clear from the relative dimensions of these molecules and the location of the primary site of proteolysis at the base of the stalk (Fig. 5.7a) that this inhibition of CD23 proteolysis at the membrane by IgE cannot be via a steric mechanism. Rather, an allosteric mechanism, perhaps involving stabilization of the trimeric structure, must be envisaged (Gould et al. 1991, 1997). In contrast to these downregulatory effects on IgE synthesis mediated by mCD23, the upregulatory function of oligomeric sCD23 fragments depends upon their interaction with CD21, which will now be considered.

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Counterreceptor for CD23 (CD21) CD21 (CR2) is a glycosylated membrane protein that consists of a tandem array of homologous domains known as short consensus repeats (SCR), a constituent domain of many complement-related proteins. Each SCR unit consists principally of β-sheet structure, and the whole molecule adopts an extended flexible structure about 40 nm long (Gilbert et al. 2006). It is shown schematically in Fig. 5.7b with the N-linked glycosylation sites indicated and associated with CD19, which together form a signal transduction unit (Fearon 1998; Fearon & Carroll 2000). The crystal structure of the two N-terminal domains, D1–2, in complex with C3d has been solved, revealing the two domains folded back on each other in a bent conformation (Szakonyi et al. 2001). CD23 binding has been localized to D1–2 and D5–8, and shown to involve the recognition of carbohydrate chains attached to domains 5 and 6 (Aubry et al. 1994). Furthermore, only certain glycoforms of CD21 appear to be recognized by CD23 (Aubry et al. 1992), and this may be functionally important if differences in glycosylation exist within the population. However, the carbohydrate specificity of CD23 is not at all understood; an interaction with galactose has been reported (Kijimoto-Ochiai et al. 1994), but recent attempts to detect binding to a range of carbohydrate moieties by NMR were unsuccessful (Hibbert et al. 2005). Functionally, CD23 and CD21 may thus form an adhesion pair, mediating interactions between B and T cells as shown in Fig. 5.7b and contributing to IgE synthesis (Bonnefoy et al. 1993). This same molecular pairing may be involved in the rescue of germinal center B cells from apoptosis, and also antigen presentation since follicular dendritic cells express both CD23 and CD21 (reviewed in Gould et al. 2003). However, in addition to adhesion interactions between cells, trimeric sCD23 could also cross-link CD21, as shown in Fig. 5.7c, and deliver a growth signal to a B cell (as do anti-CD21 antibodies). For other CD21 ligands such as C3 fragments, only multivalent forms cause B cell proliferation, whereas monovalent forms inhibit (Fearon & Carter 1995). Furthermore, the potential of sCD23 to interact with both mIgE and CD21 provides a mechanism for the specific upregulation of IgE; B cells committed to IgE synthesis will express both mIgE and CD21 and thus, as shown in Fig. 5.7d, trimeric sCD23 may cross-link these molecules at the cell surface. This is analogous to antigen–C3d complexes cross-linking IgM and CD21 and rescuing human B cells from apoptosis (Mongini et al. 2003). Since sCD23 is trimeric, and mIgE dimeric with respect to CD23 binding, the potential exists to form an extended network in the membrane, which may be essential as a signaling platform. The discovery of CD21 as a counterstructure for CD23 placed the “low-affinity receptor for IgE” in an entirely new light, and at last provided a mechanism for some of the multifarious

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activities of this molecule, both IgE-dependent and IgEindependent. On account of its role in the regulation of IgE levels, CD23 is clearly a potential target for intervention in allergy therapy, but more needs to be learned about its interaction with CD21 before this becomes a feasible proposition.

Other counterreceptors for CD23: integrins Increased levels of sCD23 have been reported in a variety of inflammatory conditions such as rheumatoid arthritis, and this observation led to the discovery of a specific interaction between sCD23 and the CD11b and CD11c components of the integrin adhesion molecule complexes CD11b–CD18 (CR3) and CD11c–CD18 (CR4) on human blood monocytes (Lecoanet-Henchoz et al. 1995). The interaction of sCD23 with these molecules is Ca2+-dependent, involves carbohydrate, and is partially inhibited by IgE (Lecoanet-Henchoz et al. 1995); sCD23 thus behaves here as a typical C-type lectin. The functional effect of this interaction is the release of proinflammatory mediators such as nitric oxide and cytokines including tumor necrosis factor (TNF)-α (Lecoanet-Henchoz et al. 1995; Aubry et al. 1997). Synthesis of proinflammatory cytokines in human monocytes has since been shown to be mediated by sCD23 interaction with another integrin, αvβ3, the vitronectin receptor (VnR) (Hermann et al. 1999). sCD23 clearly plays a role in the regulation of monocyte activation, and inhibition of these integrin interactions may provide a way to control inflammation; anti-CD23 antibodies have been shown to have such an effect in a mouse model for human rheumatoid arthritis (Plater-Zyberk & Bonnefoy 1995). Yet another integrin-mediated function of sCD23 has recently been reported, namely the sustained growth and survival of human pre-B cells as a result of sCD23 binding to αvβ5, another member of the VnR family (Borland et al. 2007). The nature of this interaction has been investigated in more detail. In common with the other integrins to which sCD23 binds, there is no involvement of an RGD sequence (which CD23 lacks), nor even the reverse (DGR) motif in the C-terminal tail of CD23 (Lecoanet-Henchoz et al. 1995; Hermann et al. 1999; Borland et al. 2007). In fact the αvβ5 binding site in sCD23 was found to include the tripeptide sequence RKC, in which the two basic residues (Arg-Lys) play a critical role (Borland et al. 2007). These amino acids are located in a disulfide-constrained loop near the N-terminal end of the lectin domain, close to the stalk and quite distinct from the IgE and CD21 (D1–2) binding sites (see Fig. 5.6b). The two basic residues are clearly exposed in the NMR and crystal structures and available for integrin binding, but the complete interaction site is likely to be more extensive. There is therefore still no counterstructure identified for the reverse RGD motif, although a peptide containing this sequence taken from the C-terminal tail of human CD23 (as well as another from the base of the stalk) were found to

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interact with major histocompatibility complex (MHC) class II molecules (Kijimoto-Ochiai & Noguchi 2000). The interaction of membrane-bound CD23 with MHC class II molecules has been observed experimentally in relation to receptor endocytosis and recycling on B cells (Karagiannis et al. 2001; Kijimoto-Ochiai et al. 2004). However, an early report of the inhibition of IgE-dependent cytotoxicity against parasites by preincubation of eosinophils with the reverse RDG peptide sequence, or anti-peptide antibodies (Grangette et al. 1989), implies that an adhesion interaction between this motif and another, as yet unidentified, counterreceptor may exist and influence CD23 activity on these cells.

The e-binding protein: galectin-3 The ε-binding protein (or Mac-2) was the original name for a protein found to be associated with mast cells and macrophages, with the ability to bind to both IgE and FcεRI (Liu 1993). It is now known as galectin-3, a member of the galectin family of carbohydrate-binding proteins but with a unique feature, namely a region of tandem proline- and glycine-rich repeats attached to the N-terminus of its carbohydrate recognition domain; it also oligomerizes, including the formation of pentamers (Liu 2005). Its specificity, in common with other members of the family, is for β-galactosecontaining oligosaccharide chains, by which means it can bind both IgE (Zuberi et al. 1994) and FcεRI (Frigeri et al. 1993) to activate mast cells and basophils, presumably by crosslinking receptor-bound IgE, FcεRI, or both. Recent experiments with galectin-3-deficient mice have demonstrated impaired mediator release from mast cells (Chen et al. 2006) and a role for this molecule in airway inflammation and bronchial hyperresponsiveness in a murine model of asthma (Zuberi et al. 2004). Both extracellular and intracellular activities are thus implicated, but whatever the mechanisms involved, it is likely that the particular glycoforms expressed by the proteins to which galectin-3 binds are important (Robertson & Liu 1991). Galectin-3 is one of a heterogeneous group of proteins known collectively as “histamine-releasing factors” (HRF) that appear to play a role in late-phase allergic reactions. Not all HRF bind to IgE, but those that do recognize only particular variants, which may be either different glycoforms or splice variants with additional C-terminal “tailpieces”; the nature of these interactions are poorly understood (for review see Kleine Budde & Aalberse 2003).

The IgE network and prospects for therapeutic intervention Since the discovery of IgE 40 years ago, a network of interacting proteins has been uncovered, revealing a number of

IgE and IgE Receptors

potential targets for therapeutic intervention. Some of these are now being exploited, though none more successfully than IgE itself: omalizumab, a humanized anti-IgE monoclonal antibody is now in clinical use. Here we briefly indicate some recent approaches that have been taken based on knowledge of the structures and interactions of IgE and its receptors. A full discussion of the associated functional studies is outside the scope of this chapter. Inhibition of the IgE–FcεRI interaction is clearly a promising target for intervention, not only for its potential to block the activation of mast cells and basophils but also the process of FcεRI-dependent antigen presentation. Disengagement of IgE from FcεRI also serves to downregulate receptor expression levels and, furthermore, a monoclonal antibody directed against IgE, such as omalizumab, can also act on mIgE-expressing B cells (Chang 2000). All these factors may contribute to the profound reduction in circulating IgE levels and anti-inflammatory effects observed with omalizumab (Holgate et al. 2005). The precise location and extent of the epitope recognized by omalizumab has not been defined, but it lies in the Cε3 domain, and antibody binding blocks both FcεRI and CD23 interactions, thus also inhibiting facilitated antigen presentation by both receptors (Owen 2002). Of course the antibody epitope and the receptor-binding sites may not physically overlap, since the steric bulk of the antibody Fab region may preclude receptor binding, but given that conformational changes occur in the IgE molecule on FcεRI engagement, anti-IgE may prevent FcεRI binding by inhibiting essential conformational changes. The precise mechanism of action of omalizumab on IgE thus remains to be determined. Peptides and peptide-based small-molecule inhibitors that bind to either IgE or FcεRI have been pursued both before and since knowledge of the individual structures and the complex became available (reviewed in Sutton & Gould 1993; Sutton et al. 2000; Sondermann & Oosthuizen 2002). Phage display methods have also been used to identify peptide inhibitors (Nakamura et al. 2001). NMR and crystallographic analyses of a family of peptides discovered by phage display, with affinity for FcεRI and inhibitory activity, revealed that they bound in way that mimicked key features of the hydrophobic interactions utilized by IgE, although the peptides had no sequence homology with the ε-chain sequence (Stamos et al. 2004). An alternative approach is to employ IgE-derived domains (e.g., Cε2–Cε3; Hellman 1994) or shorter peptides (e.g., Cε3 sequences; Wang et al. 2003) to elicit an anti-IgE response. One early example of this was the use of a peptide derived from the Cε4 domain (Stanworth et al. 1990), a location far removed from the receptor-binding site (Fig. 5.3b). At the time it was not clear how antibodies directed to this region could affect receptor binding, but we now know that in the bent structure of the Fc region this Cε4 peptide includes a loop that makes contact with the Cε2 domain (see Fig. 5.4),

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and so such anti-IgE antibodies would clearly affect the ability of the IgE molecule to undergo conformational change. Small molecules that similarly prevent allosteric change in the IgE molecule may also be effective. The conformational change in the CC′ loop of the receptor is another potential target. This peptide, a key region of interaction with IgE, has also been synthesized in a constrained cyclic form, shown to mimic its structure in the native receptor domain, and found to inhibit mast cell activation (McDonnell et al. 1996). The entire sFcεRIα fragment has also been shown to inhibit passive cutaneous anaphylaxis reactions in vivo in mice either in this form (Naito et al. 1996) or as an antibody-like fusion protein with sFcεRIα in place of the Fab regions (HaakFrendscho et al. 1993; Vangelista et al. 2002). CD23 and its array of interaction partners present a complex target for therapeutic intervention. As mentioned above, clinical trials of the anti-CD23 antibody lumiliximab demonstrated sustained suppression of IgE levels (Rosenwasser & Meng 2005). Such an antibody can cross-link mCD23 and deliver a downregulatory signal for IgE synthesis, as discussed above. Alternative strategies to target IgE upregulation by sCD23 (Fig. 5.7d) may be envisaged now that the interaction sites for IgE and CD21 are known. sCD21 fragments also have the potential to interfere with the sCD23–CD21 interaction, and inhibition of IgE synthesis has been reported for such a molecule (Frémeaux-Bacchi et al. 1998) following earlier studies of immune response suppression by soluble chimeric proteins containing the extracellular domains of CD21 (Hebell et al. 1991). Stabilization of mCD23 to prevent the release of sCD23 might present yet another therapeutic strategy (Ford et al. 2006), but we still lack a sufficiently detailed understanding of the trimeric structure of the molecule and its stalk region to design such an agent. Our knowledge of the structures and functions of IgE and its receptors has advanced dramatically in the past few years, and we can expect similarly detailed descriptions of the structures of the IgE receptor and counterreceptor complexes to emerge in the years ahead. These structural data, together with a better understanding of the molecular mechanisms of the allergic response, offer prospects for developing specific small-molecule inhibitors. However, monoclonal antibodies, in particular the anti-IgE omalizumab, are proving themselves to be exciting prospects as new therapeutic agents, clearly validating IgE and its receptor interactions as targets for intervention in allergic disease.

Acknowledgments The authors wish to thank the Medical Research Council, the Wellcome Trust, and Asthma UK for supporting their work in this field. All are members of the MRC and Asthma UK Centre for Allergic Mechanisms of Asthma.

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IgE and IgE Receptors

Sutton, B.J. & Gould, H.J. (1993) The human IgE network. Nature 366, 421–8. Sutton, B.J., Beavil, R.L., Beavil, A.J., Young, R.J. & Gould, H.J. (1997) The structural basis of IgE–receptor interactions in the allergic response. In: Roberts, A.M. & Walker, M.R., eds. Allergic Mechanisms and Immunotherapeutic Strategies. John Wiley & Sons, Chichester, UK, pp. 17–31. Sutton, B.J., Beavil, R.L. & Beavil, A.J. (2000) Inhibition of IgE– receptor interactions. Br Med Bull 56, 1004–18. Szakonyi, G., Guthridge, J.M., Li, D. et al. (2001) Structure of complement receptor 2 in complex with its C3d ligand. Science 292, 1725– 8. Vangelista, L., Cesco-Gaspere, M., Lorenzi, R. et al. (2002) A minimal receptor-Ig chimera of human Fc epsilon RI alpha-chain efficiently binds secretory and membrane IgE. Protein Eng 15, 51–7. Vangelista, L., Soprana, E., Cesco-Gaspere, M. et al. (2005) Membrane IgE binds and activates FcεRI in an antigen-independent manner. J Immunol 174, 5602–11. Vercelli, D., Helm, B.A., Marsh, P. et al. (1989) The B-cell binding site on human immunoglobulin E. Nature 338, 649–51. Wan, T., Beavil, R.L., Fabiane, S.M. et al. (2002) The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nat Immunol 3, 681–6. Wang B., Rieger, A., Kilgus, O. et al. (1992) Epidermal Langerhans cells from normal human skin bind monomeric IgE via FcεRI. J Exp Med 175, 1353–8. Wang, C.Y., Walfield, A.M., Fang, X. et al. (2003) Synthetic IgE peptide vaccine for immunotherapy of allergy. Vaccine 21, 1580–90. Weis, W.I. & Drickamer, K. (1994) Trimeric structure of a C-type mannose-binding protein. Structure 2, 1227–40. Weis, W.I., Drickamer, K. & Hendrickson, W.A. (1992) Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 360, 127–34. Weskamp, G., Ford, J.W., Sturgill, J. et al. (2006) ADAM 10 is a principal “sheddase” of the low-affinity immunoglobulin E receptor CD23. Nat Immunol 7, 1293–8. Williams, A.F. & Barclay, A.N. (1988) The immunoglobulin superfamily: domains for cell surface recognition. Annu Rev Immunol 6, 381–405. Wurzburg, B.A., Garman, S.C. & Jardetzky, T.S. (2000) Structure of the human Cε3-Cε4 reveals conformational flexibility in the antibody effector domains. Immunity 13, 375–85. Wurzburg, B.A., Tarchevskaya S.S. & Jardetzky, T.S. (2006) Structural changes in the lectin domain of CD23, the low-affinity IgE receptor, upon calcium binding. Structure 14, 1049–58. Yokota, A., Yukawa, K., Yamamoto, A. et al. (1992) Two forms of the low-affinity Fc receptor for IgE differentially mediate endocytosis and phagocytosis: identification of the critical cytoplasmic domains. Proc Natl Acad Sci USA 89, 5030–4. Young, R.J., Owens, R.J., Mackay, G.A. et al. (1995) Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants. Protein Eng 8, 193–9. Yu, P., Kosco-Vilbois, M., Richards, M., Köhler, G. & Lamers, M.C. (1994) Negative feedback regulation of IgE synthesis by murine CD23. Nature 369, 753–6. Zheng, Y., Shopes, B., Holowka, D. & Baird, B. (1991) Conformations of IgE bound to its receptor FcεRI and in solution. Biochemistry 30, 9125–32.

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Immunoglobulin Gene Organization and Expression and Regulation of IgE Hannah J. Gould and David J. Fear

Summary The allergic response is triggered by allergen-induced cross-linking of immunoglobulin E (IgE) antibodies attached to their highaffinity receptor, FcεRI, on mast cells in the target organ. The immediate response (anaphylaxis) is caused by the sudden release of stored mediators, notably histamine. Cytokines, chemokines, and proteases, simultaneously released, act in concert with newly generated arachidonic acid metabolites to initiate a cascade of inflammatory reactions (allergic inflammation) at the site of allergen provocation, thereby engendering chronic inflammation in some individuals. IgE is synthesized and secreted by B cells and sequestered by FcεRI on mast cells, Langerhans cells, and dendritic cells in tissues. These cells constitute the first line of defense against pathogens in the environment; allergies are seen as the price paid for the emergence of this protective effect during the evolution of mammals. Several processes – local B-cell synthesis of IgE antibodies, the transport of IgE carried on its low-affinity IgE receptor (CD23) by cells into tissues, the homing of mast cells to tissue, and the upregulation of FcεRI by IgE in situ – account for the greater abundance of IgE in tissues than the circulation. Were it not for the sequestration of IgE antibodies in tissues, they would be more dangerous than they actually are, for cross-linking of FcεRI on circulating basophils could then cause systemic rather than local anaphylaxis. How then is IgE regulated and how does it come to be concentrated in the target organs of allergy? This chapter outlines what is known about the mechanisms responsible for the regulation and anatomic distribution of IgE. We discuss the underlying principles of antibody structure and immunoglobulin gene organization and expression, including V(D)J recombination, receptor revision, somatic hypermutation, class switch recombination, and the epigenetic mechanisms governing gene expression by chromatin remodeling and nuclear packaging. We survey the role of antibodies in immune defense; the constitution of the antibody repertoire; the development of B cells and their differentiation into memory B cells and immunoglobulin-secreting plasma cells; and cell trafficking. All these factors converge on the regulation of IgE

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

in the broader context of the immune system and pathologic manifestations. We point to the evidence that lymphoneogenesis may be an important stage in the pathogenesis of allergy. A more comprehensive understanding of IgE regulation should inspire new approaches to the therapy of allergic disease.

Innate and adaptive immunity The adaptive immune system, the branch of the immune system that deploys antibodies in immune defense, evolved in cartaliginous fish some 500 million years ago. The origins of this development were the evolution of the recombinationactivating genes, RAG1 and RAG2, and of activation-induced cytidine deaminase (AID). Both contributed to the extraordinary diversity and adaptability of the antibody repertoire, RAG1/RAG2 through V(D)J recombination and receptor editing and receptor revision, and AID through somatic hypermutation (SHM) and class-switch recombination (CSR). Innate immunity, which preceded adaptive immunity in evolution, relies on pattern-recognition receptors (PRRs). These are encoded in the genome of all cells in the body (though specifically expressed only in certain types of cells), and evidently evolved under selection pressure to confer protection against pathogens expressing pathogen-associated molecular patterns (PAMPs). In the course of evolution, innate and adaptive immunity coevolved to act synergistically in immune defense. We consider here some examples of synergy in relation to IgE.

Antibody repertoire: specificity and function The antibody repertoire of mammals is estimated to comprise 1011, antigen specificities, sufficient for the recognition of essentially any antigen (Janeway & Travers 2003). This number far exceeds the coding capacity of the inherited genome and indeed the capacity of the cell nucleus. To overcome this obstacle, pieces of the genes, one each from two or three of the families encoding the variable (V)-region sequence of the antibody are joined (V(D)J recombination)

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to create combinatorial diversity, simultaneously changing the sequences at the junctions to generate junctional diversity. Further diversity is generated by the random pairing of heavy (H) and light (L) chains. Finally, somatic mutations are introduced into regions of the gene encoding the antigenbinding site (i.e., SHM). The antibody function is changed by recombination of the expressed H-chain V-region sequence, which encodes the antigen-binding site, with the H-chain constant (C)-region sequence of another antibody “class,” while preserving antigenic specificity (i.e., CSR). All B cells first express IgM, in which VH is linked to Cμ. Other CH genes are arrayed downstream of Cμ on the same chromosome, and the expressed VH sequence is recombined with one or a series of these during the lifetime of the B cell or B-cell clone, to express an antibody of another class (IgG, IgA or IgE). Each antibody class is recognized by cognate receptors, expressed differentially on different types of antibody effector cells that vary widely in abundance according to their locations in the body. This diversifies the antibody’s effector functions. Each B cell expresses a single antibody in terms of antigen specificity and class, at any given time, although both the V regions and H-chain CH region may change in that cell and in its progeny (the clone) during the course of an immune response. The whole population of B cells embodies the complete antibody repertoire of an individual at any given time. Specific antibodies of one class or another can be concentrated in different anatomic compartments, e.g., IgM and IgG autoantibodies in the target organs of autoimmune disease and allergen-specific IgE antibodies in the target organs of allergy.

B-cell development and differentiation B cells and T cells descend from a common lymphoid stem cell in the fetal liver or adult bone marrow. While in these locations, the B cells go through an ordered program of gene rearrangements to establish the primary antibody repertoire. H-chain genes are rearranged first in pro-B cells. The H chain is expressed with a surrogate L chain on the cell surface, which generates a signal for κ L-chain gene rearrangement in pre-B cells. Failure of this rearrangement leads to λ L-chain gene rearrangement. If H and L chains are compatible and a functional immunoglobulin is expressed on the cell surface, as a potential antigen receptor, the B cell is subject to negative selection. Cells that produce autoantibodies (estimated to be 40–60% of the total number) undergo receptor editing, whereby one of the remaining V-region sequences is recombined with the C-region sequence to generate a new specificity. If all the V regions are used up in this process, the B cells undergo programmed cell death (apoptosis). The B cells that escape negative selection migrate from the liver or bone marrow into the circulation.

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The circulating B cells are routed through lymphoid tissue, which in birds and mammals is configured to facilitate SHM, the positive selection of B cells expressing antibodies with the highest antigen affinity, and amplification of the successful clones (“affinity maturation”), and CSR. The proliferating cells differentiate into memory B cells or immunoglobulinsecreting plasma cells. The majority of B cells that have failed to undergo positive selection in the germinal centers undergo apoptosis. The continuous outpouring of B cells from the marrow ensures that dead and dying cells are replaced and the antibody repertoire is renewed. The topography of lymphoid tissue, the trafficking of cells through various subcompartments, and the dynamics of “germinal center reactions,” SHM and CSR, have long been subjects of intense interest (Przylepa et al. 1998; Tarlinton & Smith 2000). Dendritic cells capture and process antigens in the periphery and migrate to the spleen and lymph nodes that drain the various anatomic sites. There they trap, and present, antigenic peptides to cognate T cells. The activated T helper cells interact with cognate B cells and stimulate proliferation, accompanied by SHM. The resulting B-cell clones are the founders of the germinal centers, where the cells compete for antigen presented by follicular dendritic cells (FDCs). FDCs are formed from their stromal cell precursors in lymphoid tissue during the immune response. FDCs acquire antigens by displaying antibody receptors (FcR) and complement receptors (CR2 or CD21) on their surface. The FcRs bind antigen–antibody complexes, while CD21 binds to fragments of the C3 component of complement (e.g., 3dg, which has first been covalently coupled to antigens during the complement cascade) to present antigen to the B cells. The FDCs provide survival signals, which lead to clonal amplification of B cells expressing the antigen receptors of highest affinity. Following affinity maturation, some of the selected B cells differentiate into long-lived memory B cells or immunoglobulin-secreting plasma cells. The B cells that participate in the germinal center reactions are derived in the first instance from naive B cells from the primary antibody repertoire. However, memory B cells can undergo affinity maturation within the germinal centers, and also after recirculating through the lymphoid tissue. Germinal center reactions afford a mechanism for boosting the affinities in the primary antibody repertoire and tailoring the antibody for its required function.

Plasma cell differentiation Plasma cells are dedicated to the task of synthesizing and secreting thousands of copies per second of clonospecific antibodies (Schibler et al. 1978). Up to half of all protein synthesis may go into the formation of these antibodies. Differentiation of B cells into plasma cells is seldom recognized as an important element in the regulation of IgE; we hold this to

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Plasma cell

Antigen receptor signaling

BLIMP1

BCL6

PAX5 XBP1 Regulates: Ig CD19, AID expression

Regulates: ER production Ig secretion

Responses to cytokines and AgR signals

Cessation of cell cycle

Cell cycle progression SHM

High-level Ig secretion

CSR

Fig. 6.1 Transcription factors involved in plasma cell differentiation. The transcription factor BLIMP is the master regulator of plasma cell differentiation. It is inhibited by BCL-6, which is downregulated by antigen receptor (AgR) signaling. This allows expression of BLIMP1, which represses PAX5, required for the B-cell transcription program. Inhibition of PAX5 allows the expression of XBP1, which, together with BLIMP1, switches the cell to the plasma cell program. ER, endoplasmic reticulum. See text for definition of other abbreviations.

be a serious oversight, in terms of our understanding of IgE regulation, and also therefore of the search for therapeutic targets. Newly generated plasma cells migrate from the lymphoid tissue into the bone marrow, where stromal cells provide a survival niche for a limited population, renewed during each immune response (Bernasconi et al. 2002). Memory B cells can also differentiate into plasma cells at sites of antigen chalFig. 6.2 Structure of the immunoglobulin classes expressed in mammals. Variable domains are shown in light blue and constant domains in dark blue. Glycosylation sites are represented by hexagons. The positions of interchain disulfide bonds are represented by black lines. IgG, IgD, and IgA each have a flexible “hinge region” linking the Fab and Fc regions, which is replaced by an extra immunoglobulin domain in IgM and IgE.

IgG

lenge. The terminal differentiation of B cells into nondividing plasma cells involves extensive changes in gene expression and cell morphology, in which transcription factors play a critical part (Shapiro-Shelef & Calame 2005) (Fig. 6.1). The master regulator of plasma cell differentiation is Blymphocyte maturation protein 1 (BLIMP1). BLIMP1 switches off transcription factors vital to the B-cell precursors, including paired box gene 5 (PAX5), required for the expression of Igα (a signaling subunit associated with membrane immunoglobulins), CD19 (a signaling subunit associated with CD21), and B-cell linker protein (BLNK). This in turn leads to cessation of the cell cycle, repressing genes such as MYC and genes required for cell signaling through the antigen receptor, for T cell–B cell interactions, and for CSR and SHM. BLIMP1 switches on the expression of positive transcription factors, such as octamer-binding protein (OCT)-1 and interferon regulatory factor (IRF)-4, required for high-level immunoglobulin H- and L-chain gene transcription. It also induces a switch from the expression of membrane-bound to secreted immunoglobulins, at the level of differential splicing and chain termination of the H-chain mRNA precursor. PAX5 represses X-box binding protein 1 (XBP1), and the expression of XBP1 in the absence of PAX5 leads to the expression of genes involved in targeting secretory vesicles to the plasma membrane. XBP1 also acts to increase the size of the endoplasmic reticulum, a prominent feature of plasma cells, required for the high rate of immunoglobulin mRNA translation. BLIMP1 itself is suppressed by B-cell lymphoma (BCL)-6, which is present at high levels in germinal center B cells, but declines during their differentiation into plasma cells. BCL-6 is required for maintaining the B-cell phenotype by inhibiting BLIMP1 expression. Signals from the antigen receptor lead to the degradation of BCL-6, thereby initiating the plasma cell differentiation program.

Antibody structure and function L chains comprise two, and H chains (in IgD, IgG, and IgA) four or (in IgM and IgE) five immunoglobulin domains (Fig. 6.2). Immunoglobulin domains consist of two β-pleated sheets, formed by antiparallel β-strands, connected by loops. Intrachain and interchain disulfide bonds stabilize this structure. The two N-terminal domains of the H chain and of the L IgM

IgD

IgA

IgE

Fab L Fc H

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chain are aligned and associated, forming the two arms (the Fab region) of the antibody, while the remaining H-chain domains are aligned and paired to form the stem (the Fc region). The N-terminal domains of the H and L chains make up the antigen-binding site, and the Fc contains the receptor and complement-binding sites. Because each B cell is committed to the synthesis of one particular H and one L chain at any given time, the two antigen-binding sites are identical. Although there are two parent chromosomes, only one immunoglobulin locus is generally expressed, due to “allelic exclusion” (see below). The antigen-binding sites or complementarity determining regions (CDRs) in the H- and L-chain V domains are formed by three adjacent loops in the three-dimensional structure at the distal end of the V domains. The DNA sequences encoding the CDRs are particularly prone to SHM, increasing diversity without compromising the immunoglobulin framework regions of the V domain. Cell-receptor and complement binding sites face outwards from the Fc region of the antibody. All antibodies can be expressed in two functional forms, one membrane-bound, the other secreted, differing from one another at their C-termini. The membrane form, which is first expressed, lacks three C-terminal amino acids of the secreted chain, and instead has amino acids making up a linker, membrane, and cytoplasmic sequence. This anchors the antibody in the cell membrane, where the antibody is associated with its two signaling subunits, Igα and Igβ, to become an antigen receptor (Venkitaraman et al. 1991). The cytoplasmic sequence differs between isotypes and may engage in distinct activities in the B cells (Obendorfer et al. 2006) Antibodies are conducted through the cell membrane by leader sequences at the N-termini of the H and L chains, which are cleaved off during transit. Signaling through the antigen receptor complex is crucial for the viability of B cells (Achatz et al. 1997). The binding of antigens can result in cell proliferation, anergy, or apoptosis, depending on the stage of B-cell development and strength of the signal. The antigen receptor also functions in antigen presentation to cognate T helper cells. Receptor-bound antigen is internalized by endocytosis and digested by proteases in the endoplasmic reticulum, and certain of the resulting fragments are transported to the cell surface by major histocompatibility complex (MHC) class II antigens, where they are recognized by the T-cell receptor. The secreted form of the antibody is essential for recruitment of effector cells and for complement activation, leading to elimination of the antigens.

IgE structure and function IgE differs in both structure (see Chapter 5) and function from other antibody classes. Its characteristic properties are related to its affinity for FcεRs (Ka ∼ 1010 M−1 for FcεRI and ∼ 108 M−1 for CD23). It thus binds to FcεRI 102–105 times more

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FceRI

Mast cell

APC Allergen MHC II CD23

lgE

Fig. 6.3 Function of IgE in the first line of immune defense. Two important functions of IgE in mucosal tissues lining the body surfaces and cavities are exerted by antigen-presenting cells (APCs) and mast cells. APCs express the IgE receptors FceRI (in Langerhans cells and dendritic cells) or CD23 (in B cells). IgE antibodies bound to these receptors capture and process allergens and present antigenic fragments to T helper cells in a major histocompatibility complex (MHC) class II antigen-restricted manner. This culminates in the production of more of the same antibodies (positive feedback). Mast cells express very high levels of FceRI, responsible for sequestering most of the IgE, secreted by B cells, in mucosal tissues. Allergen binding to this IgE triggers mast cell activation, releasing the mediators of immediate hypersensitivity and initiating the cascade of allergic inflammation.

strongly than IgG or IgA to their own receptors (Sutton & Gould 1993; Gould et al. 2003). The affinity for CD23 is “low” only in relation to FcεRI, since it has approximately the same affinity as that of IgG for FcγRI; IgG binds to other FcγR forms with much lower affinity. The high affinity of the IgE–FcεRI interaction is important for its function as the first line of defense, with antigen-presenting cells and effector cells, in tissues (Fig. 6.3). Free IgE, even at very low concentrations, can saturate FcεRI on cells which, in contast to FcγR-bearing cells, can consequently exert immediate hypersensitivity to antigens. Mast cells migrate to mucosal tissues and the peritoneal cavity, where they express exceptionally high levels (> 105 molecules/cell) of FcεRI (Prussin & Metcalfe 2006). The presence of IgE-secreting plasma cells in the same tissues (KleinJan et al. 2000; Smurthwaite et al. 2001), and/or the transport of IgE into tissues by CD23-bearing cells, may contribute to this high expression, because IgE protects its receptors against proteolytic cleavage at the cell surface (Lee et al. 1987; MacGlashan 2005). For all these reasons IgE is well adapted to its barrier function in tissues. CD23 expressed on B cells functions in IgE antibodydependent antigen presentation (Kehry & Yamashita 1989; Pirron et al. 1990; Heyman et al. 1993; van der Heijden et al. 1993; Squire et al. 1994; Gould et al. 1997), while CD23 expressed on monocytes and eosinophils functions in IgE antibody cell-mediated cytotoxicity against tumor cells (Karagiannis et al. 2007). CD23 also participates in the transport of IgE to

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

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

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

L2 Vl2

L Vl-30

Jl1

Cl1

Jl2

Cl2

Jl4

Cl4

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

L3 Vk-3

L Vk-40

Jk1–5

Ck

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

L3 VH-3

LH VH-50

DH1–25

JH1–6

Cm

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

D J genes genes

Cm Cd

3’E (Ca1) enhancer Cg3 Cg1

Ye

Ca1

Yg

3’E (Ca2) enhancer Cg2 Cg4 Ce

Ca2

(d)

Germline Ie promoter

Se

Ce1–4 M1,2

Mouse IgH locus Ei (Em) enhancer V genes

D genes

J genes

Cm Cd

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

Ca

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CACAGTG Heptamer GTGTCAC

Vk

VH

GGTTTTTGT CACTGTG Nonamer 12 Heptamer CCAAAAACA GTGACAC

ACAAAAACC Nonamer TGTTTTTGG

23

12

23

23

23

D

12

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

l chain

Jk

k chain

JH

H chain

12

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

L1 V1

Jl

Ln Vn

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

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

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

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

RAG1/RAG2 binds to the 12 bp spacer

DNA–RAG complex recruits the 23 bp spacer

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

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

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

Coding joint

Signal joint

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

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

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

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

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

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

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Transitions at C-G T

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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IeGLT

B cell

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IL-4 CD4 T cell

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Se

IL-4

P P

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AP1

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NK-kB ID2+PAX5

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

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

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

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

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

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

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

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

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IgD+

IgD– BcI6+ Tfh IL-21

+

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IgG1 BcI6

Th2 IL-4

IgG1– IgE+ IgG1+ PB IgG1+ MC

IgE+ PB

IgE+ PC

IgG1+ PC

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

Local IgE regulation in the target organs of allergy

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

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

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

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

Positive feedback

Allergen

CD23

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IgE synthesis

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IgE synthesis

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

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

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

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

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7

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

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

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

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

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

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

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

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

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

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

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

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

Environmental Factors in IgE Production

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

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

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

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

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

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

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

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

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

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

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

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

Airway epithelium IL-10 CD80

CD28

TH1

–

Proinflammatory IL-B, Rantes GM-CSF, others

TH0 APC

TCR

IL-4 IL-13

B cell Antigen DEP

YY

Y

YY

DEP + ROS

Y Specific lgE

YY

TH2

Y

MHC-II

Y

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

Interactions with allergen Diesel exhaust

Y

Diesel exhaust and gases

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

YY

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

Environmental Factors in IgE Production

Y

Y Mast cell

Plasma cell

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

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

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

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

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

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

Environmental Factors in IgE Production

Reactive oxygen species/ oxidative stress

Sensor/detector ASK1?

Keap 1 ?

MAPKs, e.g. JNK

NK-kB kinases

Nrf2

AP-1 RE

NK-kB RE

ARE

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

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

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

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

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

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

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

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

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

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

Environmental Factors in IgE Production

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

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

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

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

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

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

Microbial ligands

TLR2/TLR1 or TLR6

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

TLR3

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

TLR4

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

TLR5

Flagellin

TLR7, TLR8

Guanosine and uridine-rich single-stranded RNA

TLR9

Bacterial and viral DNA

TLR11

Profilin (Toxoplasma gondii )

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

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

Environmental Factors in IgE Production

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

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LPS

Viruses TLR4 EC Mast cell

NKT

Treg CD4+CD25+

BEC

Cell recruitment

mDC

Low High dose of LPS Th1

IL-13

IL-4 (early)

Th2

Increased suppressive function

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

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

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

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

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

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

Environmental Factors in IgE Production

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

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

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

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

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

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

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

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

LPS Viruses

DEP LPS Viruses ETS

DEP DEP Metals Ozone ETS Viruses Parasites

–

+

+ CD80 + CD28 Th0

Th1 +

–

LPS Parasites

– IL-4 IL-13

IL-10

YY

Y

Y

Y

YY

LPS

+

YY

–

+

B cell

Th2

MHC-II + TCR Treg

Y

APC

YY

Y

Y +

+

Mast cell

Plasma cell Antigen Y Specific lgE

DEP Viruses

DEP Metals LPS Viruses

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

Environmental Factors in IgE Production

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